Trade related infections: farther, faster, quieter

Modern global trading traffics large volumes of diverse products rapidly to a broad geographic area of the world. When emergent infections enter this system in traded products their transmission is amplified. With truly novel emergent infections with long incubation periods, such as Human Immunodeficiency Virus (HIV) or variant Creutzfeld Jacob Disease (vCJD), this transmission may silently disseminate infection to far distant populations prior to detection. We describe the chronology of two such "stealth infections," vCJD and HIV, and the production, processing, and distribution changes that coincided with their emergence. The concept of "vector products" is introduced. A brief case study of HIV incursion in Japan is presented in illustration. Careful "multisectoral" analysis of such events can suggest ecologically critical pathways of emergence for further research. Such analyses emphasize the urgency of implementing safety measures when pathogens enter globally traded products.

Introduction
The global trading system today speeds unprecedented volumes of product to unparalleled numbers of markets throughout the world. Coincident with its growth has been the unwelcomed emergence and dissemination of new infections in human populations. This article will examine linkages between these two phenomena through the optic of "stealth" infections -emergent infections with long, silent incubation periods. When such pathogens are carried in a globally traded product (vector product), spectacular geographic amplification can occur prior to the onset of clinically recognizable illness. Examples of these trade-related emerging infections caused by vector products include variant Cruetzfeldt Jacob disease (vCJD) and HIV/AIDS. Vector products can be defined as those products that carry pathogens. It should be noted, however, that "vector" in infectious disease parlance usually refers to living organisms, such as mosquitoes. The use of the term here with regards to product is expedient, albeit non-conforming to normative use.
The drivers for broad marketing of the "vector products" in these two pandemics were 1) promotion and growth of global demand for beef and animal feed and 2) promotion and growth of global demand for human anticoagulant factors for medical treatment. These market drivers provoked a marked "ramping up" of production in pace and capacity for the vector product. The production of these vector products involved pooling of large amounts of biological material. In each case novel pathogens emerged and were disseminated over a broad geographic area prior to their clinical detection.

Mad Cow Disease and vCJD
Global trade trends for the products implicated in the transmission of HIV and vCJD reflect similar scales of increase in volume and dollar amounts during the decade prior to the detection of human diseases.
The incubation period of vCJD is variable, estimated at up to 15-30 years [1]. The incubation period of BSE (Bovine Spongiform Encephalopathy), or mad cow disease, in cattle is also on the order of years, which has complicated culling interventions [2]. Mad cow disease was first detected in British herds in 1986. Wide-scale trade embargoes did not occur until 2000, although some products, such as bull semen, were embargoed as early as 1996, when BSE was first linked to human vCJD [3].
In the case of beef and bone meal, the suspected source of BSE, exports of the implicated product doubled in 1989, from approximately 15,000 tons in 1988 to more than 30,000 tons ( Figure 1) [4]. The surge in exports was at least partly due to precautions taken as a result of concerns about potential disease risk. Meat and bone meal (MBM), a side product of animal slaughter, has long been appreci-ated as a source of protein for animal feed. In fact, it is speculated that BSE was introduced in cattle when scrapieinfected material from sheep was pulverized into cattle feed years ago [5]. Thus when BSE surfaced in the mid-1980s, the UK banned the use of ruminant-derived meat and bone meal in ruminant diets in 1988. However, the feed was still permitted for non-ruminants. The ban caused the price of the British MBM feed to drop, making it more attractive for international trade. Once the feedstuffs moved through Europe, they were used for a variety of purposes, including feed for ruminants; unlike in the UK, in continental Europe, bone meal was a legal commodity for all animal feeds.
Meanwhile, the value of UK beef exports more than doubled between 1985 and 1995 ( Figure 2) [6]. A superficial glance at the data would suggest a ramping up of production; however, the UK beef industry, like most industries, is more complex. In fact, as of 1995, the production of beef in the UK had been static for two decades at about UK Exports of Meat-and-Bone Meal, in tonnes, 1985 -1991* Figure 1 UK Exports of Meat-and-Bone Meal, in tonnes, 1985 -1991*. * legal for swine and poultry feed at the time; above scenario also replicated in other countries. Source: Butler, 1996 [4]. Year Tons one million tons annually [7]. But consumption of beef in the domestic market was declining, partly associated with changing lifestyles. In 1980 Britons consumed 21.3 kg per capita; in 1993 this fell to 17.0 kg per capita. In response, consumer pricing for beef declined, while production and distribution costs remained largely static. Consequently, trade had, by 1995, become an important factor, representing 21 to 23 percent of the total market [7].
Prices fell both in response to declining domestic consumption and the implementation of the General Agreement on Tariffs and Trade (GATT) reforms, which proposed liberalization of agricultural trade through reduced border protection, domestic support payments and export subsidization [7]. The GATT agreements committed economies to reduce tariffs on imports, and in effect, there was price pressure to be competitive to keep their market share at home, to become more efficient.
With margins in the supplying industries squeezed [7], there was market pressure for consolidation of the previ-ously fragmented production system. Farms of 50 cows or more increased from 6,400 to 9,000 between 1986 and 1991.
These changes in processing may have permitted prions to cross the species barrier from ruminant to human. Rendering had been a "batch" procedure in Europe and North America, relying on very high solvent extraction for the production of tallow. In the 1970's, a continuous system of rendering was introduced that relied on vacuum at relatively low temperatures and produced high quality tallow. This efficient process saved costs to the industry. After the BSE crisis, EU studies of abbattoir practices suggested that the processes in place in the United Kingdom in the 1990's were ineffective in deactivating prions [8].

Amplification and Transmission through Trade
The geographic distribution of BSE is broad. If in fact the emergence of this agent occurred only in Britain and bone meal and animal feed contamination occurred only from this source in the late 1980's it is impressive that OIE (World Organisation for Animal Health) lists only four countries currently confirmed as BSE-free [9]. By 2004, in addition to the UK, 22 countries had notified the OIE of cases of BSE in farmed cattle. Cases have been reported largely in Europe but also in North America and Asia [9]. The UK may not in fact have been the sole origin, given a recent report which suggests France had undetected BSE epidemics on a large scale during the same time period [10].
The BSE/vCJD emergence has also impacted the human blood supply. Based on the experience with HIV/AIDS, blood banks are concerned that an agent that cannot be screened for may enter the global blood supply, amplifying infectious transmission. This concern has been heightened by the occurrence of vCJD apparently related to blood transfusion [11]. Thus exclusion criteria in the United States and blood donation fractionation in the UK reflect measures to mitigate this risk.

HIV/AIDS
By the end of 2003 UNAIDS estimated that almost 40 million people were living with HIV/AIDS [12]. While human sexual contact is the major global route of infection of HIV, the history of the pandemic also documents the wide-ranging infection due to infected blood products. Again, innovations in product in response to a global market were apparently correlated with amplification and dissemination of product. In the early 1980's, when AIDS was described in the first cluster of young gay men in San Francisco, ongoing concern about the risk of hepatitis in pooled blood for manufacture of blood factors had spurred producers to a search for a means of screening their product for hepatitis virus. However, viral deactivation processes were not generalized in the industry when HIV emerged; with a profitable international market, the new lyophilized blood product was globally traded.
The commercial sector for the production of blood factors is most developed in the United States. Data from the U.S. Census Bureau attests to a growing trade in blood product from the mid-1970s to the late 1980's, from approximately 50 million USD in 1975 to 325 million USD in 1988. We now know that half of the hemophiliacs in the U.S. were infected with HIV through this route, and an untold number of hemophiliacs worldwide. In some countries, such as Japan, this was probably the primary route of entry into the population. By the end of the 1990's, there were 400 commercial centers for plasmapheresis operating in the U.S. These centers, which employ paid donors, provided 60% of the worldwide requirement for plasma [13].
By mid 1982 the possible link between AIDS and the blood supply was reported in the CDC's Morbidity and Mortality Weekly Report, and was widely known and accepted the following year with the occurrence of cases in hemophiliacs living in geographically dispersed areas. But retooling the fast-growing industry of factor production posed substantial difficulties, for several reasons. 1) The science of HIV/AIDS disease was still early in its evolution, and the data were not therefore clearcut 2) the plasmapheresis industry utilized largely compensated donors (who were often higher risk for HIV/AIDS) as a basis for obtaining plasma, 3) the most heavily impacted group, homosexual males, were wary of discrimination in measures adopted. The initial self-exclusion strategies, for example, did not ask donors about homosexual sexual practice for fear of discrimination.
In early 1983 both the voluntary and commercial sectors had taken some measures for reducing the participation of high risk donors in plasmapheresis. But the Inquiry on the Blood System in Canada, published by Health Canada's Krever Commission in 1997, notes, "There is evidence, however, that the voluntary sector refused to stop collecting in high risk areas, though its blood donor recruiting officials no longer targeted high risk individuals, and that the commercial sector also continued to operate in such areas" [13]. Viral inactivation methods had been in development since the early 1970's to try to cut down on hepatitis transmission in blood. However, the industry leaders considered such steps proprietary information for production and so the work towards successful strategies was not shared across the corporate competitors. In 1984 the major producers had all been licensed to distribute heat-treated products to cut down on the threat of hepatitis and AIDS infection.
As with any product, blood products can be subject to a recall, which is initiated when the public's health is at risk due to contamination of the product. This option was available to the US Food and Drug Administration as soon as the risk of HIV transmission in blood factors became known in March of 1983. An Institute of Medicine study convened years later to review the history was critical of the absence of a cogent, strong recall policy [14].
By 1985 the ELISA screening test for HIV came into use. This, coupled with heat treatment of the factor product, has markedly reduced the risk of HIV transmission in blood. However, global spread of the virus, in part facilitated by the global trade in factor VIII, had already occurred in the 1980's. This contributed to the profound global reach of the epidemic seen today. The experience of Japan illustrates this point.

Japan, Factor VIII and HIV/AIDS
In 1994, the Tenth International AIDS Conference was held in Japan, focusing on subgroups, such as hemophiliacs, that had previously received less attention [15]. Although Japan had been characterized by some as reluctant to acknowledge the introduction of HIV to Japan via "sexual tourism," the virus did initially hit Japan through the blood supply. According to the World Federation of Hemophilia, the majority of Japanese hemophiliacs are thought to have been affected during 1983-1985 by non-heat-treated factor concentrates imported from the US. In fact, by 1985 the US provided 90 percent of imported factor concentrates in Japan [13], and the Japanese AIDS Research Group reported that the causative agent of AIDS was found in factor VIII concentrates [13]. Of 5,000 hemophiliacs in Japan, approximately 2,000 had been infected with HIV/AIDS by 1997 according to the Krever Report. The occurrence of HIV/AIDS through factor concentrates became known in Japan as "Yakugai" AIDS (meaning "AIDS due to the harmful effect of medicine").
As factor concentrates were manufactured by pooling plasma from many donors, the entire pool could be contaminated by a single unit from an HIV-infected donor. Dr. Michael Rodell, former vice-president of the Armour Pharmaceutical Company, estimated that four infected persons could contaminate the entire world supply of factor VIII [13].
The Ministry of Health and Welfare in Japan regulates the national blood system by licensing the use of blood products. In 1983 the Japanese AIDS Research Group, the central technical group advising the Ministry on prevention and control policy, reportedly considered a proposal from the US fractionator Travenol to allow the importation of heat-treated (virus-inactivated) factor VIII. At that time, a new product could receive regulatory approval without undergoing clinical trials if it could be classified as a "change to manufacturing method having no effect on the effective ingredients" [13].
The Ministry rejected the idea, apparently mistrusting the reliability of the US FDA tests and fearing side-effects from heat-treated concentrates [13]. Thus, while US reports in 1983 showed that HIV could be contracted from contaminated factor concentrates, the Ministry continued the import of non-heat-treated factor concentrates, with the stipulation that they be accompanied by a certificate that they did not contain plasma from donors at high risk of contracting HIV/AIDS [13]. The use of imported nonheat-treated concentrates increased in 1984 and peaked in 1985 [13]. Although heat-treated factor concentrates were approved for use in 1985 -because the Ministry recommended that physicians continue to prescribe non-heat-treated factor concentrates (and blood product manufacturers continued to distribute them without warning labels about the risk of AIDS) -many hemophiliacs continued to use non-heat-treated concentrates through 1986 [13]. A further exacerbating problem was the fact that some health care providers neglected to inform their hemophiliac patients of their HIV infection, allowing their sexual partners to become infected [16].
This tragedy in Japan was due to a combination of factors in government, industries, and medical communities [17]. The former chief of the Office of Biologics at the Ministry of Health and Welfare, the head of the AIDS Research Group, and three Green Cross executives were indicted for misconduct and/or professional negligence. Still, the "Yakugai AIDS" scandal is not over, and trials continued through 2004.

Conclusion
The following similarities are thus apparent for infections with very long incubation periods in the modern era of trade and travel: 1) global forces such as market demand or GATT provisions that favor increased exports also favor consolidation, which may set the stage for streamlining processing of product 2) such streamlining of production for efficiency and cost savings may have a role in emergence of new infections when biological materials are the basis for product formulation 3) the emergence of new pathogens that are disseminated through trade and travel creates a "science gap" where rapid research is imperative to find and implement agent-specific safety strategies before extensive infection occurs 4) once clinical disease is manifest widespread dissemination of infection has occurred and risk can be mapped using product specific trade data. This mapping may allow timely institution of surveillance.
Among the most sobering lessons from these two emergent diseases are the consequences of the division between public and private science and the division between the world of health and the world of trade. When heat treatment processes were developed for factor VIII, they were not shared across the industry in an expeditious fashion. Urgent information on product risk and on safety innovations is crucial to public safety and timely public health intervention in disease.
The "sectors" of global trade and global health have few working links. Information flow between the international agencies, the World Trade Organization and World Health Organization, while improving, is not routine. At the regional and national level, intersectoral communications between the trade and health communities is also troublesome. The European Union stands in contrast to this generalization, with extensive collaboration in dis-ease surveillance among its members [18]. The regional initiatives on emerging infections of such regional trading groups as the Asia Pacific Economic Cooperation (APEC), ASEAN and others are encouraging. At the national level, formal collaboration between ministries of commerce and health on product investigation or tracing is sparse.
British authorities were prescient in their implementation of the "Offal Ban" in 1988 [19]. While the risk of vCJD from BSE food animals was not established, they took precautionary measures to limit exposure which were, in retrospect, prudent. At the same time, the issue of potential "opportunistic exporting" of animal feed by the UK onto the international market, in response to the bans imposed domestically on such feed, has been raised. If precautionary recall of factor VIII had been fully implemented in 1986, it is clear that hundreds of cases of HIV may have been averted, and the geographic extent of human infection limited. Currently the costs and effectiveness of the "precautionary principle" in commerce are under discussion [20]. Accurate and complete description of these two global emergent diseases (through mathematical modeling and other research) will be important in informing those discussions.
In summary, two novel infections with long incubation periods, HIV/AIDS and BSE/vCJD, are illustrative in describing the wide geographic dissemination and urgent consequences of emergent infections associated with globalized trade.


s2fieldsofstudy: Biology Chemistry

text:
Rac-GAP-dependent Inhibition of Breast Cancer Cell Proliferation by β2-Chimerin*

β2-Chimerin is a member of the “non-protein kinase C” intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. β2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that β2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of β2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G1 cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of β2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to β2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G1/S progression by β2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.

␤2-Chimerin is a member of the "non-protein kinase C" intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. ␤2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that ␤2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of ␤2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G 1 cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of ␤2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to ␤2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G 1 /S progression by ␤2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.
Chimerins represent a family of four closely related GAPs 1 (GTPase-activating proteins) for small GTPases that were orig-inally characterized as high affinity intracellular receptors for the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters (1)(2)(3)(4). Structurally, chimerins possess a C1 domain highly homologous to those of PKC isozymes (the DAG/phorbol ester binding site) and a C-terminal GAP domain. The ␣2and ␤2-chimerins also have a N-terminal Src homology 2 domain of unknown function, which is not present in the splice variants ␣1-(or n-) and ␤1-chimerins (5,6). Very little information is available regarding the regulation, expression, and function of ␤2-chimerin or the other chimerin isoforms as well as their role in proliferation mechanisms and cancer progression. We have been focusing our attention on ␤2-chimerin, because there is emerging evidence that this isoform is directly regulated by phorbol esters (4,7) as well as tyrosine-kinase receptors that couple to DAG generation. 2 Importantly, early studies in gliomas have suggested a potential role for ␤2-chimerin as a tumor suppressor (8) but its relevance in other cancer models is still unknown.
In vitro studies have shown that the C-terminal domain of chimerins is capable of accelerating GTP hydrolysis from the small GTPase Rac1 without affecting the activity of RhoA or Cdc42 GTPases (9,10). Our recent studies in COS cells revealed that ␤2-chimerin decreases cellular Rac-GTP levels and inhibits the elevation of Rac-GTP levels caused by epidermal growth factor (EGF) (10,11). Rac GTPase is known to act as a molecular switch, cycling between an active GTP-bound state (Rac-GTP) and an inactive GDP-bound state (Rac-GDP). This switch is regulated by three groups of molecules: 1) guanine nucleotide exchange factors, such as Vav and Tiam-1, that promote its conversion to the active GTP-bound form; 2) guanine nucleotide dissociation inhibitors; and 3) GAPs, which stimulate intrinsic GTPase activity, thus leading to Rac inactivation (12,13). Active Rac interacts with various effectors to initiate downstream signaling events that control the dynamics of actin cytoskeleton reorganization, migration, adhesion, and gene expression (14 -17). Rac and Rac-guanine nucleotide exchange factors play key roles in the control of various aspects of malignant transformation and the metastatic cascade in various models, including breast cancer cells (18 -20). Several laboratories (21)(22)(23)(24)(25) have proposed a role for Rac in the control of mitogenesis through its ability to regulate G 1 /S transition and cyclin D1 expression. Moreover, Rac and other members of the Rho GTPase family are overexpressed in human tumors such as in breast cancer (26,27) and hyperactivation of Rac leading to a higher rate of cell proliferation has been found in cellular models of human breast cancer (28). Targeted expression of an activated Rac mutant in mammary epithelium causes mam-* This work was supported by Grants RO1-CA74197 (National Institutes of Health) and RPG-97-092-06-CNE (American Cancer Society) (to M. G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  mary gland lesions (29). In addition, there is strong evidence that Rac effectors such as p21-activated kinase 1 are dysregulated in breast cancer cells (30). Collectively, these findings suggest critical implications of Rac in tumorigenesis, particularly in models of breast cancer.
In this paper, we investigated the expression of the DAG/ phorbol ester receptor ␤2-chimerin in breast cancer and its role in proliferation. We have found that ␤2-chimerin mRNA levels are strikingly reduced in breast cancer cell lines and tissues. By means of adenoviral delivery into MCF-7 breast cancer cells, we have found that ␤2-chimerin, but not the mutated forms lacking Rac-GAP activity, causes a significant impairment in G 1 /S cell cycle progression due to a reduction in the expression levels of cyclin D1. The effect of ␤2-chimerin is strictly dependent on its ability to inhibit Rac function via the C-terminal GAP domain, suggesting the possibility that Racmediated control of cell proliferation is modulated by DAGregulated pathways.

EXPERIMENTAL PROCEDURES
Human Breast Non-malignant and Cancer Cell Lines-Human breast cancer cell lines MCF-7, T-47D, MDA-MB231, MDA-MB-435, MDA-MB-468, Hs578T, and human breast immortalized non-malignant MCF-10A cells were purchased from ATCC and cultured as recommended by the provider. The human non-malignant breast cell line HMT-3522 and its malignant derivative T4-2 were cultured as previously described (31). MCF-7-Tet-On cells were purchased from Clontech and cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 g/ml G418.
Examination of ␤2-Chimerin mRNA Levels in Human Breast Cells and Tissues-For tissue RNA, 10 pairs of high quality human breast cancer tissue total RNA and matched-normal tissue total RNA (from the same patient) were purchased from Clinomix Biosciences, Inc. (Watervliet, NY). All 10 patients were diagnosed as infiltrating ductal carcinoma (IDC) at different stages including two Stage I IDCs (samples 1-2), two Stage II IDCs (samples 3-4), two Stage III IDCs (samples 5-6), and four Stage IV IDCs (samples 7-10). Total RNA was prepared using TRIzol and reversibly transcripted using SuperScript TM II Reverse Transcriptase (Invitrogen). ␤2-Chimerin mRNA levels were determined either by standard PCR (30 cycles) using the following primers: 5Ј-TGATCTCAAGAGGATCAAGAA-3Ј (forward) and 5Ј-TTG-GAATAGGTATCATATGTG-3Ј (reverse), which specifically amplify a 297-bp fragment of ␤2-chimerin. Primers used for real-time PCR (Q-PCR) are described elsewhere (32). The real-time PCR reactions were plated in triplicate and performed in 384-well plates using the ABI 7900HT sequence detection system (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization (32).
Generation of Adenoviruses (AdVs)-AdVs were generated with the AdEasy TM adenoviral vector system (Stratagene). Generation of the ␤2-chimerin and ␤-GAP AdVs was described elsewhere (10,32). For the generation of ⌬EIE-␤2-chimerin adenoviral construct, a XhoI-MluI insert comprising the mutant ⌬EIE-␤2-chimerin (10) was ligated into pShuttle-CMV-HA. A similar strategy was used for the generation of an AdV for the mutant I130A-␤2-chimerin (33). A control LacZ-AdV was generated from pShuttle-CMV-LacZ (provided by the kit) and therefore has the same backbone as the ␤2-chimerin AdVs. For adenoviral infections, MCF-7 cells in 6-well plates growing in serum-free DMEM were infected with various AdVs for 16 h. AdVs were removed after extensive washing, and experiments were performed 48 h later.
Cell Proliferation and Cell Cycle Analysis-Cell proliferation was assessed by BrdUrd incorporation and by the MTS assay (CellTiter 96® Aqueous One solution cell proliferation assay, Promega). After overnight infection (16 h) with the different AdVs, cells were washed once with PBS and incubated in serum-free DMEM for 24 h and then cultured in DMEM supplemented with 10% FBS for another 24 h. BrdUrd (Sigma) was then added into the medium for 30 min (final concentration: 0.2 mM). Cells were then collected by trypsinization, washed with PBS, and fixed with 70% ethanol for BrdUrd incorporation analysis using flow cytometry (35). For the MTS assay, after overnight adenoviral infection in 10-cm dishes, cells were collected using trypsin, counted, and then seeded onto 96-well plates (1 ϫ 10 4 cells/well in 100 l of DMEM supplemented with 10% FBS). MTS was added after 24, 48, or 72 h, and absorbance was measured at 490 nm (36). For cell cycle analysis, after overnight (16 h) adenoviral infection, cells were washed once with PBS, incubated in serum-free DMEM for 24 h, and then cultured in DMEM supplemented with 10% FBS for another 24 h. Cells were then collected by trypsinization and analyzed using flow cytometry as previously described (37).
Rac-GTP and Cdc42-GTP Pull-down Assays-After overnight infection (16 h) with different AdVs, cells were incubated in serum-free DMEM for 24 h and then stimulated with EGF (100 ng/ml, 1 min). Alternatively, after adenoviral infection, cells were cultured in 10% FBS DMEM for an additional 24-h period. Rac-GTP and Cdc42-GTP levels were determined with a "pull-down" assay using the PBD (p21-binding domain) of p21-activated kinase, as previously described (10,11), and using either anti-Rac or anti-Cdc42 antibodies for detection, respectively.
Statistical Analysis-Data were analyzed using either a Student's t test or one-way analysis of variance (ANOVA) with Scheffe's test. A p value of Ͻ0.05 was considered statistically significant.

Reduced Expression of ␤2-Chimerin in Human Breast Cancer
Cells and Tissues-The expression of ␤2-chimerin in normal and breast cancer cells is unknown. Using standard PCR analysis, we found high levels of ␤2-chimerin mRNA in non-malignant immortalized MCF-10A cells. On the other hand, in all of the cancer cell lines examined, the ␤2-chimerin transcript was barely detected or dramatically reduced (Fig. 1A). The results were confirmed by a quantitative analysis using Q-PCR. Indeed, ␤2-chimerin mRNA was not detected in MCF-7 and Hs578T cells and it was very low in T-47D, MDA-MB-231, and MDA-MB-435 cells. Only MDA-MB-468 cells showed significant levels of ␤2-chimerin transcript, although much lower than MCF-10A cells (Fig. 1B). Similarly, whereas ␤2chimerin mRNA was readily detected in the non-malignant breast cell line HMT3522 (31), it was barely detectable in its malignant derivative (T4-2) (Fig. 1, A and B). We next examined ␤2-chimerin mRNA levels in a small sample of human breast cancer tissues and their corresponding matched-normal tissues (from the same patient). It was found that the expression of ␤2-chimerin mRNA in normal tissues was highly variable. However, among the 10 patients, the ␤2-chimerin transcript was significantly lower in the cancer tissues of 7 patients (Fig. 1C). Together, these results reveal a significant reduction of ␤2-chimerin expression in human breast cancer.
␤2-Chimerin Reduces Cyclin D1 Expression and Inhibits pRb Phosphorylation-Because expression of ␤2-chimerin in MCF-7 cells leads to G 1 /S arrest, we next assessed the effect of ␤2-chimerin on pRb phosphorylation. The expression of ␤2chimerin dose-dependently reduced pRb phosphorylation (Fig.  4A). Notably, ␤2-chimerin significantly inhibited the expression of cyclin D1. ␤2-Chimerin also reduced the expression of cyclin A and caused a slight increase in cyclin E levels. On the other hand, cells infected with control LacZ-AdV (100 m.o.i.) showed no obvious alterations in cyclin expression and pRb phosphorylation. Infection of MCF-7 cells with ␤2-chimerin-AdV did not cause any significant changes on the expression of cyclin-dependent kinases 2, 4, and 6 (data not shown). Infection of MCF-7 cells with the ␤-GAP-AdV also led to a reduction in cyclin D1 levels and pRb phosphorylation (Fig. 4A). However,

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
the Rac-GAP inactive mutant, ⌬EIE-␤2-chimerin, did not impair cyclin D1 expression or pRb phosphorylation (Fig. 4B). Taken together, these results suggest that the inhibition of G 1 /S transition by ␤2-chimerin via its ␤-GAP domain involves the reduction of cyclin D1 and pRb phosphorylation levels.
Inhibition of Rac by ␤2-Chimerin in MCF-7 Cells-␤2-Chimerin has specificity for the Rac GTPase both in in vitro GAP assays and in COS-1 cells but does not affect RhoA or Cdc42 activity (10,11). EGF (100 ng/ml, 1 min) caused a 3.3 Ϯ 0.5-fold (n ϭ 3) increase in Rac-GTP levels in MCF-7 cells, which was significantly impaired by the expression of ␤2 chimerin. ␤2-Chimerin also reduced Rac-GTP levels in MCF-7 cells growing in 10% serum. The effect was proportional to the m.o.i. used for infection, and it was not observed with the GAP-inactive mutant, ⌬EIE-␤2-chimerin (Fig. 5A). A densitometric analysis of the ␤2-chimerin effect on serum-induced activation of Rac is presented in Fig. 5B. A striking correlation was observed between the inhibitory effect of ␤2-chimerin on Rac activity and the reduction in cyclin D1 levels by different m.o.i. of the ␤2-chimerin-AdV (r ϭ 0.95).
Ectopic Expression of Cyclin D1 Rescues the Anti-proliferative Effect of ␤2-Chimerin-To further explore the link between Rac and cyclin D1 in our experimental model, we expressed cyclin D1 using a retroviral approach (Fig. 6A). Interestingly, the ectopic expression of cyclin D1 using the D1-RetroV significantly rescued the anti-proliferative effect of ␤2-chimerin, whereas the control retrovirus (V-RetroV) did not (Fig. 6B).
We then determined whether the expression of other active Rho-GTPases could rescue the effect of ␤2-chimerin. MCF-7 cell lines stably expressing constitutively active Cdc42 (V12Cdc42) or RhoA (V14RhoA) were generated (Fig. 7A). Similar to control MCF-7 cells, HA-V14RhoA-MCF-7 cells were highly sensitive to ␤2-chimerin for the inhibition of cell proliferation, reduction of cyclin D1, and Rb dephosphorylation (Fig.  7, A and B). Unexpectedly, in cells expressing active Cdc42,

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
adenoviral delivery of ␤2-chimerin was unable to inhibit cell proliferation, cyclin D1 expression, and pRb phosphorylation (Fig. 7, A and B). To further examine the mechanisms involved in the protective effect of V12Cdc42, we determined Cdc42-GTP levels in response to EGF (100 g/ml, 1 min). A 3.1 Ϯ 0.6-fold (n ϭ 3) increase in Cdc42-GTP levels was observed in response to the growth factor, which was not affected by the ␤2-chimerin-AdV, even at the highest m.o.i. used (100 plaque-forming units/cell) (Fig. 7C). Interestingly, we found that basal Rac-GTP levels were elevated in HA-V12Cdc42-MCF-7 cells (Fig. 7,  D and E), which probably explain the protective effect of active Cdc42 on ␤2-chimerin-induced inhibition of cyclin D1 levels, pRb phosphorylation, and cell proliferation.
A Hyperactive ␤2-Chimerin Mutant Is a Potent Inhibitor of Cyclin D1 Expression and Proliferation-Based on structural predictions gained from the recently solved structure of ␤2chimerin, we generated an AdV encoding for a ␤2-chimerin mutant locked in the constitutively active conformation. This mutant, I130A-␤2-chimerin, was shown to have constitutive Rac-GAP activity when expressed in COS-1 cells by bypassing lipid activation (33). An AdV encoding for I130A-␤2-chimerin was generated and used to infect MCF-7 cells. We optimized conditions to achieve similar low levels of expression as those observed in the non-malignant breast cell line HMT3522 cells (as detected by Q-PCR, Table I). In this case, we used a lower m.o.i. and shorter expression times (16 h instead of 40 h) and the levels of the mutant I130A-␤2-chimerin in MCF-7 cells were well below the detection levels using Western blot. Under these experimental conditions, the wild-type ␤2-chimerin still showed very high levels of expression by Western blot and caused a ϳ25% reduction in Rac-GTP levels (Fig. 8A). Remark-

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
ably, even if I130A-␤2-chimerin was expressed at very low levels, it caused a 51% reduction in Rac-GTP levels (Fig. 8A). Moreover, I130A-␤2-chimerin markedly reduced cyclin D1 levels (Fig. 8B) and impaired cell proliferation (Fig. 8C). DISCUSSION Understanding the functional properties of Rac-GAPs is relevant, because Rac is a key player in the process of malignant transformation and metastasis (12,18,30). The two most relevant findings in the present study are that ␤2-chimerin expression is down-regulated in breast cancer and that the expression of this Rac-GAP in MCF-7 breast cancer cells impairs G 1 /S cell cycle progression by reducing cyclin D1 levels and Rb phosphorylation. Inhibition of proliferation by ␤2-chimerin in MCF-7 cells is dependent on the ␤2-chimerin GAP activity, and indeed, a functionally active GAP domain is required for the anti-mitogenic effect. Our results suggest that Rac activity is critical for G 1 /S progression in breast cancer MCF-7 cells.
Analysis of ␤2-chimerin mRNA levels revealed that breast cancer cells have significantly lower levels than non-malignant cells. This effect is particularly striking when we compare the non-malignant HMT3522 cell line with its malignant derivate T4-2 cell line. Moreover, studies using matched pairs of RNA samples from breast cancer patients revealed that ␤2-chimerin is significantly down-regulated in 70% of tumor samples. Members of the Rho GTPase family such as Rac and Rho are overexpressed in human tumors, particularly in breast cancer (26,27). Rac activity was found to be elevated in transformed cells, as recently described in v-Src-transformed fibroblasts, and inhibition of Rac function using dominant-negative Rac mutants dramatically reduced the ability of v-src to transform NIH 3T3 cells (39). Small GTPase hyperactivation may be the consequence of enhanced upstream inputs and/or reduced activity of GAPs, as suggested by Mira and co-workers (28) in breast cancer cell models. Various mechanisms can account for the elevated upstream inputs including receptor hyperactivation and/or enhanced activation of Rac-guanine nucleotide exchange factors, such as Tiam1 and Vav (20,40). On the other hand, the relative contribution of the down-regulation of Rho GAPs in cancer progression and their potential roles as tumor suppressors has not been extensively studied. For example, a recent study (41) has found that the Rho/Cdc42 GAP DLC2 is

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
significantly underexpressed in 18% human hepatocellular carcinoma. It is conceivable that the down-regulation of ␤2-chimerin in breast cancer cells may contribute, at least in part, to the progression of the disease. Early studies in glioma models have identified ␤2-chimerin as a gene that is significantly down-regulated in high-grade gliomas compared with normal brain and low-grade astrocytomas (8). Down-regulation of ␤2chimerin in advanced stages of the disease could contribute to the enhanced proliferation and metastatic dissemination of glioma cells due to dysregulation of Rac activity. Along the same lines, we have recently found using tissue microarrays that ␤2-chimerin expression is reduced by ϳ60% in benign duodenal adenomas and ϳ80% in duodenal adenocarcinomas when compared with normal tissues. 3 ␤-GAP significantly inhibits cell migration as well as tumor growth, invasiveness, and metastatic dissemination in vivo (32), suggesting that specific inhibition of Rac by ␤2-chimerin may impinge on various steps of malignant transformation. Although more extensive studies would be required to establish whether this Rac-GAP may serve as a prognostic marker, this body of evidence suggests that down-regulation of ␤2-chimerin expression may contribute to breast cancer progression. This may also be relevant in tissues that express high levels of ␤2-chimerin, including brain, pancreas, and intestine. Adenoviral delivery of ␤2-chimerin, ␤-GAP, or I130A-␤2chimerin, but not ⌬EIE-␤2-chimerin, significantly impairs proliferation and elevations in Rac-GTP levels in MCF-7 breast cancer cells, suggesting an essential role for chimerin Rac-GAP activity in these effects. ␤2-Chimerin also impairs heregulin ␤1-induced Rac activation and proliferation in breast cancer cells. 4 This highlights the potential relevance of ␤2-chimerin as a general negative regulator of growth factor-mediated mitogenic responses. Moreover, we have recently observed that ␤2-chimerin RNAi in HeLa cells leads to a significant potentiation of EGF-induced Rac activation. 2 Our results also emphasize the importance of Rac in cell cycle control, as previously reported using constitutively active and dominant-negative Rac1 mutants (42)(43)(44). A dominantnegative N17Rac1 mutant impairs serum-induced DNA synthesis in fibroblasts and has been reported to causes cell growth arrest in G 1 (21) or G 2 /M (45). Although the specificity of dominant-negative Rac mutants may be questioned, our experiments revealed that the inhibition of Rac activity with a specific Rac-GAP leads to G 1 /S arrest in MCF-7 breast cancer cells, an effect that strongly correlates with the reduction in cyclin D1 and pRb phosphorylation. Rac regulates cyclin D1 expression, probably at multiple levels, depending on the experimental condition and cell type. For example, dominant-negative and constitutively active forms of Rac1 regulate cyclin D1 promoter activity in smooth muscle cells through Rac-dependent generation of reactive oxygen species (24). A role for the NF-B pathway downstream of Rac has also been described in NIH 3T3 cells (23). Regulation of the cyclin D1 messenger by Rac at a translational level has also been reported (21,25). Activated Rac is capable of enhancing pRb phosphorylation and E2F-mediated transcription of genes required for S phase entry and DNA replication (46). It has also been proposed that Rac integrates signals from specific integrins and growth factors to promote the synthesis of cyclin D1 and tumor cell survival (24,25,47). The insensitivity of V12Rac1-expressing MCF-7 cells to ␤2-chimerininduced reduction in cyclin D1 levels and Rb phosphorylation, as well as the rescue of the ␤2-chimerin effect by ectopic expression of cyclin D1, further supports the Rac-cyclin D1-G 1 /S progression link.
An emerging paradigm is that ␤2-chimerin can be regulated by cell surface receptors, as it is well known for Ras-GAPs (48). Receptors coupled to the generation of the lipid second messenger DAG, such as the EGF receptor, control the activity of ␤2-chimerin both by positional and allosteric mechanisms, 2 which substantiates the concept of DAG divergence via the activation of "non-PKC" pathways. These DAG-regulated mechanisms, as well as the selectivity of ␤2-chimerin for the Rac GTPase, have been further validated by the recently solved three-dimensional structure of this Rac-GAP (33). Our hypothesis is that, in the context of receptors such as the EGF or platelet-derived growth factor receptor, ␤2-chimerins represent a DAG-regulated negative loop that self-limits Rac activation and Rac-mediated responses. Our focus now is to elucidate the molecular basis of such lipid regulation, which will provide further insight into the receptor-mediated control of chimerin function and G 1 /S cell cycle progression in breast cancer and other diseases.


extfieldsofstudy: Biology Medicine

text:
RNA in human sperm

We have yet to develop a fundamental understanding of the molecular complexities of human spermatozoa. This encompasses the unique packaging and structure of the sperm genome along with their paternally derived RNAs in preparation for their delivery to the egg. The diversity of these transcripts is vast, including several anti-sense molecules resembling known regulatory micro-RNAs. The field is still grasping with its delivery to the oocyte at fertilization and possible significance. It remains tempting to analogize them to maternally-derived transcripts active in early embryo patterning. Irrespective of their role in the embryo, their use as a means to assess male factor infertility is promising. (Asian J Androl 2005 Jun; 7: 115-120)

Introduction
Mature spermatozoa serve as a repository for information regarding both genetic and environmental influences. They seed some of the many forms of male factor infertility. Within the past few decades, there has been a decline in human male fertility [1][2][3][4]. The direct causes of this reduction remain enigmatic and controversial, but some work suggests that increased environmental and systemic exposure to pesticides, herbicides, estrogenic compounds, heavy metals and reactive oxygen species [5][6][7] may play a causative role. Concurrent with and perhaps contributory to the decrease in male fertility, there has been a corresponding increase in the incidence of testicular cancer and cryptorchidism [8,9]. The medical costs associated with treating the rising number of infertile men and men with a fertility-affecting pathology is mounting. These theories remain speculative because little is known about the genetic and molecular basis of infertility.
Of the genes reported to correlate with male factor infertility, most have been identified via non-systematic, or limited surveys; the focus has been on specific genes that were of interest to an individual investigator rather than a genome-wide approach [10]. For example, single nucleotide polymorphisms in one of the genes involved in repackaging the male haploid genome, prm2 [11] were found in infertile males; one of which leads to a truncated protein. Decreased levels of the estrogen receptor [12] in spermatozoan nuclear matrices was also found to occur in ideopathically infertile men. Studies using animal models have pointed to a number of factors that when deficient or inappropriately expressed, lead to infertility [13][14][15][16][17][18]. Using a more comprehensive approach, recent systematic analyses of the human Y chromosome have identified a number of candidate genes for male factor infertility. For example, deletions identified in the non-recombining region of the Y chromosome lead to spermatogenic failure [19,20]. Other testis-specific defects have been demonstrated in men with deletions of the Y chromosome encompassing one or more genes [21]. Some of the genes in this region have since been determined to not have any clinical relevance with regard to infertility [22], while other factors demonstrate a clear relationship to an infertile phenotype. It is reasonable to assume that the majority of idiopathic male factor infertilities are likely multifactorial disorders [23][24][25][26]. As shown by the recent transcriptional profiling of the CREM knock out mouse model [27] this is reflected by substantial changes in the presence or absence of various multiple members of the affected pathways. Determining exactly how all of these genes are involved, however, remains necessary.

RNA in sperm
It has long been held that the tightly packaged chromatin within mature spermatozoa is transcriptionally inert [28]. Despite this, RNA was observed in the mature sperm nucleus of Scolopendrium [29] and in rodents and other species [28][29][30][31][32][33]. In situ hybridization from normal fertile human ejaculate localized both beta-actin and prm2 mRNAs to the head region [34], suggesting that this RNA was a unique sperm head component. Spermatozoa transcript complexity was first addressed by analyzing a series of randomly selected cDNA clones [35]. Sequence comparison showed that 11 of the hybridizing cDNAs were unique within the expressed sequence tag (ESTs) and non-redundant databases whereas five showed no similarity to any of the sequences in the database. Additionally, one was a member of the SINE, i.e., short interspersed repetitive element family, e.g. Alu and another contained a CAn repetitive element comprised of a repeating string of CA nucleotides. Together these stud-ies independently confirmed that spermatozoa contain a wealth of both known and unknown protein-encoding and non-coding RNAs. The presence of this suite of RNAs is intriguing when we consider that mature sperm have little if any cytoplasm [36]. These methods of characterization proved a rather laborious means to profile the set of transcripts present in the mature spermatozoa. Since then, other large-scale strategies have been employed.

Molecular characterization of sperm transcripts
Characterizing the transcript complexity of the spermatozoan has been refined by the use of microarrays. For example, using mRNA from both testes and ejaculate spermatozoa, a suite of cloned EST microarrays were probed [37,38]. Stringent precautions were employed to avoid somatic cell contamination. This included two sequential centrifugations through a 40:80 discontinuous gradient of percoll followed by treatment with Triton-X 100 plus sodium dodecyl sulphate (SDS) to remove even a hint of residual somatic cells. cDNA probes from the pooled poly(A+) RNA fraction and the single total RNA fraction were then hybridized to a series of six microarrays containing 27 016 unique ESTs. All transcripts isolated from human sperm were present in testes, but all testes transcripts were not present in sperm. The pooled testes probe hybridized to 26 % of the ESTs whereas the pooled poly(A+) RNA fertile spermatozoa probe identified 12 % of the ESTs. The total RNA spermatozoal probe from the single fertile male ejaculate hybridized to 10 % of the ESTs. The spermatozoal sequences comprised a discrete subset of those identified with the testes probe. As expected the population of RNAs represented by the poly (A+) RNA fraction was similar to that of the total RNA fraction.
To assess variance, different preparations of sperm RNA from different individuals have been compared. Representative results are summarized in Figure 1. Spermatozoal RNAs were isolated and array specific labeled probes were constructed. Each probe was then individually hybridized to an array spotted with 1.176 unique ESTs, shown in panel A. Panels B and C demonstrate a greater than 90 % concordance, between individuals, of both positively hybridized ESTs (+ve: 132 of ~170 ESTs), as well as with those ESTs that failed to hybridize (-ve: 938 or about ~1015 ESTs). This supports the notion that a core set of invariant fertile transcripts will be identified.
Subsequent data analysis from these and other stud-. 117 .
ies has revealed that hydrolases, many of which are found in the acrosome and DNA binding proteins, associated with the extensive restructuring of the nucleus during spermiogenesis [39] are among the largest protein-encoding groups identified. In comparison, when queried by cellular component, the largest protein-encoding groups were the plasma membrane, nucleus and cytoplasm. A series of spermatozoal transcripts have been identified that are concordant with mRNAs known to participate in fertilization and embryonic development. A comparison of this suite of sperm RNAs with those present in human and mouse oocyte cDNA libraries revealed that several sperm derived transcripts essential for early development were not present in the oocyte. These encode a series of proteins associated with fertilization (i.e., SP-40, sulfated glycoprotein 2, calmegin and several heat shock response products) that are important for embryo development [40]. Their presence in human testes and sperm and their absence in the unfertilized egg as assessed by RT-PCR and the delivery of some of these RNAs from the sperm to the egg upon fertilization has been confirmed [41]. This has led to the obvious yet unanswered question, do spermatozoal RNAs, encode unique function(s) in the developing zygote and/or embryo ( Figure 2A)? Perhaps they help sustain viability of the embryonic genome?
Of the ribonucleic species delivered to the oocyte at fertilization, a large proportion of them are of low molecular weight and are shown to be in the anti-sense orientation ( Figure 2A) [42]. A total of 68 different siRNAs have been identified in human spermatozoa (http:// compbio.med.wayne.edu/Sperm_RNAi.htm) of which 13 show significant similarity with those previously implicated in RNA-mediated regulation [42]. Could these function in a manner similar to small interference RNAs (siRNAs) like lin-4 and let-7? These are well known as regulators of development and differentiation in Caenorhabditis elegans regulating the timing of larval development [43,44] and the transition from late larval to adult stages [45,46], respectively. The small sperm RNA transcripts show a wide tissue distribution including a number of early embryonic tissues. Several of the transcripts are implicated in embryonic development including DKK2, TIA and fat-3. For example, dickkopf2 (DKK2) is known to inhibit the WNT signaling pathway [47][48][49][50] regulating cell fate and pattern generation dur-  Spermatozoa deliver not only their genomic contribution to the embryo, but also many RNA transcripts; some of which are not present among the stored maternal RNAs. Among these are transcripts thought to be involved in early development, as well as anti-sense micro-RNAs that may play a role in early pre-translational regulation in the embryo. (B): The majority of male factor infertility is idiopathic, as sperm otherwise appear normal. RNA isolated from ejaculate spermatozoa from these men could be used to clinically assess putative genetic or environmental disturbances. This will not only shed light on key genetic processes that are integral to spermatogenic differentiation, but also provide avenues for therapy. . 118 .
ing embryogenesis whereas, fat-3, is perhaps a key player in the differentiation of mesoderm, somites and neurons in mammals [51], as well as growth and neuronal signaling in C. elegans [52].
With such a large number and diversity of transcripts found in mature spermatozoa, it is tempting to draw comparisons with maternal RNAs, stored in the oocyte. The importance of these in the early patterning and development in Drosophila [53] and Xenopus [54,55] embryos is well documented. In mammals, their importance was first only inferred [56] and later confirmed through the discovery of specific genes and by experiments examining the onset of zygotic transcription [57,58]. As sperm have been shown to deliver RNA to the oocyte, the presence of antisense RNAs within this pool could provide a means to regulate early parental-based gene activity, by selectively targeting maternally or paternally derived RNAs for degradation. Furthermore, with siRNAs being known to persist through several cell divisions, it is possible that these transcripts could help regulate early genetic activity through to the multi-cell embryo.

Sperm RNA as a clinical tool
The application of microarray technology to spermatozoal RNA isolated from ejaculate presents a unique opportunity to globally address the mechanisms that control the differentiation of the male gamete during normal, perturbed and diseased states ( Figure 2B). They provide a useful molecular record to assess environmental insult and/or genetic status since spermatogenesis is highly sensitive to environmental exposures including chemical, thermal and biological agents [59]. Recently two independent studies have used RNA profiling techniques to address the relationship of motility and the RNA population between normal and motility impaired sperm [60,61]. Interestingly several transcripts were identified that varied in a significant manner between the normal and motility impaired samples. These included testis specific protein 1 and lactate dehydrogenase C transcript variant 1 [61]. This clearly points to the potential of this strategy to be used as a clinical assay to provide a panoramic view of testis gene expression [37] that can be difficult to achieve from a testicular biopsy. Defining the "fertile male fingerprint" would have a significant impact on diagnosis, treatment and counseling. Realization would signal a major advance in the field of andrology.


extfieldsofstudy: Medicine Biology

text:
N-Terminal Arm of Mcm1 Is Required for Transcription of a Subset of Genes Involved in Maintenance of the Cell Wall

ABSTRACT The yeast Mcm1 protein is a member of the MADS box family of transcription factors that interacts with several cofactors to differentially regulate genes involved in cell-type determination, mating, cell cycle control and arginine metabolism. Residues 18 to 96 of the protein, which form the core DNA-binding domain of Mcm1, are sufficient to carry out many Mcm1-dependent functions. However, deletion of residues 2 to 17, which form the nonessential N-terminal (NT) arm, confers a salt-sensitive phenotype, suggesting that the NT arm is required for the activation of salt response genes. We used a strategy that combined information from the mutational analysis of the Mcm1-binding site with microarray expression data under salt stress conditions to identify a new subset of Mcm1-regulated genes. Northern blot analysis showed that the transcript levels of several genes encoding associated with the cell wall, especially YGP1, decrease significantly upon deletion of the Mcm1 NT arm. Deletion of the Mcm1 NT arm results in a calcofluor white-sensitive phenotype, which is often associated with defects in transcription of cell wall genes. In addition, the deletion makes cells sensitive to CaCl2 and alkaline pH. We found that the defect caused by removal of the NT arm is not due to changes in Mcm1 protein level, stability, DNA-binding affinity, or DNA bending. This suggests that residues 2 to 17 of Mcm1 may be involved in recruiting a cofactor to the promoters of these genes to activate transcription.

The MADS box family of proteins contains a highly conserved DNA-binding and dimerization domain, named after the founding members, Mcm1, Agamous, Deficiens, and serum response factor (SRF) (34,36,46,51). MADS box proteins have been identified in most eukaryotic organisms, and these proteins are involved in the transcriptional regulation of a wide array of cellular and developmental processes (3,26,34,46,49). Many MADS box proteins are able to bind DNA as dimers with high affinity on their own in vitro. However, these proteins often interact with other DNA-binding cofactors to regulate transcription in vivo. Their interaction with different cofactors helps specify the DNA target sites bound by these proteins and thus the genes that they regulate. The conserved 80-amino-acid MADS box, DNA-binding, and dimerization domain is often sufficient for the interaction with these cofactors (6,10,37,45). However, many MADS box proteins also appear to use regions outside of the conserved domain to interact with cofactors. For example, the 28-amino-acid-long myocyte enhancer factor (MEF) domain of MEF2 proteins in animals is found immediately C terminal to the MADS box domain and enables these proteins to heterodimerize with other members of the MEF2 family (32). In plants, the I region and K box outside the MADS box are involved in homo-and heterodimerization (11,16,39).
Mcm1 is a MADS box transcriptional regulatory protein in the yeast Saccharomyces cerevisiae. The 286-amino-acid-long Mcm1 protein binds to a 10-bp palindromic sequence, frequently called the CArG box [CC(A/T) 6 GG] (31). In combination with different cofactors, Mcm1 bound to CArG sites activates or represses the transcription of a variety of genes, including those involved in cell type determination, arginine metabolism, pheromone response, mating, and cell cycle progression (20,27,29,36). It is also involved in Ty-mediated transcription and minichromosome maintenance (36,52). Mcm1 was recently found to bind to yeast autonomous replicating sequences (designated ARS) and initiate replication (8,9). Mcm1 is an essential protein in yeast, and the conserved MADS box domain (residues 18 to 96; designated Mcm1-18-96) is sufficient to maintain viability, DNA binding, dimerization, interaction with several cofactors, and transcriptional regulation of many of its target genes (6,10,28). However, deletion of the nonessential N-terminal extension (residues 2 to 17) of Mcm1 causes cells to be sensitive to high-salt levels, indicating that regions outside the conserved MADS box are important for this function (24).
In this paper, we investigate the role of the Mcm1 N-terminal (NT) arm in regulating cellular osmotolerance. We have found that deletion of the NT arm confers strong sensitivity to CaCl 2 . Deletion of the arm also confers sensitivity to cell walldisrupting agents like calcofluor white (CFW) and alkaline pH. Importantly, we show here that the NT arm of Mcm1 is required for the proper regulation of a subset of genes that encode components of the cell wall, such as YGP1 (for yeast glycoprotein 1). Our results suggest that the NT arm of Mcm1 may be needed for interaction with another cofactor that is required for the proper regulation of these cell wall genes.

MATERIALS AND METHODS
Plasmids and strains. Yeast strain JM01 (MAT␣ leu2-3,112 trp1-1 ura3 his3-11,15 ade2-1 can1-100 mcm1::LEU2/pSL1574-CEN/ARS MCM1 URA3) was used for all the in vivo assays (28). Plasmids with different mcm1 mutations were transformed into strain JM01 and selected on synthetic medium lacking histidine. Colonies that had lost the wild-type MCM1 on plasmid pSL1574 were selected by growth on 5-fluoroorotic acid plates. Mutations of the putative Mcm1 and Rlm1 sites in the YGP1-lacZ reporters were constructed by site-directed mutagenesis using the Stratagene QuikChange kit. Plasmids used in this study are listed in Table 1. Sequences of the oligonucleotides used for creating probes, construction of plasmids, and site-directed mutagenesis are available on request.
Biological assays. JM01 strains transformed with wild-type or mutant mcm1 on HIS3 plasmids were grown overnight in liquid SD-His-Leu medium (28). The concentration of the cells in each culture was normalized after their optical density at 600 nm was measured. Threefold serial dilutions (each, 3 l) of the normalized strains were spotted onto yeast extract-peptone-dextrose (YEPD) medium supplemented with the indicated concentrations of calcofluor white, KCl, sorbitol, dithiothreitol (DTT), and CaCl 2 . The pH of alkaline YEPD medium was adjusted to 8.5 with NaOH before being autoclaved. The ␤-galactosidase activities of YGP1 promoter-lacZ fusion reporters were determined in strain JM01 cotransformed with wild-type or mutant derivatives of pJM231 (MCM1 HIS3 CEN) and the different lacZ reporter plasmids (2 URA3). Cells were grown to log phase in minimal medium lacking leucine, histidine, and uracil; liquid ␤-galactosidase assays were performed as described previously (20).
Protein purification. The Mcm1 proteins used in the in vitro DNA-binding and DNA-bending studies were purified from Escherichia coli BL21 cells transformed with pTBA25 or pTBA22 or maltose-binding protein fusion expression vectors with sequences encoding Mcm1 residues 1 to 96 and 18 to 96, respectively. The proteins were purified from 50-ml cultures as described previously (2). Protein concentrations were determined by Bradford assays, normalized, and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Northern blot analysis. Yeast strain JM01 was transformed with wild-type and mutant derivatives of pJM231 (MCM1 HIS3 CEN) or pAB76 (MCM1 HIS3 2), and cultures were grown at 30°C in SD-His-Leu medium to mid-log phase (optical density at 600 nm, ϳ0.6). The cells were split into two aliquots. One half was resuspended in dropout medium supplemented with 1.4 M NaCl and grown at 30°C for 1.5 h, while the other half was harvested after growth in medium without a high salt level. Total RNA was extracted from both samples by hot acid-phenol extraction (5). RNA was separated by molecular weight on a 1.5% agarose gel containing formaldehyde and transferred to a Nytran membrane (Schleicher and Schuell) by capillary transfer. The membrane was prehybridized in a 10-ml hybridization solution (50% formamide, 5ϫ SSC [1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 50 mM NaPO 4 [pH 7.0], 1ϫ Denhardt's reagent, 0.1% SDS, 100-g/ml sheared single-stranded salmon sperm DNA).
DNA probes to open reading frames (ORFs) were generated by PCR and labeled with [␣-32 P]dCTP by random priming (MegaPrime DNA Labeling Kit, Amersham). After overnight hybridization to labeled DNA probes and washing with 2ϫ SSC and 0.1% SDS at 42°C, specific mRNAs were detected by exposure to a phosphorscreen and imaging on a Molecular Dynamics phosphorimager.
Western blot analysis. Cultures of yeast strain JM01 transformed with C-terminal V5 epitope tagged full-length MCM1 and mcm1-⌬2-17 were grown to mid-log phase in SD-His-Leu medium. Proteins were extracted by glass bead lysis, and the protein concentration was determined with the Bio-Rad Protein Assay kit. Proteins were separated on 12% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, probed with mouse antibody directed against the V5 epitope (Invitrogen), and detected with sheep anti-mouse immunoglobulin G horseradish peroxidase (Amersham Pharmacia). The blot was stripped and probed with rabbit anti-Rpa1 antibodies (a gift from Steve Brill) and goat anti-rabbit immunoglobulin G horseradish peroxidase (Bio-Rad). Proteins were detected with the ECL Western blot detection kit (Amersham Pharmacia Biotech). Protein levels were quantitated with FluorChem software. To determine the rate of degradation of Mcm1, cells were grown to mid-log phase, and protein synthesis was arrested by the addition of cycloheximide to a final concentration of 20 g/ml (14). Cells were harvested every 30 min for 4 h after the addition of cycloheximide. Protein extraction and detection were performed as described above.
Electrophoretic mobility shift assay (EMSA). Oligonucleotides with P(PAL) or YGP1 M1M2 sites were end labeled with [␥-32 P]ATP using polynucleotide kinase and purified using QIAGEN nucleotide removal columns. The labeled oligonucleotides were incubated at 90°C for 5 min with a threefold excess of the matching strand and allowed to anneal by being cooled slowly to 25°C. The purified Mcm1-1-96 and Mcm1-18-96 proteins were incubated for 1 h at room temperature with the labeled DNA probes in 20 mM Tris (pH 7.6), 5 mM MgCl 2 , 0.1 mM EDTA, 0.1% NP-40, 5% glycerol, 10-mg/ml bovine serum albumin, 10-mg/ml single-stranded DNA, and labeled oligonucleotide pairs (300 cpm/l) in a total volume of 24 l at room temperature for 1 h. All protein dilutions were made in 50 mM Tris (pH 7.6), 500 mM NaCl, 1 mM EDTA, 10 mM ␤-mercaptoethanol, and 1-mg/ml bovine serum albumin. Samples were loaded on a 6% polyacrylamide gel (run in 0.5ϫ Tris-borate-EDTA buffer for 1 h at 200 V). Gels were dried after electrophoresis, exposed to a phosphor screen, and scanned with a Molecular Dynamics Phosphorimager. Circular permutation assay. A circularly permuted plasmid, pDA192, with a 80-bp fragment containing the R1, M2, and M3 sites from the YGP1 promoter cloned into the SacI-XbaI polylinker site of pGD579 (2) was digested with BamHI, NheI, HindIII, or EcoRI to yield probes with the R1-M2-M3 region located at different positions along the length of each DNA fragment. EMSAs were performed using position-permuted fragments and purified Mcm1-1-96 and Mcm1-18-96. The bend angle for each protein-DNA complex was calculated using the Thompson and Landy relationship (48).
ChIP assay. Transformants of strain JM01 with wild-type and mutant derivatives of MCM1 with or without the V5 epitope tag were used for chromatin immunoprecipitation (ChIP) assays. Proteins bound to DNA were cross-linked by the addition of 1.35 ml of 37% formaldehyde to 50 ml of log-phase cultures and shaking at room temperature for 15 min. The cells were then harvested and lysed with glass beads. The crude lysate was sonicated to shear the protein-bound DNA to approximately 500-bp-long fragments. After cell debris was removed by centrifugation, the total chromatin was precleared by nutation with protein G-agarose for 1 h and centrifugation. The supernatant was then incubated with the anti-V5 antibody overnight at 4°C. Protein G-agarose was added to immunoprecipitate the sample. Reversal of formaldehyde cross-links was carried out as described previously (22). The DNA fragments precipitated with the V5-tagged Mcm1 proteins were detected by PCR using primers that annealed to the target promoter regions. Each PCR used three sets of primers: one set to amplify the positive control for Mcm1 binding, another for the test promoter, and a third for the negative control.

Identification of genes requiring the NT arm of Mcm1 for activation.
Deletion of the NT arm of Mcm1 (residues 2 to 17) makes cells osmosensitive (24). We therefore reasoned that the NT arm may be required for the transcriptional activation of genes required for tolerance to high salt levels. To identify potential Mcm1-activated salt response genes, we scanned the promoter regions of genes that showed increased expression under high-salt conditions for sites that are similar to the consensus sequence ccywwwxxrg (where y ϭ T/C, w ϭ A/T, x ϭ any base, and r ϭ A/G) that was derived from mutational analysis of an Mcm1-binding site (2,50). Among the 570 saltinduced genes that were induced twofold or more in the microarray analysis, 163 genes had one or more putative Mcm1binding sites in the 500-bp region upstream of their start codon (50). Among this group, we focused on genes that contained multiple Mcm1-binding sites or that were known to be involved in the osmotic stress response. To test if the NT arm of Mcm1 was required for the regulation of the selected salt-induced genes, we compared their expression by Northern blot analysis in strains containing wild-type Mcm1 or Mcm1-⌬2-17 grown under high-and low-salt conditions. Deletion of Mcm1 residues 2 to 17 resulted in a decrease in the mRNA levels of YGP1, PNC1, DAK1, and YHR087W (Fig. 1). The most dramatic effect was observed for YGP1, in which both the noninduced and salt-induced transcription decreased to Ͻ10% of wild-type levels. In addition to causing a decrease in expression for some genes, the deletion of the NT arm caused an increase in the expression of URA1 and SIT1. This suggests that the NT arm might be required for Mcm1 to function as a repressor at  (21,43). Taken together, these results indicate that Mcm1 residues 2 to 17 are required for the activation of a specific subset of genes that are induced by salt.
Among the genes that were assayed, deletion of the NT arm had the greatest effect on YGP1, which encodes a protein that is secreted from protoplasts during cell wall regeneration. Ygp1 is highly glycosylated, and its synthesis is induced in response to glucose, nitrogen, phosphate starvation, and cell wall disruptions (12,35). We therefore wanted to determine if other stress-induced genes that were coinduced with YGP1 under these conditions also required the Mcm1 NT arm for activation. YGP1 is one of 25 genes that are regulated by Rlm1, another MADS box transcription factor (18). Twenty of these genes had putative Mcm1 sites in the 1-kb region upstream of their start sites. However, Northern blot analysis showed that only the salt-induced expression of SPS100 was dependent on the presence of the Mcm1 NT arm ( Fig. 1 and data not shown). Since YGP1 is induced in response to disruption of the cell wall, we also examined the promoters of genes that are induced in response to various cell wall stresses (25). Among the 80 genes categorized as the cell wall compensatory cluster, 38 had putative Mcm1-binding sites in their promoters. The expression of 15 of these genes was assayed under high-salt conditions in Mcm1 and Mcm1-⌬2-17 strains by Northern blotting. Among them, only SPI1 and YBR071W expression was dependent on the presence of the NT arm by Northern analysis ( Fig. 1 and data not shown).
Deletion of the NT arm causes cell wall defects. Deletion of the Mcm1 NT arm causes sensitivity to increased NaCl levels (24). Since the Mcm1 NT arm is required for the proper regulation of a subset of cell wall biosynthesis genes, we were interested in determining if this mutation affected sensitivity to other cell wall related stresses. While the mcm1-⌬2-17 strain was very sensitive to YEPD medium with 0.35 M CaCl 2 (Fig. 2B), it was only mildly sensitive to 1.3 M KCl (Fig. 2C) and hardly sensitive to 0.4 M LiCl (data not shown).
Since the NT arm of Mcm1 was required for the proper expression of genes that encode components of the cell wall, we tested cells with mcm1-⌬2-17 for sensitivity to several cell wall-disrupting agents. The drug CFW is a fluorescent dye that binds to chitin in the cell wall and is thought to weaken the cell wall by preventing microfibril formation (15,33,38). CFW sensitivity is commonly used as an indicator of defects in cell wall biogenesis. Deletion of the Mcm1 NT arm conferred a strong sensitivity to CFW (Fig. 2D). Deletion of the Mcm1 NT arm also caused the cells to be sensitive to alkaline pH (Fig. 2E). However, the mutant was not sensitive to other agents that are commonly used to monitor defects in the cell wall, such as 2 M sorbitol, 50 mM DTT, and acidic pH ( Fig. 2F and G and data not shown). These data suggest that Mcm1 is required for the transcriptional regulation of a subset of cell wall genes that are required for CFW and high pH resistance but not DTT or sorbitol resistance.
CFW interacts with chitin in the yeast cell wall, and exposure to CFW induces chitin synthesis (40)(41)(42). CHS1, CHS2, and CHS3 encode the catalytic subunits of chitin synthetases. There are three putative Mcm1-binding sites in the 1-kb region upstream of the CHS2 start codon. However, Northern analysis showed that CHS1, CHS2, and CHS3 mRNA levels were not altered by the Mcm1 NT arm deletion, suggesting that the CFW-sensitive phenotype is not caused by improper expression of these genes (data not shown).
Effect of Mcm1 point mutations on YGP1 transcription and CFW and alkaline pH tolerance. Although there are multiple phosphorylated isoforms of Mcm1, residues S2 and T8 in the Mcm1 NT arm are the two major phosphorylation sites in the protein (24). Under normal conditions, residue S2 in the NT arm is phosphorylated with nearly 100% efficiency and a serine-to-alanine mutation at position S2, which mimics the constitutively dephosphorylated form of S2, has a salt-sensitive phenotype. This result suggested that phosphorylation of S2 was required for the induction of Mcm1-dependent salt response genes. Therefore, it is possible that phosphorylation of residues in the NT arm also has a role in YGP1 regulation. We tested this hypothesis by making alanine (A) and aspartate (D) substitutions of S2, T8, and T10 to mimic constitutive dephosphorylation and phosphorylation, respectively, of these residues. If differential phosphorylation states of the NT arm were important for the activation of YGP1, either A or D substitutions would be expected to have antagonistic effects on its transcription. However, Northern analysis showed that both the A and D substitutions of residues in the NT arm only resulted in slightly lower levels of the YGP1 transcript (Fig. 3A). Different combinations of these mutations also did not significantly affect YGP1 expression. In contrast, point mutations (V34A, T35A, S37A, K40A, and T66A) in the MADS box domain of Mcm1 that were previously shown to decrease DNA-binding affinity or bending and thus affect transcriptional and data not shown). We next tested the effects of the amino acid substitutions in the NT arm and MADS box domain on resistance to cell wall-disrupting agents. Although deletion of the Mcm1 NT arm conferred a pronounced growth defect on rich medium supplemented with CaCl 2 (Fig. 2B), single amino acid substitutions in the NT arm or MADS box domain were not sensitive to CaCl 2 (data not shown). However, although significantly less than the 243-fold decrease in growth caused by deletion of the NT arm, A and D substitutions of residues S2, T8, and T10 caused 3-to 27-fold decreases in growth on medium with CFW or alkaline pH ( Fig. 4A and B). Alanine replacements of other residues in the arm that are not phosphorylated also made the cells sensitive to CFW. Since deletion of the NT arm showed a much stronger phenotype than individual point mutations in the arm, it appears that the entire arm is important for its function.
We also examined the relative contribution of different residues in the MADS box domain toward CFW and alkaline pH tolerance. A comparison of the mutant strains showed that the same regions in Mcm1 are important for tolerance to both CFW (Fig. 4A) and alkaline pH (Fig. 4B). In general, the mutations had a more severe growth defect on CFW than high pH, underscoring the sensitivity of the CFW phenotype over the pH phenotype. Interestingly, the mutations that were the most defective in these assays mapped to three locations on the crystal structure of the Mcm1 dimer bound to DNA. One class of mutants, exemplified by L60, L61, V62, and V63, consists of residues along the dimer interface. It is not surprising that mutations of these residues affect Mcm1-mediated functions, most likely by adversely affecting Mcm1 homodimerization. The second class of mutants, represented by V81A, G86A, and R87A, form a hydrophobic groove on the surface of Mcm1 (Fig. 4C). In the crystal structure of the Mcm1-␣2-DNA complex, this region of Mcm1 interacts with residues in the ␣2 protein and it is possible that Mcm1 uses these same residues to interact with another cofactor required for induction of the cell wall-associated genes identified (47). The third class of mutants, I23A, F48A, and E49A, are positioned near the NT arm in the crystal structure (Fig. 4D) (23). This mutation also suppressed the effect of the NT arm deletion. We therefore wanted to determine if the T35A mutation was able to suppress the effects of the Mcm1 NT arm deletion on sensitivity to cell wall-disrupting agents. On its own, the T35A Mcm1 mutant grew as well as the wild-type MCM1 strain on medium with CaCl 2 (Fig. 5). In contrast, this mutant was defective in growth on plates with CFW and alkaline pH, although not to the same extent as mcm1-⌬2-17. As was observed for the NaCl-sensitive phenotype, the T35A mutant was able to partially suppress the CaCl 2 -sensitive phenotype of the Mcm1 NT arm deletion. Similarly, the T35A mutation partially suppressed the growth defect of mcm1-⌬2-17 on alkaline pH and very weakly on CFW.
Since YGP1 transcription was induced with high salt levels and was adversely affected by the deletion of the Mcm1 NT arm, we wondered if the T35A mutation suppressed the effect of Mcm1-⌬2-17 on expression of the gene. However, Northern blot analysis showed that both Mcm1-T35A and Mcm1-⌬2-17,T35A caused decreases in YGP1 expression (Fig. 5B). Taken together, these data suggest that the mechanism of Mcm1-dependent regulation of genes required for growth on CFW and with alkaline pH and CaCl 2 is different than Mcm1 regulation of YGP1.
The YGP1 promoter is regulated by two MADS box proteins, Mcm1 and Rlm1. Since deletion of the Mcm1 NT arm showed the largest effect on the transcription of YGP1, we decided to analyze the YGP1 promoter to understand the mode of regulation of this gene by Mcm1. To monitor expression of the gene, we fused the 700-bp promoter region 5Ј to the YGP1 ORF to the lacZ gene. The reporter was strongly expressed in a wild-type strain, but its expression was decreased almost 300 fold in an mcm1-⌬2-17 strain (Fig. 6). This result showed that the regulatory elements that responded to the deletion of the Mcm1 NT arm were located in this region of the YGP1 promoter. Sequence inspection of the YGP1 reporter predicted four potential Mcm1-binding sites, which we called M1, M2, M3, and M4. To determine if the putative Mcm1-binding sites were functional, these sites were mutated individually and in combination in the context of the YGP1-lacZ reporter, and their effect on YGP1 expression was measured by assaying ␤-galactosidase activity in cells with wild-type Mcm1. Individual disruption of each of these sites caused a partial decrease in expression of the reporter, indicating that these sites are required for regulation of YGP1 and are likely to be functional Mcm1 targets. The relative effect of each of the mutants varied, suggesting that these sites may be bound by Mcm1 with differing affinities. Mutation of all four Mcm1-binding sites (M1, M2, M3, and M4) resulted in a significant decrease in activation, similar to ␤-galactosidase levels obtained by deletion of the NT arm.
In addition to the Mcm1-binding sites, there is a putative binding site for the Rlm1 MADS box protein in the YGP1 promoter. Rlm1 is activated through phosphorylation by the Mpk1 MAP kinase in response to cell wall integrity signaling (13). Northern blot analysis of a strain with constitutively activated Rlm1 showed that YGP1 expression was decreased, suggesting that Rlm1 functions as a repressor of this gene (18). However, mutation of the Rlm1-binding site in the YGP1-lacZ fusion reporter caused a slight decrease in expression both on its own and in combination with mutations in the Mcm1 sites (Fig. 6). This result suggests that Rlm1 may have a direct role in activating YGP1 expression under the noninducing growth condition used. promoter. The protein with NT arm deletion bound to the consensus P(PAL) and YGP1 probes with the same affinity as the wild-type protein (Fig. 8A). This indicates that the arm is not required for binding to these sites in vitro.
Although deletion of the NT arm did not affect Mcm1 binding in vitro, it was possible that the mutations affect the ability of the protein to bind its target sites in vivo. We therefore tested the in vivo DNA-binding affinity of Mcm1-⌬2-17-V5 and Mcm1-V5 to the YGP1 promoter by ChIP assays in which Mcm1-bound DNA was immunoprecipitated with the ␣-V5 antibody. We probed for the presence of Mcm1-regulated STE2 and YGP1 promoters and, as a control, the Mcm1-independent ACT1 promoter using primers sets that were specific to each promoter (Fig. 8B). The deletion of Mcm1 residues 2 to 17 did not affect DNA binding to Mcm1-regulated genes that do or do not require the NT arm for transcriptional regulation. In contrast, the T35A mutation of Mcm1 caused a dramatic decrease in binding of Mcm1 to the YGP1 promoter (Fig. 8B). This accounts for the poor transcription of this gene in this mutant background (Fig. 5B). The ChIP data also showed that Mcm1 is bound to the YGP1 promoter in the absence of salt stress. This indicates that the Mcm1 NT arm is not required for binding to the YGP1 promoter in vivo.  (2). When the Mcm1 dimer binds to DNA, it also induces a bend in the DNA that is important for activation (2,7,44). Mutations that change the bending angle result in a decrease in transcriptional activation. It is therefore possible that deletion of the NT arm affects DNA bending by the protein and thereby decreases the expression of the NT arm-dependent genes. However, using EM-SAs with circularly permuted fragments, we determined that the apparent bending angles induced by Mcm1-1-96 and Mcm1-18-96 binding to the R1M2M3 YGP1 probe were almost identical, suggesting that the decrease in transcriptional activation of YGP1 was not due to changes in the DNA-bending angle (Fig. 9).

DISCUSSION
The N-terminal arm of Mcm1 has a role in regulating genes that are required for tolerance to NaCl (24). In addition to the NaCl-sensitive phenotype previously associated with deletion of the arm, we have observed other biological phenotypes, including sensitivity to CaCl 2 , high pH, and CFW. We also determined that the NT arm of Mcm1 is required for transcriptional activation of a set of genes, including cell wall biogenesis and transport genes that were not earlier known to be regulated by Mcm1. In addition to the requirement for the NT arm, mutations in the MADS box domain that are defective in the transcriptional activation of other subsets of Mcm1-regulated genes also affect the transcription of many of these newly identified Mcm1 targets. Among the genes that we identified, only the promoter region of YGP1 appeared to be bound by Mcm1 by genome-wide location analysis (17). Four of these genes, YBR071W, SPI1, PNC1, and YGP1, are induced under various cell wall-damaging conditions (25). YBR071W is an uncharacterized ORF that appears to be part of the cell wall integrity pathway (25). YGP1 and SPI1 are components of the cell wall (12,19). SPS100, which has 50% overall identity to YGP1, is a spore wall component but is also induced under stress. The increased sensitivity of the mcm1-⌬2-17 strain to NaCl, CaCl 2 , high pH, and CFW is likely caused by decreased transcription of these and other cell wall genes that are regulated by Mcm1.
The Mcm1 protein has strong in vitro DNA-binding affinity for the promoters of a subset of cell wall-and membraneassociated genes, such as GFA1, HSP150, PMA1, and PIS1 (23). Given that we identified other cell wall-associated genes that were sensitive to the Mcm1 NT arm deletion, it was possible that these genes were regulated by a similar mechanism. However, Northern blot analysis indicated that expression of these genes was not affected by deletion of the Mcm1 NT arm (data not shown). Thus, the Mcm1 arm-dependent genes we identified are distinct from other cell wall-and membraneassociated genes that may be regulated by Mcm1. It is likely that transcriptional regulation of the two groups of genes, although both mediated by Mcm1, occur through distinct mechanisms.
Most of the genes that we have identified showed a decrease in expression in strains containing the Mcm1 NT arm deletion. This result suggests that Mcm1 is functioning as an activator of these genes. However, two of the NT arm-dependent genes we identified, SIT1 and URA1, showed an increase in expression in the deletion mutant, suggesting that Mcm1 is functioning as a repressor of these genes. It is possible that depending on the promoter, Mcm1 can function either as an activator or a repressor of the salt induced genes. This would be similar to regulation by the Mcm1-ArgR complex, which functions to both activate arginine catabolic genes and repress anabolic genes in the presence of arginine in the medium (30). However, we were unable to detect the direct binding of either Mcm1 or Mcm1-⌬2-17 to these promoters by ChIP analysis, suggesting that Mcm1 may indirectly regulate the expression of these genes through the Mcm1 NT arm-dependent expression of a repressor protein that targets these genes (data not shown).
The decreased transcription of YGP1 in the mcm1-⌬2-17 mutant was not due to a decrease in Mcm1 protein level or stability. The deletion of the Mcm1 arm also did not decrease DNA-binding affinity or change the apparent DNA-bending angle, both of which contribute to transcriptional activation by Mcm1 (1). Based on our data, we hypothesize that the Mcm1 NT arm may be required to recruit or stabilize binding of another transcription cofactor that binds to the YGP1 promoter to activate its transcription. Transcription of YGP1 was earlier shown to be regulated by Rlm1, a type II MADS box protein (18). In a strain with constitutively activated Rlm1 there was a decrease in YGP1 expression, leading to the conclusion that Rlm1 was a repressor of this gene (18). However, mutational analysis of the regulatory sites in the YGP1 promoter suggests that Rlm1 may also have a minor role in the activation of YGP1. This model is supported by Northern analysis, which showed that deletion of RLM1 resulted in a decrease in YGP1 transcript compared to the wild-type strain (18). We have shown here that Mcm1, a type I MADS box protein, has a direct role in regulating the expression of this gene. Interestingly, this is the first report of a gene that is jointly regulated by both type I and type II MADS box proteins in yeast. Since Rlm1 is known to regulate YGP1 transcription, there is a possibility that Rlm1 may interact with the NT arm of Mcm1. Among the 25 genes that are known to be regulated by Rlm1, 20 also have putative Mcm1binding sites (18; our data). While the expression of some of these transcripts appear to be affected by mutations in the Mcm1 MADS box domain that decrease Mcm1 DNA-binding affinity, we found that only YGP1 and SPS100 transcription are dependent on the NT arm ( Fig. 1 and data not shown). If the Mcm1 NT arm is important for interaction with its cofactor,

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ABRAHAM AND VERSHON EUKARYOT. CELL this result suggests that Rlm1 is unlikely to be the cofactor that Mcm1 is interacting with at the YGP1 promoter. Also, since mutagenesis of the Rlm1-binding site, R1, in the YGP1-lacZ reporter showed only a minor decrease in activation (Fig. 6), there is little evidence for the role of Rlm1 as the cofactor recruited by the Mcm1 NT arm. Residues S2 and T8 in the Mcm1 NT arm are heavily phosphorylated and a mutation of S2 to alanine, which partially mimics the unphosphorylated form of these residues, had a salt-sensitive phenotype that was similar to deletion of the NT arm (24). Since the deletion of the NT arm also causes cells to be sensitive to CFW, high pH, and CaCl 2 , we expected that amino acid replacements of residues S2, T8, and T10 may have similar phenotypes on these media. However, none of the point mutations affected growth on CaCl 2 and had relatively mild effects on growth on CFW and high pH in comparison to the NT arm deletion. Interestingly, mutations of these residues to either alanine or aspartate, which partially mimic constitutive dephosphorylation or phosphorylation, respectively, showed similar sensitivity to CFW and alkaline pH ( Fig. 4A and B). This suggests that the phosphorylation state of these residues may have little to do with resistance to CFW and alkaline pH. Alternatively, it is also possible that the aspartate mutation does not effectively mimic the phosphorylated form of these residues.
The T35A mutation in the MADS box domain of Mcm1 suppresses the growth defect of the NT arm deletion under high-salt conditions (23). We found that this mutation had a similar effect on suppressing the NT arm-sensitive phenotypes on media with CFW, high pH, and CaCl 2 but did not restore growth to the level of wild-type Mcm1. Residue T35 of Mcm1 contacts the phosphate backbone of the DNA and a T35A mutation decreases Mcm1 in vitro DNA-binding affinity (1,45). As expected from the decreased DNA-binding affinity, the T35A mutation caused a significant decrease in YGP1 transcript level. Although the T35A mutation partially suppressed the growth defects caused by the deletion of the Mcm1 NT arm, it did not cause an increase in YGP1 expression (Fig. 5). Therefore, the ability to grow in medium with CFW, high pH, and CaCl 2 is not simply due to restoring expression of YGP1. This result implies that the Mcm1 NT arm may have different mechanisms of regulating YGP1 and other genes required for resistance to various cell wall stresses. This suggests that there are other Mcm1 NT arm-dependent genes that are required for resistance to these growth conditions. It is unclear how the T35A mutation suppresses the sensitivity to cell wall stresses caused by deletion of the NT arm. It is possible the T35A mutation prevents interaction with other Mcm1 cofactors or decreases Mcm1 binding to weak sites in the genome, causing an increase in the level of Mcm1 that is available to interact with cofactors at the cell wall gene promoters.
The Mcm1 point mutations in the NT arm and the MADS box domain appear to be more sensitive to CFW than growth under high-pH conditions, while hardly affecting sensitivity to CaCl 2 . One model to account for this differential sensitivity would be that genes conferring resistance to high pH and CaCl 2 have other activators in addition to Mcm1. For example, Mcm1 may be the primary activator for NT arm-dependent genes that confer CFW resistance, causing cells with the NT arm deletion to be very sensitive to the presence of this com-  While deletion of the NT arm made cells sensitive to CaCl 2 , in contrast to the CFW and high pH phenotypes, growth on CaCl 2 was not sensitive to any of the point mutations in the MADS box domain, suggesting that a very different mechanism may be at play in regulating genes that confer resistance to this growth condition. It is possible that another cofactor that binds strongly to these promoters and may be tethering Mcm1 to the DNA mainly via interactions with its NT arm. Thus, DNAbinding defects in Mcm1 may not have significant effects on activation of these genes as long as Mcm1 is tethered to the promoter through interactions with this cofactor.