Altered Histone H1 Stoichiometry and an Absence of Nucleosome Positioning on Transfected DNA*

The packaging of DNA with histones to form chromatin represents an important and powerful mechanism to regulate gene expression. Critical aspects of chromatin-specific contributions to gene regulation have been revealed by the comparison of the activities from DNA regulatory elements examined both as transiently transfected reporters and stably integrated reporters organized as chromatin. Using the mouse mammary tumor virus (MMTV) promoter as a model, we probed the structural differences between transiently transfected and stably integrated DNA templates. We demonstrated that all four core histones and the linker histone (H1) are associated with the transient template. However, whereas the core histones were present at a similar stoichiometry between the transient and the stable templates, we found that linker histone H1 molecules are fewer on the transient template. By using supercoiling assay, we show that the transient template shows intermediate levels of nucleosomal assembly. Overexpression of H1 resulted in repression of MMTV transcriptional activity and reduced accessibility to restriction endonucleases on the transient MMTV promoter. However, the addition of exogenous H1 failed to impose a normal chromatin structure on the transient template as measured by micrococcal nuclease digestion pattern. Thus, our results suggest that while transiently transfected DNA acquires a full complement of core histones, the underrepresentation of H1 on the transient template is indicative of structural differences between the two templates that may underlie the differences in the mechanisms of activation of the two templates.

The packaging of DNA with histones to form chromatin represents an important and powerful mechanism to regulate gene expression. Critical aspects of chromatin-specific contributions to gene regulation have been revealed by the comparison of the activities from DNA regulatory elements examined both as transiently transfected reporters and stably integrated reporters organized as chromatin. Using the mouse mammary tumor virus (MMTV) promoter as a model, we probed the structural differences between transiently transfected and stably integrated DNA templates. We demonstrated that all four core histones and the linker histone (H1) are associated with the transient template. However, whereas the core histones were present at a similar stoichiometry between the transient and the stable templates, we found that linker histone H1 molecules are fewer on the transient template. By using supercoiling assay, we show that the transient template shows intermediate levels of nucleosomal assembly. Overexpression of H1 resulted in repression of MMTV transcriptional activity and reduced accessibility to restriction endonucleases on the transient MMTV promoter. However, the addition of exogenous H1 failed to impose a normal chromatin structure on the transient template as measured by micrococcal nuclease digestion pattern. Thus, our results suggest that while transiently transfected DNA acquires a full complement of core histones, the underrepresentation of H1 on the transient template is indicative of structural differences between the two templates that may underlie the differences in the mechanisms of activation of the two templates.
Transcriptional regulation of eukaryotic promoters is extensively studied by using transient transfection reporter assays. Although these assays have given abundant information on transcription factor binding sites and the effects of various treatments on transcription, it is undetermined how closely they emulate the activity of endogenous promoters. One of the main concerns about using transient transfection to assay promoter activity is the lack of proper chromatin architecture on this DNA (1). This seems particularly important given the impact of chromatin, histone modifications, and chromatin remodeling proteins on transcriptional regulation.
Using the glucocorticoid-responsive mouse mammary tumor virus (MMTV) 2 promoter as a model promoter, previous work has demonstrated very clear differences between the stably integrated, replicating copies of the promoter and transiently transfected promoter DNA (2). The MMTV promoter is activated by the hormone-bound glucocorticoid receptor (GR). Although both the stable and the transient MMTV promoters are dependent on hormone for transcriptional activity, accessibility to restriction endonucleases on the promoter is hormoneinducible on the stable template and constitutively accessible on the transient template. This architectural difference was attributed to the lack of proper nucleosomal organization on the transient template as assayed by micrococcal nuclease digestion (2). Recent work using MMTV promoter has demonstrated other such differences between the two templates. BRG1 chromatinremodeling complex was shown to be necessary for activation of the stably integrated promoter but not the transient promoter (3). Overexpression of NFB relA subunit leads to repression of GRmediated transcriptional activity only from the stable promoter and not from the transiently transfected promoter (4). More recently, we demonstrated that cooperative binding between two site-specific transcription factors, GR and NF1, on the MMTV promoter occurs only on stably transfected, and not on the transiently transfected, promoter (5). Differences in transcriptional activity between the templates have not been limited to MMTV and have been used to study MyoD-activated genes (6).
In this study, we initiated a detailed investigation of the structural differences between these two templates. We demonstrate that the stoichiometry of all the core histones is similar in both these templates but the linker histone H1 is underrepresented on the transient template. Linker histone H1 acts as a generalized transcriptional repressor on transiently transfected MMTV promoter when overexpressed. Under conditions of H1 overexpression, there is alteration of the chromatin conformation as measured by reduced accessibility of transient template to restriction endonucleases. These conformational changes on the transient template do not result in regular nucleosomal organization. cDNA (from 1471.1 cells) cloned in pCDNA3.1 mammalian expression plasmid, which overexpresses H1.3 with a FLAG tag at the C-terminal end of the protein.
Cell Culture and Transfections-C127 is a mouse mammary carcinoma cell line, and 1471.1 is its derivative that maintains ϳ300 copies of a bovine papilloma virus with the MMTV LTR fused to chloramphenicol acetyltransferase (CAT) reporter gene (3). Both these cell lines were grown at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 g/ml penicillin/ streptomycin, and 10 mM HEPES. Cells were treated with a synthetic glucocorticoid dexamethasone (10 Ϫ7 M). Transient transfections were performed by the Lipofectamine PLUS method (Invitrogen) as described by the manufacturer.
Nucleosome Assembly Assay-Relaxed form of plasmid DNA (topoisomerase-treated) was prepared by treatment with Escherichia coli topoisomerase I. C127 cells were transfected with supercoiled, topoisomerase-treated, linear pLTRluc plasmid for 24 h. Isolated nuclei and proteinase K were treated overnight. Genomic DNA was purified by phenol chloroform extractions and ethanol precipitation. The genomic DNA was digested with KpnI (no KpnI site in pLTRluc) for ease in loading the gel. Two micrograms of DNA was electrophoresed on a 0.8% agarose tris-phosphate-EDTA gel (8) with 50 g/ml chloroquine for 48 h in the dark and blotted. The blot was probed with vector-specific probe (ϳ1.0-kb XbaI-AvaI fragment of the luciferase gene).
DNA Replication Assay-1471.1 cells were transfected with pLTRLuc for 48 h, and genomic DNA was isolated by proteinase K digestion followed by phenol chloroform extractions and ethanol precipitation. Genomic DNA was digested to completion using DpnI restriction enzyme. Purified DNA was subjected to PCR using Oligo-344 (5Ј-tta agt aag ttt ttg gtt aca aac t-3Ј) and Luc-2 (5Ј-ATT TTA CCA ACA GTA CCG GAA TGC-3Ј) primer pair (to assay transient template) or Oligo-344 and CAT-2 (5Ј-GAA CGG TCT GGT TAT AGG TAC ATT GA-3Ј) (for stable template). The PCR products were electrophoresed on a 1.5% agarose gel.
Luciferase Assay and CAT Assays-Cells were grown in 6-well plates and were washed twice with phosphate-buffered saline and lysed on the plate with 500 l of reporter lysis buffer (Promega, Madison, WI) per well. The plates were scraped, and lysates were pelleted by centrifugation at 20,800 ϫ g for 1 min at room temperature. Twenty microliters of the lysate was added to 100 l of luciferase substrate, and the activity was monitored. Relative light units were normalized to the total protein measured. For CAT assay, 20 l of the lysate was used following the manufacturer's direction (Promega).
Western Blot Analysis-Nuclei were isolated as described previously (9). Acid-soluble proteins were isolated from nuclei as previously described (11). Histone proteins (40 g) were electrophoresed using a 14% SDS-polyacrylamide gel. The gels were either stained with Coomassie Blue or transferred to a nitrocellulose membrane (Amersham Biosciences) and probed with H1 antibody (Upstate Biotechnology) or FLAG antibody (Sigma).
In Vivo Restriction Enzyme Accessibility Assay-Nuclei were isolated as previously described (9) and digested with SstI (100 units/ml), FokI (400 units/ml), DdeI (1000 units/ml), and HaeIII (1000 units/ml). DNA was purified and then digested to completion with HaeIII or AlwN1. The samples were amplified by reiterative primer extension using 32 P-labeled luc-2 primer, and the products were separated on 6% denaturing polyacrylamide gels. The gels were exposed to phosphorimaging screens for analysis. The accessibility to restriction enzymes was calculated as percent in vivo enzyme cleavage of in vitro enzyme digestion.
Micrococcal Nuclease Analysis-Isolated nuclei were resuspended in 200 l of wash buffer containing 1 mM CaCl 2 and digested with 20 units/ml micrococcal nuclease (Worthingtom Biochemicals, Lakewood, NJ) for 5 min at 30°C (12). The reaction was stopped by adding 40 l of 100 mM EDTA, pH 8.0, 10 mM EGTA, pH 7.5. The samples were purified and digested to completion with PstI prior to Southern blot. Ten micrograms of micrococcal nuclease-digested DNA was separated on a 2% agarose gel and transferred to Hybond Nϩ membrane (Amersham Biosciences). The blot was probed with a 32 P-end-labeled 84-bp PCR product corresponding to the nucleosome B region of the MMTV promoter (amplified using nucB-1 and nucB-2, 5Ј-GGT TAC AAA CTG TTC TTA AAA CGA GGA T-3Ј and 5Ј-CAG AGC TCA GAT CAG AAC CTT TGA-3Ј primer pairs). The blot was exposed to phosphorimaging screens for analysis.

RESULTS
Nucleosome Assembly on Transiently Transfected DNA-To understand the chromatin architecture of transiently transfected DNA, we first wanted to assess whether nucleosomes associate with plasmid DNA introduced into the mammalian cell. Previous work demonstrated that transiently transfected Transiently Transfected DNA Architecture DNA does not assume proper nucleosomal organization as assayed by micrococcal nuclease digestion (2,13). It was also shown that using the DEAE-dextran method of transfection for 24 h, relaxed DNA was converted to supercoiled DNA (14), which is indicative of nucleosome deposition. Nucleosome deposition on a circular DNA creates changes in superhelical densities. We wanted to assess changes in linking number of the plasmid DNA after transfection under our experimental conditions. To analyze nucleosome assembly on transient template, we performed supercoiling assay with genomic DNA from C127 cells transfected with topoisomerase-treated (relaxed) and untreated (supercoiled) pLTRluc. As a control we also transfected linear DNA (pLTRluc digested with a single cutter SstI restriction enzyme) for 24 h. The various species of plasmid DNA with different linking numbers were separated on chloroquine-agarose gels to analyze their superhelical distribution. In the lane with supercoiled DNA transfection (Fig. 1, lane 1), there appear to be some faint bands in between the highly supercoiled and the relaxed form of the plasmid DNA that are not in the original introduced plasmid (Fig. 1, lane 4). This suggests some intermediate levels of nucleosomal assembly on the plasmid when introduced in the cell. However, when we introduce relaxed pLTRLuc into cells, we do not observe significant changes from the relaxed DNA originally transfected (Fig.  1, compare lanes 2 and 5). We know that relaxed replicating plasmids assume supercoiling using cell-free extracts indicating nucleosome deposition (15,16). So the lack of supercoiling on transiently introduced relaxed DNA implies that assembly on this DNA is not similar to what is observed on the stably integrated DNA.
Association of Histones with the Transiently Transfected DNA-The results from the supercoiling assay suggest that there is some nucleosomal organization on a transiently transfected DNA. We next wanted to investigate the association of histones with the transiently transfected DNA and to understand if there are differences in the stoichiometry between the histones associated with this template when compared with the stable template. We utilized an experimental setup as previously described (2) followed by chromatin immunoprecipitation assay. In this setup, 1471.1 cells that have ϳ300 copies of the MMTV promoter fused to the CAT reporter gene on stably replicating episome were transfected with pLTRluc construct, which has the MMTV promoter fused to the luciferase reporter gene. This allows us to assay transcription and chromatin remodeling of both templates in the same cell. Under these conditions, earlier work demonstrated that whereas both the templates were hormone-inducible for transcriptional activity, accessibility to restriction endonucleases is hormone-inducible on the stable template and constitutive on the transient template. This architectural difference was attributed to the lack of proper nucleosomal organization on the transient template as assayed by micrococcal nuclease digestion.
Using the above mentioned experimental setup, we performed ChIP assay with cells transfected with pLTRLuc for 24 h. To avoid the cytoplasmic portion of the transfected DNA, we performed ChIP assay with isolated nuclei. To assess the association of histones on both templates from the same immunoprecipitated samples, we fragmented the DNA with HaeIII restriction endonuclease. HaeIII enzyme digests DNA in the linker regions on the stably integrated MMTV-CAT gene, releasing an ϳ3-nucleosome length of DNA that encompasses the nucleosome A and B region and part of the coding region of both these reporter genes (17) (Fig. 2A). By using reporter-specific primers, we can analyze the differences between these two templates. We immunoprecipitated nuclear lysate with all the core histones and linker histone H1 antibodies. We found all the histones associated with the transient template (Fig. 2B). Interestingly, the stoichiometries of core histones as assayed by ChIP/input ratio were similar on both the transient and stable templates (Fig. 2B). This is intriguing given the lack of micrococcal ladder with the transiently transfected DNA (2) and intermediate levels of nucleosome assembly. The association of histones with the transient template was detected as early as 4 h after transfection (data not shown). Surprisingly, when we compared ChIP/input ratio of histone H1 on these two templates, we found that there were half the number of histone H1 associated with the transient template when compared with the stable template (Fig. 2, C and D). Because the comparison of histone binding to the two templates is done in the same cell, the effect of transfection on histone binding is not a concern. Taken together, these results suggest that there are similar amounts of core histones associated with the transient template when compared with the stable template but the linker histone is underrepresented on the transient template.
Because the transiently transfected template behaves like the stable template with respect to association of core histones, we wanted to rule out the possibility of DNA replication on this template. Although pLTRluc does not have a eukaryotic origin of replication, we still wanted to exclude any repair-induced DNA replication. To analyze DNA replication on the transient template, we isolated genomic DNA from 1471.1 cells transfected with pLTRLuc DNA for 48 h. Genomic DNA was subjected to digestion by restriction enzyme DpnI, which is methylation-dependent (G m ATC). The transfected plasmid DNA that is bacterial in origin is digestible, The DNA was electrophoresed on 0.8% agarose tris-phosphate-EDTA gel with chloroquine 50 g/ml for 48 h in the dark. As controls, supercoiled, topoisomerase-treated, and linear (digested with Sst1) pLTRluc (lanes 4 -6; P, plasmid control) were also loaded on the gel. The gel was blotted and probed with 32 P labeled with luciferase-specific probe.
Transiently Transfected DNA Architecture FEBRUARY 22, 2008 • VOLUME 283 • NUMBER 8 but if this DNA undergoes replication in a eukaryotic cell then the DNA becomes resistant to DpnI digestion due to the loss of methylation. The MMTV promoter sequence has DpnI site in the nucleosome B region (Fig. 2E). When PCR was performed using template-specific primers encompassing the DpnI site, we failed to observe resistance to DpnI digestion upon transfection (Fig. 2F). In contrast and as expected, the stable template showed complete resistance to DpnI (Fig. 2F). This suggests that transiently transfected DNA did not undergo replication even after 48 h of transfection. Another point we wanted to ensure in our ChIP analysis is that it is not limited to a pool of transiently transfected DNA that is associated with octamer proteins. So we compared the ratio of transient template to stable template in ChIP input samples and total genomic DNA and found the ratio to be similar in both these cases (data not shown).

Overexpression of H1 Leads to Repression of Transcriptional
Activity-Our data so far show that histones are associated with the transient template; however, there is less histone H1 represented on the transient template when compared with the stable template. Because H1 is implicated in nucleosomal organization and higher order chromatin structure (18 -20), we wanted to determine whether insufficient levels of H1 in the cells were the cause for the structural differences between these two templates. We overexpressed H1 in 1471.1 cells co-transfected with pLTRLuc DNA (Fig. 3A). We observed repression of both basal transcription and glucocorticoidmediated transactivation of the MMTV promoter (Fig. 3B), consistent with the idea that H1 is a generalized transcriptional repressor. We also observed repression of transcription from pGL3-control vector with the SV-40 promoter fused to luciferase reporter gene (data not shown). When we analyzed transcription from the stable template by performing CAT assay, overexpression of H1 had very minimal effect on GR-mediated transcriptional activation of the promoter (Fig. 3C). Therefore, H1 overexpression results in generalized repression of transcription from a transiently transfected DNA.
Overexpression of H1 Alters the Chromatin Architecture of the Transient Template-Next we wanted to understand the structural effect of histone H1 overexpression on the transiently transfected DNA. Using the MMTV promoter, it had been previously shown that while the stable template undergoes chromatin remodeling upon hormone induction as evidenced by increased accessibility to restriction enzymes, the transient template is constitutively accessible to restriction enzymes (2). Given our results thus far, it seems that the constitutive accessibility to restriction enzymes may be due to fluidity in nucleosomal positioning on the transient template. We wanted to examine whether overexpression of H1 has any effect on the constitutively open chromatin organization of the transiently transfected DNA. To achieve this, we assayed the accessibility of SstI restriction enzyme, which has a cleavage site in the nucleosome B region (Fig. 4A). As expected, in the absence of H1 overexpression we did not observe changes in accessibility to SstI upon hormone treatment (Fig. 4B, lanes 1 and 2). Inter- Purified DNA was digested to completion using DpnI enzyme. Using the primers shown in the schematic, the region was amplified by PCR and the product was electrophoresed on 1.5% agarose gel. As a control, total genomic DNA (uncut with DpnI) was amplified with the same primer sets.
estingly, when H1 was overexpressed SstI accessibility was reduced, indicating a conformational change (Fig. 4B, lanes 3  and 4). This reduced SstI cutting was the same under both control and hormone treatment conditions. This is consistent with the transcriptional activity of the MMTV promoter in which we observed reduction in both basal and hormone-induced transcription on the transient template (Fig. 3B). Although the basal cutting in the presence of H1 overexpression mimicked what is observed on the stable template, the hormone-induced hypersensitivity associated with the stable template was not evidenced on the transient template. Similar to the SstI enzyme, accessibility to other restriction enzymes such as the Fok1 (Ϫ162) (Fig. 4B, lanes 5-8, and 4D) and the DdeI (Fig. 4C, lanes  1-4, and 4D) in the nucleosome B region on the transient template was reduced upon H1 overexpression. To understand whether this change in conformation is restricted to the nucleosome B region of the MMTV promoter, we analyzed accessibility to enzymes with sites outside the nucleosome B region (Fig. 4A), such as HaeIII (Fig. 4C, lanes 5-8, and 4D) and Fok1 (ϩ50) (Fig. 4B, lanes 5-8, and 4D) and we found reduced cutting upon H1 overexpression. Thus, overexpression of H1 results in conformational change on the transient template that is evidenced all along the promoter and possibly the entire plasmid. Our results thus far suggest that the transiently transfected DNA has higher nucleosomal mobility that may be restricted by binding of H1 under conditions of H1 overexpression. H1 Overexpression Does Not Alter Nucleosomal Organization on the Transiently Transfected DNA-To determine whether overexpression of H1 results in alteration of nucleosomal organization on the transient template, we performed micrococcal nuclease assay. For this assay we used C127 cells transfected with pLTRluc cotransfected with vector or the H1 construct to analyze the transient template, and we used 1471.1 cells transfected with vector or the H1 construct to examine the stable template. As expected, we observed ordered chromatin organization on the stable template ( Fig. 5A and 5B, lane 4) as evidenced by the regularly spaced ladder generated by the micrococcal nuclease enzyme. Unlike the stable template and consistent with the previous results (2), we failed to detect the nucleosomal ladder (Fig. 5B, lane 2) on the transient template. However, interestingly, we observed no change in the pattern of micrococcal nuclease digestion on the transient template upon H1 overexpression (Fig. 5B, lanes 1 and 2). This suggests that overexpression of H1 does not result in the ordered nucleosomal organization on the transient template. Therefore, we conclude that under conditions of H1 overexpression, excess H1 probably binds to all accessible sites on the plasmid in a very random way, thus reducing the nucleosome fluidity on the DNA but not organizing the nucleosomes.

DISCUSSION
Functional differences in transcriptional activity between MMTV promoters embedded in chromosomes and within  Purified DNA was digested to completion with HaeIII or AlwN1. Five micrograms of DNA were amplified by linear PCR using Taq DNA polymerase and 32 P-labeled primer as depicted in A. The PCR products were then separated on 6% denaturing polyacrylamide gels. D, the results in B and C are quantitated as percent cutting.
transiently transfected plasmids help to illuminate contributions that chromatin make to regulate gene expression. In this study we wanted to understand the structural differences between transiently transfected DNA and DNA within chromosomes. Using a Lipofectamine method of transient transfection, we demonstrated intermediate levels of nucleosome deposition on the transiently transfected DNA. In an attempt to address the structural disparities between these two templates, we evaluated the differences in stoichiometry in association of histones using chromatin immunoprecipitation assay followed by real-time PCR. We demonstrate association of all four core histones and the linker histone with the transiently transfected DNA. Interestingly, the stoichiometry of the core histones on the transient template is very similar to what is observed on the stable template. This suggests that the histones associated with the transient template have nucleosomal composition similar to that of the stably replicating DNA. Because the introduction of supercoils on a plasmid DNA in the supercoiling assay can also be indicative of association of H3-H4 tetramer and nonhistone proteins, our result suggesting histone octamer association with the transient template is a significant one. Because transiently transfected template does not undergo replication, this octamer deposition on the DNA is replication-independent deposition. However, this association of octamers with the transient template does not lead to a proper micrococcal nuclease ladder pattern. Therefore, the absence of a micrococcal nuclease ladder pattern may suggest a random placement of octamers on the transient template. This is consistent with structural data available from earlier work suggesting that nucleosomes are deposited on transiently transfected template but the organization of nucleosomes is not similar to what is found on a replicating endogenous DNA (2,13,14).
Intriguingly, in contrast to the core histones, the linker histone association was found to be less on the transient template when compared with the stable template. This underrepresentation of histone H1 on the transient template can perhaps explain the constitutive accessibility of transient template to restriction endonucleases. This also implies that the arrangement of histone octamers on the transiently transfected DNA may be very dynamic and fluid due to fewer H1 molecules. Consistent with this idea and in contrast with the stably integrated promoter, the transiently transfected MMTV promoter does not require the BRG1 chromatin remodeling complex for GR-mediated transcriptional activation (4). In vitro studies have shown a similar role of the linker histone H1 in modulating nucleosomal remodeling by human SWI/SNF (21). However, the lack of a micrococcal nuclease ladder on a transient template cannot be associated with underrepresentation of H1 on this template, given the fact that changes in stoichiometry of H1 in vivo do not result in a loss of micrococcal nuclease ladder. Changes in H1 levels in the cells lead to alteration in the spacing between nucleosomes (22). There are cell type-specific differences in the levels of H1 per nucleosome in higher eukaryotes (20). Interestingly, the embryonic stem cells have 0.5 H1/nucleosome with a nucleosome repeat length of 189 bp (23). Moreover, recent imaging studies have demonstrated that H1 molecules in the cells are very mobile, with a residence time of 3 min compared with several hours for the core histones (24).
Based on our observations, we wanted to understand the effect of overexpression of histone H1 on a transiently transfected DNA. We found that overexpression of H1 had a generalized repressive effect on transcription from a transient template (both basal and induced). Overexpression of histone H1 has very little effect on transcription from stably integrated MMTV promoter. This selective transcriptional repression from the transient template may be due to the fact that the transient template architecture is more accessible to binding of factors. This allows H1 to bind at random sites and inhibit transcription. Consistent with this idea, transcription profiling of H1 knock-out cells indicates effects on very few genes when compared with the wild-type cells in mice (25). Along with the transcriptional repression on transiently transfected DNA, H1 overexpression resulted in structural changes of the template as evidenced by reduced restriction enzyme accessibility. These data are consistent with a mechanism by which H1 stabilizes histone octamer-DNA interactions by reducing its mobility on the DNA (26), thus reducing the accessibility of restriction enzymes to DNA. Despite the structural changes on the transient template upon H1 overexpression, the nucleosome arrangement was not regularized. This result is consistent with the idea that limiting amounts of H1 in the cells is not a reason for improper nucleosome positioning on the transient template. Similarly, the random binding of H1 on the transiently transfected DNA, which reduces the histone octamer fluidity, is not sufficient to organize a phased array of nucleosomes as observed on the stable template.
Taken together, our results suggest that transiently transfected DNA is associated with histone octamers that are randomly positioned on the DNA. Apart from this random positioning, the nucleosome arrangement is dynamic and fluid, possibly due to the underrepresentation of histone H1 on this template. Overexpression of H1 diminishes accessibility of the DNA to transcription machinery as assessed by reduced transcription and restriction endonuclease access. Our results imply a more basic role of H1 in DNA replication-mediated chromatin organization. Histone H1 is known to deposit on newly replicated DNA immediately after the core histone deposition (27). Because the nucleosome assembly proteins are so closely linked with the DNA replication machinery, there is immediate deposition of nucleosomes on both the copies of DNA. We propose that in the case of transiently transfected DNA, the large circular DNA molecule is presented to the nuclear pools of histones all at once, which results in random deposition and positioning of octamers on the template. Coupled with the reduced levels of H1, the newly deposited octamers may have few restrictions on their mobility relative to the octamers within the chromosomes. Therefore, random organization of octamers on transient template depicts a scenario in which DNA replication and nucleosome deposition steps are uncoupled in vivo.