Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment
In order to mount effective adaptive responses against a diverse range of pathogens and antigens , the immune system has to generate sufficient numbers of mature peripheral T cells that express functional T cell receptors ( TCRs ) . T cell development is a complex and highly regulated process that involves multiple stages of selection and maturation , both within the thymus and after thymic export . In the thymus , productive rearrangement of the TCR β-chain in CD4−CD8− double negative ( DN ) thymocytes drives progression to the CD4+CD8+ double positive ( DP ) stage ( Starr et al . , 2003 ) . After rearrangement of the TCR α-chain , DP thymocytes express mature TCRs which are then used to survey self-peptide/MHC complexes presented by specialized epithelial cells in the thymic cortex ( cTECs ) ( Klein et al . , 2014 ) . A small percentage of DP thymocytes receive positively selecting TCR signals which promote their survival , in part through upregulation of IL-7Rα ( Sinclair et al . , 2011 ) . Positively selected thymocytes become committed to either the CD4 or CD8 single-positive ( SP ) lineage and migrate to the thymic medulla , where they undergo further negative selection mediated by interactions with antigen-presenting cells such as dendritic cells ( DCs ) or AIRE-dependent medullary thymic epithelial cells ( mTECs ) ( Hogquist et al . , 2005; Klein et al . , 2014 ) , during which thymocytes expressing self-reactive TCRs either undergo apoptosis or are diverted to alternative fates , such as becoming regulatory T cells ( Tregs ) ( Stritesky et al . , 2012 ) . As SP thymocytes undergo maturation , expression of the surface marker CD24 is decreased while CD62L expression is upregulated . As such , SP thymocytes may be further divided into two phenotypically distinct populations , often referred to as semi-mature ( CD62LloCD24hi ) and mature ( CD62LhiCD24lo ) . These phenotypic changes are associated with an important functional difference: semi-mature SP thymocytes are susceptible to apoptosis upon TCR stimulation , whereas mature SP thymocytes are not and respond instead by proliferating ( Sprent and Kishimoto , 2002; Weinreich and Hogquist , 2008 ) . In addition , only mature SP thymocytes upregulate sphingosine-1-phosphate receptor ( S1PR1 ) , which is required for egress from the thymus ( Matloubian et al . , 2004; Weinreich and Hogquist , 2008 ) . The process of SP thymocyte maturation , from entry into the SP compartment to thymic egress , has been estimated to take 4–5 days ( McCaughtry et al . , 2007 ) . New T cells , also referred to as recent thymic emigrants ( RTEs ) , undergo a phase of post-thymic phenotypic and functional maturation before they are incorporated into the long-lived peripheral naïve T cell pool ( Fink , 2012 ) . The transition from the semi-mature to mature stage of SP thymocyte development is marked by numerous changes in gene expression . Some of these changes , including the upregulation of S1PR1 and CD62L , are mediated by the transcription factor KLF2 ( Carlson et al . , 2006 ) . In the periphery , the process of post-thymic maturation is also associated with transcriptional changes , though on a smaller scale ( Mingueneau et al . , 2013 ) . For instance , an increase in IL-7Rα expression during this period has been shown to promote recent thymic emigrant ( RTE ) survival ( Silva et al . , 2014 ) . Proper regulation of the transcriptional program underlying late-stage SP thymocyte and post-thymic T cell maturation is thus critically important for the establishment of a normal naïve T cell compartment . Multiple genes involved in NF-κB signaling have been reported to be required for the development of mature T cells ( Guerin et al . , 2002; Schmidt-Supprian et al . , 2003; Sato et al . , 2005; Zhang and He , 2005; Liu et al . , 2006; Wan et al . , 2006; Silva et al . , 2014 ) , primarily through mechanisms related to TCR signaling and protection from apoptosis . In addition , roles for the transcriptional repressor Nkap ( Hsu et al . , 2011 ) and chromatin remodeling factor Bptf ( Landry et al . , 2011 ) have been identified in recent years . However , the transcriptional regulators controlling these stages of T cell maturation remain largely unknown . The C2H2 zinc finger family constitutes the largest class of transcription factors in mammalian genomes , and many key transcriptional regulators in immune cell development , such as Ikaros and Plzf , contain multiple C2H2 zinc fingers ( Brayer and Segal , 2008 ) . The C2H2 zinc finger fold is classically recognized to be a DNA-binding domain ( Wolfe et al . , 2000; Iuchi , 2005 ) , although it may also participate in interactions with RNA ( Brown , 2005 ) or other proteins ( Brayer and Segal , 2008 ) . In this study , we identify a C2H2 zinc finger protein , Zfp335 , as an essential regulator of T cell maturation . Zfp335 , also known as NIF-1 ( Mahajan et al . , 2002; Garapaty et al . , 2009 ) , is ubiquitously expressed and is essential for early development , with homozygous deletion resulting in embryonic lethality at E7 . 5 ( Yang et al . , 2012 ) . Here , we report that an ENU-induced mutant allele of Zfp335 results in defective accumulation of naïve T cells , largely as a consequence of impaired maturation in SP thymocytes and RTEs . We show that this maturation defect is independent of thymic selection or effects on proliferation , but is associated with reduced viability . We identify a set of Zfp335 target genes in thymocytes and present evidence that decreased Zfp335 occupancy at a subset of these targets alters gene expression in mutant thymocytes . Taken together , our findings provide evidence that Zfp335 functions as a transcription factor and key regulator of a transcriptional program required for T cell maturation .
As part of an N-ethyl-N-nitrosourea ( ENU ) mutagenesis screen ( Nelms and Goodnow , 2001 ) for lymphocyte phenotypes , we identified a variant C57BL/6 mouse pedigree with decreased frequencies of CD4+ and CD8+ T cells in peripheral blood ( Figure 1A ) , which we named bloto ( blood T cells low; allele henceforth designated blt ) . This trait was fully penetrant and occurred at a frequency consistent with recessive inheritance . Homozygotes were viable , fertile and displayed no gross external abnormalities . 10 . 7554/eLife . 03549 . 003Figure 1 . Identification of an ENU mouse mutant with a cell-intrinsic deficiency in peripheral T cells . ( A ) Frequency of CD4+ and CD8+ T cells in peripheral blood of 8-week-old heterozygous ( blt/+ ) or homozygous ( blt/blt ) mice as detected by flow cytometry . Numbers in quadrants indicate mean frequencies ± s . d . ( n = 3 mice per genotype ) . ( B ) Frequency of total splenic CD4+ and CD8+ T cells ( left ) ; percentage of CD4+ T cells with a naïve ( CD62LhiCD44lo ) or effector ( CD62LloCD44hi ) phenotype ( right ) . ( C ) Frequency of major thymocyte subsets ( left ) ; proportion of semi-mature ( CD62LloCD24hi ) and mature ( CD62LhiCD24lo ) subsets within the CD4SP thymocyte population ( right ) . ( D ) Absolute number of DN , DP , CD4SP , and CD8SP thymocytes in blt/+ vs blt/blt mice . Semi-mature and mature CD4SP thymocytes were gated as in ( C ) . CD8SP thymocytes were gated as follows: semi-mature ( TCRβhiCD62LloCD24int ) , mature ( TCRβhiCD62LhiCD24lo ) . Mature CD4SP and CD8SP thymocytes are reduced in numbers by approximately 1 . 8- and 2 . 3-fold , respectively . ( E ) Quantification of CD4+ and CD8+ naïve T cells in the spleen , gated as in ( B ) , showing a 4 . 6-fold and 7 . 8-fold decrease in CD4+ and CD8+ naïve T cells , respectively . ( F ) Ratio of blt/blt ( CD45 . 2+ ) vs wild-type ( CD45 . 1+CD45 . 2+ ) cells for splenic NK cells ( NK1 . 1+TCRβ− ) , DP , semi-mature ( semi ) and mature ( mat ) SP thymocytes and naïve splenic T cells from lethally irradiated WT CD45 . 1+ hosts reconstituted with a 1:1 mix of blt/blt and WT bone marrow cells . Data in ( B ) and ( C ) are representative of seven to eight independent experiments with matched blt/+ and blt/blt littermates and are summarized in ( D ) and ( E ) . Mice were analyzed at 8 to 10 weeks of age ( A–E ) or 8 to 12 weeks post-reconstitution ( F ) . Each symbol represents an individual mouse; small horizontal lines indicate the mean; n . s , not significant; *p < 0 . 05 and **p < 0 . 01 ( two-tailed Mann–Whitney test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 00310 . 7554/eLife . 03549 . 004Figure 1—figure supplement 1 . Similar relative decrease in blt/blt T cells in mixed chimeras vs intact mice , indicating the lack of a competitive or rescue effect by WT cells . ( A ) Ratio of blt/blt to WT mature SP thymocytes and naïve T cells normalized to the ratio in DP thymocytes from mixed chimeras ( open symbols ) , compared to the ratio of the same subsets between matched pairs of intact mice ( filled symbols ) , based on data reported in Figure 1 . ( mean ± s . d . , n = 9 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 00410 . 7554/eLife . 03549 . 005Figure 1—figure supplement 2 . Mice heterozygous for the bloto mutation do not exhibit a T cell phenotype . ( A ) Number of CD4+ and CD8+ naïve T cells from spleens of WT ( n = 6 ) and blt/+ ( n = 4 ) mice . ( B ) Ratio of blt/+ ( CD45 . 2+ ) vs WT ( CD45 . 1+CD45 . 2+ ) cells in splenic NK cells and indicated thymocyte and T cell populations from irradiation chimeras reconstituted with a mix of blt/+ and WT bone marrow as in Figure 1F . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 00510 . 7554/eLife . 03549 . 006Figure 1—figure supplement 3 . blt/blt mice exhibit a selective defect in αβ T cells . ( A ) Number of effector/memory phenotype ( CD44hiCD62Llo ) CD4+ and CD8+ T cells in the spleen . ( B ) Number of Foxp3+ CD4SP thymocytes ( left ) , splenic Foxp3+CD4+ Tregs ( center ) , and splenic iNKTs identified by positive CD1d tetramer staining ( right ) . ( C ) Number of splenic B cells ( CD19+; left ) , NK cells ( NK1 . 1+TCRβ+; center ) , and γδ T cells ( TCRγδ+; right ) . n . s , not significant; *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 ( two-tailed Mann-Whitney test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 006 Further characterization of the bloto mutant revealed a strong reduction in overall T cell frequencies in secondary lymphoid organs , especially in the CD62LhiCD44lo naïve T cell population ( Figure 1B ) . Analysis of T cell development in the thymus revealed no significant decrease in frequencies or numbers of CD4−CD8− DN or CD4+CD8+ DP thymocytes of blt/blt mice relative to heterozygous controls ( Figure 1C , D ) . However , blt/blt mice had slightly lower SP thymocyte frequencies , and subgating on semi-mature ( CD62LloCD24hi ) and mature ( CD62LhiCD24lo ) SP thymocytes showed significant underrepresentation of the mature subset ( Figure 1C ) , with an approximately twofold decrease in the numbers of both CD4 and CD8 mature SP thymocytes ( Figure 1D ) . In comparison , CD4+ and CD8+ naïve T cell numbers in the spleen were reduced about fivefold to eightfold ( Figure 1E ) , suggesting both a thymic and peripheral component to the bloto T cell developmental defect . In mixed bone marrow chimeras , lower percentages of blt/blt as compared to wild-type cells were observed in the SP thymocyte and naïve T cell populations , demonstrating that the T cell phenotype is cell-intrinsic and recapitulating the progressive developmental defect seen in intact mice ( Figure 1F ) . The decrease in blt/blt T cells in mixed chimeras was comparable to that in intact mice ( Figure 1—figure supplement 1A ) , which indicates the lack of a competitive or rescue effect by wild-type cells . The bloto phenotype is a fully recessive trait with no evidence for haploinsufficiency or a dominant negative effect , since heterozygous mice exhibited no decrease in naïve T cells compared to wild-type controls ( Figure 1—figure supplement 2A ) , and blt/+ T cells did not decline relative to wild-type cells even in a competitive mixed chimeric setting ( Figure 1—figure supplement 2B ) . Despite the strong defect in naïve T cells , we noted little difference in the number of T cells with an effector/memory phenotype ( CD62LloCD44hi ) ( Figure 1—figure supplement 3A ) . This is likely due to homeostatic expansion of surviving cells in T cell-deficient blt/blt mice , as this increase in memory relative to naïve T cells was not observed in mixed chimeras in which the effects of T cell lymphopenia were alleviated by the presence of wild-type cells ( data not shown ) . Non-conventional αβT cell lineages , such as Foxp3+ regulatory T cells and iNKTs , were also affected ( Figure 1—figure supplement 3B ) , but not to a greater degree than conventional CD4+ and CD8+ T cells . However , there were no deficiencies in other major lymphocyte lineages such as NK cells ( Figure 1F; Figure 1—figure supplement 3C ) , γδT cells , and B cells ( Figure 1—figure supplement 3C ) , suggesting that the bloto mutation has a selective effect on αβT cells . To identify the causative genetic lesion , the peripheral blood T cell deficiency was used to map the mutation in an F2 intercross to a genomic interval between 163 . 16 and 165 . 88 Mb on chromosome 2 ( Figure 2—figure supplement 1A ) . Whole-exome sequencing of DNA from an affected mouse identified a single novel single-nucleotide variant within the interval of interest: a C to T missense mutation in exon 21 of Zfp335 ( Figure 2A ) . This results in the replacement of a positively charged Arg residue at position 1092 ( henceforth referred to as R1092 ) by Trp , a bulky non-polar amino acid . 10 . 7554/eLife . 03549 . 007Figure 2 . Identification of causative missense mutation within a C2H2 zinc finger of Zfp335 . ( A ) Sequence trace analysis of the mutated codon in homozygous ( blt/blt ) compared to heterogygous ( blt/+ ) mice , showing an Arg-to-Trp substitution at position 1092 . ( B ) Linear schematic of the 13 C2H2 zinc finger ( ZF ) domains ( shaded boxes ) in Zfp335 . Asterisk indicates the R1092W bloto mutation in ZF12 ( black box ) . Diagram drawn to approximate scale . ( C ) Multiple sequence alignment of predicted Zfp335 orthologs from dog ( Canis lupus ) , pig ( Sus scrofa ) , human ( Homo sapiens ) , mouse ( Mus musculus ) , chicken ( Gallus gallus ) , and zebrafish ( Danio rerio ) . Asterisk indicates Arg residue affected by bloto mutation . Amino acids are colored according to their physicochemical properties . ( D ) Quantitative RT-PCR analysis of Zfp335 mRNA from indicated FACS-purified thymocyte subsets and naïve T cells ( n = 3–4 mice , mean ± s . d . for biological replicates ) ; ISP , immature CD8+ thymocytes identified by tlack of TCRβ expression; results are presented relative to expression of Hprt . ( E ) Western blot for Zfp335 protein in the thymocytes from wild-type ( +/+ ) and homozygous mutant ( b/b ) mice , with actin as loading control . ( F ) Immunofluorescence analysis of Zfp335 nuclear localization in mature CD4SP thymocytes; nucleus counterstained with DAPI . ( right ) Secondary antibody-only negative staining control . Scale bar: 2 μm . ( G ) Frequency of CD4+ and CD8+ T cells differentiating from blt/blt hematopoietic stem cells transduced ( Thy1 . 1+ ) with either wild-type Zfp335 ( Zfp335WT ) or control MSCV-IRES-Thy1 . 1 vector , compared to non-transduced ( Thy1 . 1− ) cells from the same mouse , 8 to 10 weeks after reconstitution of irradiated hosts . ( H ) Transduced ( Thy1 . 1+ ) cells as a percentage of indicated thymocyte and T cell subsets from irradiation chimeras that had received bone marrow retrovirally transduced with WT Zfp335 , bloto Zfp335 or control vector . Data points are connected by a separate line for individual mice . Data are representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 00710 . 7554/eLife . 03549 . 008Figure 2—figure supplement 1 . Linkage mapping of bloto mutation to a 2 . 72 Mb region on chromosome 2 containing Zfp335 . ( A ) Black bars represent B6 homozygosity and gray bars represent B6/CBA heterozygosity as determined by SNP analysis . Data from two affected and three unaffected F2 progeny shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 00810 . 7554/eLife . 03549 . 009Figure 2—figure supplement 2 . Protein sequence analysis and structural modeling of mutated C2H2 zinc finger in Zfp335 . ( A ) Amino acid sequence of ZF12 and ZF13 ( 1073–1127 a . a ) . Positions −1 , +2 , +3 , and +6 , which are thought to mediate base recognition in DNA-binding C2H2 zinc fingers , are indicated for ZF12 . R1092 ( red ) ; canonical C2H2 linker ( green box ) . ( B ) Structural model of three zinc fingers near the C-terminus of Zfp335 ( 1044–1126 a . a ) . The mutated Arg residue is highlighted in red; gray spheres represent zinc ions coordinated within the zinc finger fold . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 009 Zfp335 is a 1337-amino acid protein containing 13 predicted C2H2 zinc finger domains ( Figure 2B ) . Its role as a transcriptional regulator in neurogenesis and neuronal differentiation has recently been described ( Yang et al . , 2012 ) , but any immunological function has thus far been unknown . The R1092W mutation falls within the 12th zinc finger ( ZF12 ) near the C-terminus ( Figure 2B ) , at a position that is highly conserved across vertebrate evolution ( Figure 2C ) . Homology modeling places R1092 in the ZF12 α-helix at position +6 ( Figure 2—figure supplement 2A , B ) , one of the canonical positions mediating DNA base recognition by C2H2 zinc fingers ( Wolfe et al . , 2000 ) . The presence of a TNEKP linker between ZF12 and ZF13 ( Figure 2—figure supplement 2A ) and its similarity to the conserved TGEKP linker , a key structural feature of DNA-binding C2H2 zinc fingers ( Wolfe et al . , 2000 ) , further hint at the possibility that R1092 may play a direct role in DNA binding by Zfp335 . Zfp335 transcript levels were not decreased in blt/blt thymocytes and T cells; in fact , a slight increase was observed relative to blt/+ controls , particularly in the most mature subsets ( Figure 2D ) . Western blotting analysis of thymocytes showed no reduction in the amount of Zfp335 protein ( Figure 2E ) , indicating that the R1092W mutation had no adverse effect on protein expression or stability . Confocal imaging of sorted mature SP thymocytes showed that both wild-type and mutant Zfp335 localized to the nucleus , forming punctate foci within regions of euchromatin ( Figure 2F ) . No detectable differences in subnuclear distribution were observed . These data suggest that the bloto mutation is hypomorphic rather than null , as it results in normal levels of stable protein that can localize appropriately to the nucleus but has impaired function due to the selective disruption of ZF12 . An in vivo gene complementation test was carried out by retroviral transduction of wild-type Zfp335 into blt/blt bone marrow for hematopoietic reconstitution of irradiated hosts . The T cell development block was strongly reversed in blt/blt cells transduced with wild-type Zfp335 but not control vector ( Figure 2G , H ) , hence establishing that Zfp335R1092W was the causative mutation . Overexpression of Zfp335R1092W yielded an intermediate rescue effect ( Figure 2H ) , suggesting that supraphysiological protein expression may partially compensate for impaired function caused by a hypomorphic mutation . Interestingly , we observed that transduction frequencies for Zfp335WT in DP thymocytes ( Figure 2H ) or non-T lymphocytes ( data not shown ) were typically low ( <10% ) compared to transduction frequencies achieved with Zfp335R1092W or other genes , suggesting that overexpression of Zfp335 may have an inhibitory effect on early hematopoiesis , leading to poorer reconstitution of transduced progenitors . To further characterize the block in intrathymic development , we examined thymocyte populations by continuous in vivo bromodeoxyuridine ( BrdU ) labeling , where the percentage of BrdU+ cells indicates population turnover . After 4 days of BrdU administration , when comparing blt/blt mice to heterozygous controls , the mature SP population showed a decrease in turnover , whereas thymocyte subsets from earlier stages of development labeled with similar kinetics ( Figure 3A; Figure 3—figure supplement 1A ) . Genome-wide transcriptome analysis of sorted mature CD4SP thymocytes revealed a gene expression profile consistent with impaired SP thymocyte maturation; by showing , for instance , decreased expression of genes known to be upregulated during maturation ( Teng et al . , 2011 ) ( Figure 3B ) . By comparing staining intensities of various surface markers associated with SP maturation ( e . g . , CD24 , CD62L ) in pre-gated mature SP subsets , we also observed a trend towards a less mature surface phenotype ( data not shown ) . Taken together , these data suggest that blt/blt mice have decreased efficiency of entry into the mature SP compartment and progression through the final maturation stages within the mature SP thymocyte population . 10 . 7554/eLife . 03549 . 010Figure 3 . Zfp335R1092W-induced T cell dysregulation affects mainly mature SP thymocytes and recent thymic emigrants . ( A ) Percentage of BrdU+ cells in indicated thymocyte populations from blt/blt mice relative to blt/+ controls after 4 days of continuous BrdU labeling ( mean ± s . d . , n = 4 ) . ( B ) Gene set enrichment analysis ( GSEA ) analysis of gene expression data from blt/blt vs WT mature CD4SP thymocytes showing significant negative correlation with genes known to be upregulated during SP thymocyte maturation ( MSigDB gene set: GSE30083 ) . ( C and D ) Input-normalized fraction of total ( left panel ) or GFP+ ( right panel ) Rag1-GFP blt/+ and Rag1-GFP blt/blt cells within the total CD45 . 2+ CD4+ naïve donor population recovered from recipient spleens ( C ) and peripheral lymph nodes ( D ) at indicated time points after co–transfer with control CD45 . 2+ blt/+ cells . ( E ) Flow cytometry analysis of CD4SP cells in the thymus of blt/blt mice and blt/+ controls after 4 days of FTY720 or saline treatment . Percentage of cells in mature SP ( CD62LhiCD24lo ) gate shown . Results are quantified ( right ) for CD4SP and CD8SP thymocytes . ***p < 0 . 001 ( two-tailed Mann–Whitney test ) , data pooled from four independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 01010 . 7554/eLife . 03549 . 011Figure 3—figure supplement 1 . Impaired late-stage SP thymocyte development and early post-thymic peripheral T cell maturation in blt/blt mice . ( A ) Turnover of DN , DP , semi-mature , and mature CD4SP thymocytes assessed after 2–4 days of continuous in vivo BrdU labeling ( mean ± s . d . , n = 4 ) . ( B ) Input-normalized fraction of donor CD45 . 2+ CD4+CD62LhiCD44lo naïve T cells ( closed symbols ) or CD4SP thymocytes ( open symbols ) that were blt/blt , recovered from spleen at indicated time points after transfer of blt/blt and control peripheral lymphocytes or thymocytes to lymphoreplete CD45 . 1+ hosts ( mean ± s . d . , n = 2–4 ) . ( C ) ( left ) GFP signal in CD62LhiCD44lo CD4+ and CD8+ T cells from spleen of Rag1-GFP transgenic blt/+ vs . blt/blt mice . ( right ) Percentage of GFPhi naïve T cells , gated as shown in histograms ( mean ± s . d . , n = 6–8 ) . ( D ) Percentage of total naïve T cells , GFPhi RTEs and GFPlo mature naïve ( MN ) T cells from spleens of blt/blt mice relative to matched blt/+ littermate controls . Data obtained from mice analyzed between 7 to 10 weeks of age ( mean ± s . d . , n = 10 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 011 As described earlier , blt/blt mice have a more severe defect in the accumulation of naïve T cells compared to that of mature SP thymocytes , suggesting that Zfp335R1092W-induced dysregulation extends to events in the periphery following thymic export . To assess the impact of Zfp335R1092W on naïve T cell homeostasis , we adoptively transferred blt/blt peripheral T cells together with wild-type controls into congenic lymphoreplete hosts and assessed their relative maintenance over 7 days . Surprisingly , blt/blt T cells persisted just as well as control wild-type T cells ( Figure 3—figure supplement 1B ) , suggesting that Zfp335R1092W does not significantly impair the survival of the bulk naïve T cell population . This led us to hypothesize that the bloto defect may be largely confined to new T cells that have recently left the thymus , otherwise known as recent thymic emigrants ( RTEs ) . In adult mice , these cells comprise a relatively small percentage of total naïve T cells and may therefore not be significantly represented in measurements involving the bulk naïve T cell population . We initially assessed the ability of SP thymocytes to survive once introduced into the periphery , in essence behaving as surrogate RTEs . Significantly , blt/blt cells decayed more rapidly than co-transferred controls , hinting that RTE survival may be impaired in blt/blt mice ( Figure 3—figure supplement 1B ) . In order to study the RTE population in blt/blt mice in greater detail , we crossed the blt/blt mutant to the Rag1-GFP reporter line ( Kuwata et al . , 1999 ) . In these reporter mice , GFP signal intensity is inversely proportional to time spent in the periphery ( Boursalian et al . , 2004 ) , allowing for the identification of RTEs as GFP+ cells within the naïve T cell population . We found that the GFPhi subset was significantly overrepresented in blt/blt naïve T cells and was skewed towards cells with the highest GFP signal intensities that have most recently exited the thymus , consistent with a partial block in post-thymic naïve T cell maturation ( Figure 3—figure supplement 1C ) . To directly assess RTE maintenance in vivo , a test population of either Rag1-GFP blt/+ or Rag1-GFP blt/blt peripheral T cells was mixed with control non-fluorescent T cells and injected i . v . into congenic lymphoreplete hosts . Consistent with previous experiments ( Figure 3—figure supplement 1B ) , there was no significant decline in relative numbers of total blt/blt naïve T cells recovered from the spleen 1 , 3 , and 5 days post-transfer ( Figure 3C ) . However , the blt/blt Rag1-GFP+ population declined more rapidly than blt/+ Rag1-GFP+ controls , particularly within the first day of adoptive transfer ( Figure 3C ) . Interestingly , in contrast to the spleen , we noted a small decrease in the maintenance of total blt/blt naïve T cells , relative to blt/+ cells , that were recovered from peripheral lymph nodes . Similarly , the lymph nodes exhibited a larger decline in blt/blt Rag1-GFP+ relative to blt/+ Rag1-GFP+ T cells ( Figure 3D ) . These data suggest that some of the apparent decline in blt/blt RTEs ( Figure 3D ) may be due to less efficient short-term accumulation in lymph nodes . Nonetheless , the fact that we observe in both spleen and lymph nodes a higher rate of decay in the Rag1-GFP+ population as compared to the overall effect on the bulk naïve T cell pool strongly suggests that blt/blt RTEs survive less well than control RTEs . In terms of absolute numbers , GFPhi RTEs in blt/blt mice were reduced by a magnitude largely matching that of GFP− mature naïve cells ( Figure 3—figure supplement 1D ) , suggesting that most of the drop-off in cell numbers may have occurred at the earliest stages of RTE maturation . To test this hypothesis , we treated mice with FTY720 , a potent inhibitor of thymic egress ( Matloubian et al . , 2004 ) . This strategy ensured that new T cells , which would have otherwise been exported into the periphery , were trapped in the thymus where their accumulation could be measured . After 4 days of thymic egress blockade , blt/blt mice showed greatly impaired accumulation of SP cells with a mature CD62LhiCD24lo phenotype ( Figure 3E ) , suggesting that most of the losses in RTEs take place within a short period after they enter the periphery . These data also indicate that the cell loss is due to intrinsic defects in the maturing T cells and is not dependent on their location in a particular lymphoid compartment . In mice with a polyclonal TCR repertoire , we observed normal frequencies of positively selected DP thymocytes with a CD69hiTCRβint phenotype ( Figure 4—figure supplement 1A ) , with no differences in CD5 surface expression on post-selection thymocytes ( Figure 4—figure supplement 1B ) , suggesting that positive selection is not strongly impaired by the bloto mutation . Because compensatory TCR rearrangements may mask a potential positive selection defect , we examined thymic development in blt/blt mice expressing the class II MHC-restricted OTII TCR transgene , in which impaired positive selection would be expected to cause a dramatic reduction in CD4SP thymocytes . However , this was not observed in OTII blt/blt mice , which had only slightly lower CD4SP frequencies compared to controls and a fold reduction in CD4SP numbers ( Figure 4A ) similar to that seen in polyclonal blt/blt mice ( Figure 1D ) . 10 . 7554/eLife . 03549 . 012Figure 4 . The T cell maturation defect in blt/blt mice is not caused by altered thymic selection or Bcl2-dependent survival . ( A ) Flow cytometry analysis of major thymocyte populations from OTII TCR transgenic blt/+ and blt/blt mice , gated on total live thymocytes ( left ) ; quantification of OTII TCR-expressing Vα2+ CD4SP thymocytes from OTII blt/+ ( n = 14 ) and OTII blt/blt ( n = 14 ) mice ( right ) . ( B ) Frequency of thymocyte subsets in lethally irradiated WT B6 ( top left ) or RIP-mOVATg ( bottom left ) recipients reconstituted with T cell-depleted bone marrow from OTII blt/+ and OTII blt/blt mice; quantification of Vα2+ CD4SP thymocytes from indicated RIP-mOVATg chimeric mice ( right ) . ( C ) In vitro viability of sorted CD45 . 1− blt/+ vs blt/blt semi-mature ( left ) and mature ( right ) CD4SP thymocytes co-cultured with CD45 . 1+ WT CD4SP thymocytes . Live cells were pre-gated as annexin V− DAPI− and percentage of CD45 . 1− cells was normalized to input . ( D ) Frequency of CD4+ and CD8+ T cells in the spleen of blt/+ vs blt/blt mice expressing a human BCL2 transgene ( BCL2Tg ) under the control of the proximal Lck promoter ( left ) ; quantification of naïve T cells from BCL2Tg blt/+ ( n = 10 ) , and BCL2Tg blt/blt ( n = 10 ) mice ( right ) . Data representative of eight ( A ) , three ( B ) , two ( C ) , and six ( D ) independent experiments; n . s , not significant , ***p < 0 . 001 ( two-tailed Mann–Whitney test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 01210 . 7554/eLife . 03549 . 013Figure 4—figure supplement 1 . blt/blt mice exhibit intact positive and negative selection in the thymus . ( A ) Surface expression of CD69 and TCRβ on DP thymocytes from blt/+ and blt/blt mice with a polyclonal TCR repertoire . Positively selected DP thymocytes are CD69hiTCRβint . ( B ) CD5 surface expression on CD69hiTCRβint and CD69hiTCRβint post-positive selection populations , gated on total live thymocytes . ( C ) Quantitative RT-PCR analysis of Nr4a1 ( Nur77 ) mRNA in FACS-purified CD4 and CD8 semi-mature SP thymocytes ( mean ± s . d . , n = 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 01310 . 7554/eLife . 03549 . 014Figure 4—figure supplement 2 . Normal expression of IL-7 receptor and Bcl2 family members . ( A ) Normalized expression levels of indicated Bcl2 family genes from Affymetrix array analysis of mRNA from sorted WT and blt/blt mature CD4SP thymocytes ( mean ± s . d . , n = 3 ) . ( B ) Quantitative RT-PCR analysis of Il7r transcript in FACS-purified mature SP thymocytes and naïve T cells ( mean ± s . d . , n = 3 ) . ( C ) Surface expression of IL-7Rα on WT ( CD45 . 1+CD45 . 2+ ) and blt/blt ( CD45 . 2+ ) mature SP thymocytes and naïve T cells from mixed chimeras . Data are representative of eight mice . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 01410 . 7554/eLife . 03549 . 015Figure 4—figure supplement 3 . blt/blt naïve T cells proliferate normally in response to TCR stimulation in vitro and show no significant reduction in cycling of mature SP thymocytes . ( A ) BrdU labeling of DP and mature CD4SP thymocytes from blt/+ ( n = 5 ) and blt/blt ( n = 4 ) mice after a 4 h pulse ( mean ± s . d . , two-tailed Mann-Whitney test ) . ( B ) Percentage of indicated thymocyte subsets in S/G2/M phases of the cell cycle , as defined by >2n DNA content . Data pooled from three independent experiments ( mean ± s . d . , n = 6–7 ) . ( C ) Analysis of CFSE dilution by congenically marked WT and blt/blt CD4+ naïve T cells in mixed culture , after 3 days of stimulation with plate-bound αCD3 and αCD28 . Data are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 015 Using the OTII/RIP-mOVA model of AIRE-dependent clonal deletion ( Anderson et al . , 2005 ) , we found no evidence that blt/blt mice had altered negative selection ( Figure 4B ) . Consistent with this conclusion , mRNA expression of Nur77 , a key proapoptotic regulator induced during negative selection ( Baldwin and Hogquist , 2007 ) , was not elevated in blt/blt semi-mature SP thymocytes ( Figure 4—figure supplement 1D ) . Hence , our data indicate that altered thymic selection does not contribute to the T cell deficiency in blt/blt mice . The findings in Figure 3C , D and Figure 3—figure supplement 1B suggested decreased survival may at least in part explain why blt/blt T lymphocytes fail to accumulate normally . Consistent with this notion , blt/blt mature SP thymocytes showed a greater loss of viability in vitro over time compared to blt/+ controls , while a lesser effect was seen for blt/blt semi-mature SP cells ( Figure 4C ) . Annexin V staining and measurement of active caspase 3 in freshly isolated thymocytes did not reveal differences between blt/blt and blt/+ mice ( data not shown ) , likely because cells in the earliest stages of apoptosis are efficiently cleared in vivo ( Surh and Sprent , 1994 ) . To examine whether Bcl2-regulated apoptotic pathways were involved in promoting the loss of blt/blt T cells , we crossed blt/blt mice to a transgenic line expressing human BCL2 under control of the Lck promoter ( Sentman et al . , 1991 ) . Overexpession of BCL2 failed to rescue the defect in peripheral naïve blt/blt T cells ( Figure 4D ) , indicating that it is not due to reduced Bcl2-dependent survival . In addition , no differences in the expression of Bcl2 family pro- and anti-apoptotic genes were observed in blt/blt mature SP thymocytes ( Figure 4—figure supplement 2A ) . IL-7 is a critical regulator of naïve T cell homeostasis ( Surh and Sprent , 2008 ) ; in particular , IL-7Rα expression is induced in new T cells and is required for their survival and integration into the peripheral pool ( Silva et al . , 2014 ) . However , no significant differences in IL-7Rα expression were detected in blt/blt thymocytes and T cells , either at the transcript level ( Figure 4—figure supplement 2B ) or by surface receptor staining ( Figure 4—figure supplement 2C ) . Furthermore , survival of blt/blt thymocytes and naïve T cells in the presence of IL-7 in vitro was comparable to that of co-cultured wild-type cells ( data not shown ) , suggesting that the T cell defect is not due to the loss of IL-7R function . Lastly , Zfp335R1092W had no significant effect on the fraction of thymocytes in cell cycle , as shown by short-term BrdU labeling ( Figure 4—figure supplement 3A ) and DNA content analysis ( Figure 4—figure supplement 3B ) . Similarly , blt/blt naïve T cells were able to expand at a normal rate following TCR stimulation in vitro ( Figure 4—figure supplement 3C ) , demonstrating that blt/blt T cells are not defective in their ability to undergo proliferation . Zfp335 has recently been shown to bind a variety of gene promoters in mouse embryonic brain ( Yang et al . , 2012 ) , but its genome-wide binding characteristics and targets relevant to its function in T cell development remain to be defined . In addition , because our structure-function predictions ( Figure 2—figure supplement 2 ) led us to hypothesize a DNA-binding role for the mutated Arg , we wished to know if the R1092W mutation disrupted the ability of Zfp335 to bind to its targets in vivo . To address these questions , we performed ChIP-seq analysis of Zfp335 binding sites in total thymocytes isolated from wild-type and blt/blt mice . ChIP-seq data sets were generated using two separate polyclonal antibodies and are referred to as ‘ChIP-C’ and ‘ChIP-N’ respectively . Using data obtained from wild-type thymocytes , peak calling with a q-value threshold of <0 . 05 identified 157 Zfp335-binding regions in the vicinity of 177 genes . Genome browser inspection of ChIP-seq signal tracks confirmed that these peaks , although fairly limited in number , represented regions of significantly enriched binding intensity . Genome-wide , Zfp335 peaks were strongly enriched in gene promoters ( Figure 5A ) and located upstream of transcriptional start sites ( TSS ) ( Figure 5B ) . Zfp335-bound regions were associated with high levels of H3K4me3 , a hallmark of active gene promoters , and low levels of the enhancer-associated modification H3K27ac and the repressive chromatin mark H3K27me3 ( Figure 5C ) , consistent with Zfp335 functioning primarily as a regulator of promoter-dependent gene transcription . Zfp335 target genes were enriched for functional categories representing a diverse range of biological processes , including protein synthesis and metabolism , mitochondrial function , cell cycle regulation , RNA processing , and transcriptional regulation ( Figure 5D; Supplementary file 4 ) . 10 . 7554/eLife . 03549 . 016Figure 5 . Genome-wide analysis of Zfp335 binding sites in wild-type thymocytes based on ChIP-seq using an antibody against the C-terminus of Zfp335 . ( A ) Genomic feature annotation of Zfp335 peaks reveals strong enrichment in promoter regions ( ≤1 kb upstream of TSS ) and 5ʹ UTRs relative to genomic background . ( B ) Average profile of peak center distances from nearest RefSeq TSS for 141 Zfp335 peaks located within ±1 . 5 kb of a TSS , showing a positional preference for binding upstream of the TSS . ( C ) Average density of H3K4me3 ( blue ) , H3K27ac ( green ) and H3K27me3 ( purple ) marks for a region from −2 kb to +2 kb relative to Zfp335 peak summits , based on ENCODE histone modification ChIP-seq data for murine whole thymus . ( D ) Gene ontology analysis of genes associated with Zfp335 binding sites using GREAT . Top enriched annotation terms in the MSigDB pathway ontology are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 016 With the same q-value cutoff ( <0 . 05 ) that yielded a total of 157 binding events for wild-type thymocytes , we detected 141 peaks in blt/blt thymocytes ( Supplementary file 2 , 3 ) . By visual inspection of ChIP-seq data on a genome browser , we determined that of the 28 peaks detected in wild-type but not blt/blt thymocytes , 22 showed a convincing loss of binding while the rest were false positives due to noise at low-confidence peaks . Of the nine peaks that were called for Zfp335R1092W but not Zfp335WT , four were not true binding events , but rather signal artifacts arising from repeat regions , while the remaining five were low-confidence peaks . This strongly suggests that the bloto mutation does not lead to the gain of novel binding sites , which is consistent with our hypothesis that it is a loss-of-function hypomorph . Interestingly , we did not observe a global decrease in Zfp335 binding intensities across all target sites . Reduced binding was detected for a subset of target genes: Ankle2 , Nme6 , and Mrps5 are shown as representative examples ( Figure 6A ) . However , at other target sites , such as Rbbp5 , Polr2e , and Pes1 , Zfp335 binding did not appear to be significantly impaired ( Figure 6B ) . Only nine Zfp335-bound regions showed decreases in ChIP-seq peak intensities in blt/blt thymocytes that were robust enough to be detected across both sets of ChIP-C and ChIP-N replicates ( Figure 6C ) . We performed ChIP-qPCR to validate a selection of target genes that we had identified as differentially bound by Zfp335R1092W , vs targets that showed normal binding , and found the ChIP-qPCR data to be in agreement with the ChIP-seq-based assessment ( Figure 6D ) . Moreover , ChIP-qPCR analysis of sorted CD4SP thymocytes yielded similar results to the analysis with total thymocytes ( Figure 6—figure supplement 1A ) . 10 . 7554/eLife . 03549 . 017Figure 6 . Decreased Zfp335 binding in blt/blt thymocytes is detected for a subset of target genes . ( A ) Signal tracks showing Zfp335 occupancy at three target genes ( Ankle2 , Nme6 , Mrps5 ) for which significantly decreased binding in blt/blt relative to WT thymocytes is observed using both the C-terminus-specific antibody ( ChIP-C ) and the N-terminus-specific antibody ( ChIP-N ) . Vertical axis , fragment pileup per million reads ( normalized to library sequencing depth ) . Input , sequencing of input genomic DNA ( background control ) . ( B ) Signal tracks showing Zfp335 occupancy at three target genes ( Rbbp5 , Polr2e , Pes1 ) for which no reduction in binding is detected in blt/blt thymocytes . ( C ) Identification of nine putative differentially bound target sites and their ten associated genes from the intersection of differential peaks ( bloto < WT ) called for both ChIP-C and ChIP-N data sets . We consider Aimp1 and Tbck to be associated with a single peak as they share a bidirectional promoter . ( D ) ChIP-qPCR analysis of Zfp335 binding at selected targets to validate ChIP-seq-based assessment of differential binding in blt/blt thymocytes . ChIP enrichment was calculated as percent input; results are presented as the fold-change in ChIP enrichment for blt/blt vs WT ( mean ± s . d . , n = 3 for three independent experiments ) . ( E ) Relative Zfp335 binding and gene expression changes for target genes associated with the ChIP-C set of differentially bound regions: horizontal axis , expression fold-change ( log2 ) values from microarray analysis of blt/blt vs WT mature CD4SP thymocytes; vertical axis , score reflecting likelihood that Zfp335 binding is significantly enriched in WT relative to blt/blt thymocytes . Red circles , target genes identified as differentially bound in both ChIP-C and ChIP-N data sets ( Figure 6C ) ; black circles , target genes associated with reduced Zfp335 binding in the ChIP-C but not ChIP-N data set . Wdr47 is highlighted ( filled black circle ) as a target gene that was identified as differentially bound only in the ChIP-C data set ( Figure 6—figure supplement 1C , D ) but showed significantly downregulated expression in blt/blt thymocytes ( Figure 6—figure supplement 1B , E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 01710 . 7554/eLife . 03549 . 018Figure 6—figure supplement 1 . Analysis of correlation between changes in Zfp335 binding and gene expression in blt/blt thymocytes . ( A ) ChIP-qPCR analysis of Zfp335 binding at selected targets in sorted CD4SP thymocytes . ChIP enrichment was calculated as percent input; results are presented as the fold-change in ChIP enrichment for blt/blt vs WT for two biological replicates . ( B ) Quantitative RT-PCR analysis of Mrps5 and Rabggtb mRNA in sorted DP thymocytes ( mean ± s . d . , n = 3 ) . ( C ) GSEA plot comparing expression values of Zfp335 target genes ( TSS within ±1 kb of a Zfp335 peak ) in blt/blt vs WT mature CD4SP thymocytes ( left ) . Heatmaps depicting relative expression of the top 50 downregulated ( right ) and top 11 upregulated ( bottom left ) target genes; Wdr47 is marked with an asterisk . ( D ) ChIP-seq signal tracks showing Zfp335 occupancy at Wdr47 , a target gene for which reduced Zfp335 binding is detected in the ChIP-C but not ChIP-N data set . ( E ) ChIP-qPCR confirmation of reduced Zfp335 binding to the Wdr47 promoter . Antibody used was specific for C-terminal epitope of Zfp335 . ChIP enrichment represented as % input; matched data points for blt/+ ( filled symbols ) vs blt/blt ( open symbols ) thymocytes from three independent ChIP experiments shown . ( F ) Quantitative RT-PCR analysis of Wdr47 mRNA in sorted DP thymocytes ( mean ± s . d . , n = 2–3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 018 To understand how reduced Zfp335 binding at a subset of direct targets in blt/blt thymocytes could be biologically significant , we integrated our ChIP-seq data with gene expression profiles that we had obtained for blt/blt and wild-type mature CD4SP thymocytes . Of the ten target genes listed in Figure 6C as having significantly reduced Zfp335R1092W binding , only three ( Ankle2 , Nme6 , Cnpy2 ) were downregulated in expression by more than twofold ( Figure 6E ) . Since our ChIP-seq results were derived from total thymocytes , of which only a small percentage are mature CD4SP , one caveat was that we might have missed gene expression changes in the larger population , so we sorted DP thymocytes ( >90% of total ) and tested them by RT-qPCR . Relative to blt/+ controls , blt/blt DP thymocytes had no detectable differences in the expression of Mrps5 and Rabggtb , even though these genes exhibit strongly reduced Zfp335 binding in blt/blt thymocytes ( Figure 6—figure supplement 1B ) . This is not an unexpected finding given that transcription factors are known to bind sites which they do not functionally regulate ( Smale , 2014 ) . Nonetheless , we did observe deregulated expression of many Zfp335 target genes in our CD4SP array data , including many which did not exhibit reduced Zfp335 occupancy according to our stringent criteria ( Figure 6—figure supplement 1C ) . For example , although Wdr47 failed to meet these criteria because it showed decreased Zfp335R1092W binding in the ChIP-C but not ChIP-N data set ( Figure 6—figure supplement 1D , E ) , its expression was nonetheless significantly downregulated in blt/blt thymocytes ( Figure 6E , Figure 6—figure supplement 1C , F ) . With the exception of a handful of strongly downregulated targets , gene expression changes were usually mild . Most deregulated target genes exhibited decreased expression , though a few were modestly upregulated ( Figure 6—figure supplement 1C ) . Interestingly , this small group of upregulated targets include Zfp335 itself , which we confirmed by RT-qPCR ( Figure 2D ) , suggesting that Zfp335 participates in an autoregulatory negative feedback loop . It is probably reasonable to assume that many of these differentially expressed genes do in fact have reduced Zfp335R1092W binding at their promoters , which we failed to detect owing to limitations in ChIP-seq sensitivity and/or the inability of our approach to identify differential binding events that are not evident at the whole population level because they occur exclusively in mature CD4SP thymocytes . As a member of the C2H2 zinc finger protein family , it is highly likely that Zfp335 interacts with its genomic targets through direct binding to a specific DNA sequence motif . Using a set of high-confidence peaks from our Zfp335WT ChIP-seq data , we performed de novo motif analysis and identified a novel 22 bp bipartite motif consisting of two conserved elements separated by a variable spacer ( Figure 7A ) . The positional distribution of this putative consensus motif was unimodal and located near the centers of Zfp335 peaks ( Figure 7B ) , consistent with the hypothesis that it is the direct DNA-binding motif . Motif sites found within Zfp335 peaks showed a distinct DNase I genomic footprint ( Figure 7C ) and strong sequence conservation across evolution ( Figure 7D ) compared with motif sites outside peaks , further suggesting that it is a functional DNA-binding motif for Zfp335 . 10 . 7554/eLife . 03549 . 019Figure 7 . Identification of a novel DNA motif bound by Zfp335 . ( A ) Sequence motif identified by de novo motif search of WT Zfp335 ChIP-seq peaks . ( B ) Density histogram showing localization of motif relative to Zfp335 peaks . ( C ) DNase I genomic footprinting analysis of motif sites in Zfp335 ChIP-seq peaks ( blue ) compared with motif sites in all regions ±2 kb of TSS ( orange ) , using ENCODE DGF data for whole thymus . The 22 bp motif is marked on both sides by dashed lines . ( D ) Sequence conservation ( phyloP ) profiles around Zfp335 motif sites within ChIP-seq peaks ( blue ) vs sites in all regions ±2 kb of TSS ( orange ) . ( E ) Sequences of oligonucleotide probes used in ( F ) and ( G ) . Z1 probe sequence was derived from Zfp335 binding site at the Zfp335 promoter and contains the primary consensus motif ( capitalized , bold letters ) . For probes M1–M3 , the first half ( red ) , second half ( green ) , or both parts of the consensus motif are mutated as shown . ( F ) Gel shift assay demonstrating sequence-specific binding of Zfp335 protein to labeled Z1 probe . Nuclear extracts from 293T cells transfected with control ( − ) or FLAG-Zfp335 ( + ) expression vectors were used . Signal from Zfp335-specific complexes ( black arrowhead ) is eliminated with an excess of unlabeled Z1 competitor oligo . Relative amounts of total Zfp335 protein verified by Western blot ( bottom panel ) . Data are representative of three independent experiments . ( G ) Zfp335 binding to labeled Z1 probe in the presence of competition from unlabeled mutant oligos Z1–M1 , Z1–M2 , and Z1–M3 . Signal intensity inversely correlates with ability of mutant probe to bind Zfp335: M1 is least able to bind , followed by M2 , then M3 . Black arrowhead , Zfp335 complex . Data are representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 01910 . 7554/eLife . 03549 . 020Figure 7—figure supplement 1 . Further EMSA characterization of Zfp335-binding motif . ( A ) Gel shift assay showing Zfp335 complex formation with labeled Z1 probe but not with Pdap1 ( Pd ) probe containing the motif reported in a previous study ( Yang et al . , 2012 ) . The standard Ikaros gel shift probe ( Molnár and Georgopoulos , 1994; Cobb et al . , 2000 ) , IKbs4 ( Ik ) , was used as negative control . ( B ) Zfp335 binding to labeled Z1 probe is competed away in the presence of excess unlabeled Z1 oligo , whereas Pdap1 ( Pd ) and Ikaros ( Ik ) oligos have no effect . Data from ( A ) and ( B ) are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 020 To determine if Zfp335 was able to bind to this DNA sequence in vitro , we performed gel shift assays with labeled oligonucleotide probe containing the predicted consensus motif ( Figure 7E ) and 293T cell nuclear extracts ectopically expressing Zfp335 protein . We found that Zfp335 formed a gel shift complex with the labeled probe ( Figure 7F ) . This complex increased in abundance proportional to the amount of Zfp335 protein and was eliminated by competition with excess unlabeled probe , demonstrating that it is sequence-specific and Zfp335-dependent ( Figure 7F ) . Competition experiments with unlabeled probes containing mutations of conserved nucleotides ( Figure 7E ) showed that Zfp335 binding was abolished when both DNA elements of the bipartite motif were mutated , whereas mutations in either the first or second element had an intermediate effect , indicating that both parts of the consensus motif are required for full Zfp335 binding ( Figure 7G ) . We noted a stronger effect on competition upon mutation of the first element compared to the second , suggesting that it makes a greater contribution to Zfp335 binding ( Figure 7G ) . We also performed gel shift assays with an oligonucleotide containing the previously reported Zfp335 recognition motif ( Yang et al . , 2012 ) , but failed to detect binding above the negative control ( Ikaros binding site ) ( Figure 7—figure supplement 1 ) . Re-analysis of the embryonic brain ChIP-Seq data published by Yang et al . according to the parameters we applied to our data set ( see ‘Materials and methods’ ) identified the motif shown in Figure 7A . These observations suggest that the discrepancy between our and the previously reported motif arises from differences in motif finding strategy . Importantly , the identification of a common motif in these distinct data sets strongly suggests that this sequence element is recognized by Zfp335 in a diversity of cell types . To understand which direct targets of Zfp335 were functionally relevant to the T cell maturation defect in blt/blt mice , we focused our efforts on a set of genes for which we had clear evidence of reduced Zfp335 occupancy and mRNA expression , the most prominent of which was Ankle2 ( ankyrin repeat and LEM domain-containing protein 2 ) . Zfp335 binding to the Ankle2 promoter was effectively abolished in blt/blt thymocytes ( Figure 6A , D ) ; this was accompanied by decreased transcript expression across multiple stages of T cell development ( Figure 8A ) and consequently , a virtual absence of Ankle2 protein ( Figure 8B ) . Exogenous expression of Zfp335 significantly increased Ankle2 mRNA levels in blt/blt T cells ( Figure 8C ) , providing evidence that Zfp335 is both necessary and sufficient for Ankle2 expression . 10 . 7554/eLife . 03549 . 021Figure 8 . Ankle2 is a functional target gene of Zfp335 and its dysregulation by Zfp335R1092W contributes to the maturation defect in blt/blt T cells . ( A ) Quantitative RT-PCR analysis of Ankle2 transcript levels in indicated thymocyte and naïve T cell populations sorted from blt/+ and blt/blt mice ( mean ± s . d . , n = 3–4 ) . ( B ) Western blot for Ankle2 protein in wild-type ( +/+ ) and mutant ( b/b ) thymocytes , with actin as loading control . ( C ) Rag1-GFP blt/blt bone marrow was retrovirally transduced with either WT Zfp335 or control vector and used to reconstitute irradiated hosts . CD4+ RTEs from these chimeras were sorted into transduced ( Thy1 . 1+ ) and non-transduced ( Thy1 . 1− ) populations and analyzed for Ankle2 expression by RT-qPCR ( mean ± s . d . , n = 3 ) . ( D ) Gating strategy for spleen CD4+ naïve T cells , subdivided into GFPhi ( less mature ) and GFPlo ( more mature ) populations . Red line , Rag1-GFP+ naïve T cells; blue dashed line , Rag1-GFP+ mature SP thymocytes; grey fill , Rag1-GFP− CD45 . 1+ host naïve T cells ( background control ) . GFPlo and GFPhi T cells were then gated on Thy1 . 1 reporter expression as indicated . Flow cytometry plots shown are for chimeras reconstituted with Ankle2- or empty vector-transduced Rag1-GFP blt/blt bone marrow . ( E ) Change in the percentage of cells transduced with Ankle2 ( n = 6 ) , WT Zfp335 ( n = 5 ) or control vector ( n = 10 ) during naïve T cell maturation ( Δ%Thy1 . 1+ = %Thy1 . 1+ GFPlo − %Thy1 . 1+ GFPhi ) . A higher Δ%Thy1 . 1+ indicates enrichment of the reporter+ cells in the more mature GFPlo population compared to the less mature GFPhi population . Data are represented as Tukey box plots; *p < 0 . 05 , **p < 0 . 01 ( one-tailed Mann–Whitney test ) . Chimeras were analyzed 8 weeks post-reconstitution . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 02110 . 7554/eLife . 03549 . 022Figure 8—figure supplement 1 . Ectopic expression of other Zfp335 target genes is not sufficient to reverse the T cell maturation defect . ( A ) blt/blt bone marrow was retrovirally transduced with Ankle2 , Cnpy2 , Nme6 , Sep15 , Tbck , Wdr47 , or control vector , and used to reconstitute irradiated chimeras . The normalized ratios of %Thy1 . 1+ CD4+ naïve T cells relative to %Thy1 . 1+ DP thymocytes are shown . Overexpression of Ankle2 yielded a ratio greater than that for vector control , suggesting it is able to drive T cell maturation to some degree . Other constructs tested either showed weak or insignificant effects ( Nme6 , Wdr47 , Sep15 , Tbck ) , or may be inhibitory for maturation ( Cnpy2 ) . Each symbol represents one chimeric mouse . ; **p < 0 . 01 ( one-tailed Mann-Whitney test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03549 . 022 We were able to partially reverse the T cell maturation defect in the periphery by exogenously expressing Ankle2 in Rag1-GFP blt/blt cells ( Figure 8D , E ) , as shown by the overall increase in representation of Ankle2-transduced ( Thy1 . 1 reporter+ ) cells within the more mature Rag1-GFPlo naïve T cell subset compared to the less mature Rag1-GFPhi subset . However , overexpression of Ankle2 had a weaker effect compared to that achieved by Zfp335 ( Figure 8E ) , consistent with the idea that Ankle2 , though important , is but one of several downstream targets that are required for Zfp335-dependent T cell maturation . In addition to Ankle2 , we tested five other Zfp335 target genes for their ability to rescue the T cell maturation defect in blt/blt bone marrow chimeras , but did not detect a significant effect for any of these genes ( Figure 8—figure supplement 1 ) .
In this study , we identify a novel role for Zfp335 as an essential regulator of T cell maturation . By analyzing mice with a hypomorphic missense mutation in a C2H2 zinc finger of Zfp335 , we reveal a selective defect in the accumulation of naïve T cells resulting from a maturation block in SP thymocytes and recent thymic emigrants . In line with another recent study ( Yang et al . , 2012 ) , we have shown that Zfp335 regulates transcription by binding to promoters of target genes . We have identified a set of direct targets in thymocytes and provide evidence that Zfp335 occupancy at a small subset of target sites was significantly decreased in mutant T cells . Zfp335 target genes were enriched in categories related to protein metabolism , mitochondrial function , and transcriptional regulation . In addition , we identified a new DNA recognition motif that is bound by Zfp335 . Taken together , our findings suggest that Zfp335 acts as a novel transcription factor required for regulating expression of multiple genes required for late stage naïve T cell maturation . We provide evidence that Zfp335 regulates T cell maturation in part by promoting Ankle2 transcription . A study in Caenorhabditis elegans and HeLa cells showed a role for Ankle2 in nuclear envelope reassembly by promoting dephosphorylation of BAF during mitotic exit ( Asencio et al . , 2012 ) . As we found no significant defects in proliferation ( Figure 4—figure supplement 3 ) and did not detect nuclear envelope abnormalities in blt/blt mature SP thymocytes by immunofluorescence microscopy ( data not shown ) , it is likely that the requirement for Ankle2 is dependent on some other as-yet-unknown function of this protein . To our knowledge , this is the first time an in vivo role for Ankle2 has been reported , and a more detailed understanding of its function and mechanism of action in T cells will be an important subject for future studies . The T cell deficiency in blt/blt mice does not appear to be a consequence of defects in thymic selection , proliferation , or IL-7Rα expression , but is associated with reduced viability of mature SP thymocytes and recent thymic emigrants . Our finding that BCL2 overexpression failed to rescue the relative deficiency in blt/blt T cells , together with the unaltered expression of Bcl2 family genes ( Figure 4—figure supplement 2A ) or other well-defined pro-apoptotic genes such as members of the death receptor family ( data not shown ) , suggests Zfp335 has an indirect and likely multigenic pro-survival influence . Although our data do not provide support for altered thymic selection being an explanation for the reduced T cell numbers in blt/blt mice , we cannot exclude the possibility that the selection of some T cell specificities is affected by the Zfp335 mutation and future deep sequencing studies will be needed to fully address this issue . The intact proliferation of blt/blt T cells contrasts with the defective proliferation of human lymphoblastic cells and neuronal stem cells carrying a H1111R mutation in ZFP335 ( ZNF335 ) ( Yang et al . , 2012 ) . We suspect that this difference is a consequence of the almost complete loss of protein caused by the H1111R mutation , compared to the more subtle influence of the R1092W mutation studied here . Naïve T cell deficiencies have been observed in several mouse lines with deletions in genes related to NF-κB signaling , such as RelB ( Guerin et al . , 2002 ) , NEMO ( Schmidt-Supprian et al . , 2003 ) , c-FLIP ( Zhang and He , 2005 ) , TAK1 ( Sato et al . , 2005; Liu et al . , 2006; Wan et al . , 2006 ) , and IKK2 ( Schmidt-Supprian et al . , 2003; Silva et al . , 2014 ) . However , the expression of these genes was not altered in blt/blt T lymphocytes and we did not detect enrichment for NF-κB targets in the gene expression analysis ( data not shown ) . Two other genes known to be required for T cell maturation are the transcriptional repressor Nkap ( Hsu et al . , 2011 ) and Bptf , a component of the ISWI-containing chromatin remodeling complex NURF ( Landry et al . , 2011 ) . Similar to what we observed in blt/blt mice , the maturation block in Nkap- and Bptf-deficient T cells was not caused by altered thymic selection and could not be rescued by Bcl2 overexpression . However , neither Nkap nor Bptf showed significant expression changes in blt/blt T lymphocytes . Moreover , we did not find a strong correlation between the expression profiles of blt/blt and Bptf-deficient SP thymocytes ( unpublished observation ) . Nonetheless , this does not exclude the possibility of a partial overlap between genes regulated by Bptf and Zfp335—one potential mechanism could be that Bptf-dependent nucleosome repositioning is required for efficient Zfp335 binding to some of its target sites . A small number of studies have shown that autophagy plays a role in mature T cell survival ( Pua et al . , 2007; He et al . , 2012; Parekh et al . , 2013 ) , possibly by ensuring the clearance of excess mitochondria to minimize the harmful impact of reactive oxygen species ( ROS ) as newly mature cells transition from the thymus to the periphery ( Pua et al . , 2009 ) . Although we observed genes involved in oxidative phosphorylation enriched amongst Zfp335 targets , our gene expression data did not reveal a clear autophagy signature , and initial experiments found no significant differences in mitochondrial content or ROS levels in blt/blt mature SP thymocytes ( data not shown ) . However , we cannot rule out the possibility that other autophagy-dependent processes may be affected by Zfp335 deficiency . From our microarray analyses , we found that most gene expression changes in blt/blt T lymphocytes were subtle . In our mature CD4SP thymocyte data set , of the 1504 genes passing a p-value threshold of <0 . 05 , only 16 genes , or 1% of the total had a twofold or greater change in expression , while the vast majority ( 93% ) showed a less than 1 . 5-fold difference between blt/blt and WT . This may not be a surprising result , given that we were only able to detect strongly diminished Zfp335R1092W occupancy at a limited subset of target sites . However , due to the highly conservative criteria used and other technical limitations discussed earlier , this is most likely an underestimate of the true degree of differential binding , which may affect a broader range of target genes and account for many of the more subtle changes in gene expression . Consistent with this notion , we observed that 33 direct Zfp335 targets , representing ∼20% of the total , were differentially expressed in mature CD4SP thymocytes ( p < 0 . 05 ) , and of these genes , only four ( Ankle2 , Cnpy2 , Nme6 , Tbck ) met our differential binding criteria . Furthermore , several known or putative transcriptional regulators are among genes directly targeted by Zfp335 ( Supplementary file 4 ) or differentially expressed in blt/blt thymocytes and RTEs ( Supplementary file 1 ) , suggesting that Zfp335 operates within the context of a broader gene regulatory network . It is likely that the T cell maturation defect arises from the cumulative effects of relatively small gene expression changes within the Zfp335 regulatory network , possibly resulting in mild perturbations of multiple pathways whose functional consequences may not be individually significant but in combination may negatively impact overall T cell fitness . It is interesting to note that survival of mature naïve T cells appears to be intact , despite RT-qPCR evidence indicating that a number of Zfp335 target genes found to be downregulated in blt/blt mature SP thymocytes and RTEs ( Ankle2 , Cnpy2 , Sep15 , and Wdr47 ) are similarly downregulated in the total naïve T cell population ( Figure 8A and data not shown ) . If we extend this observation to the genome-wide level to assume that Zfp335R1092W-induced transcriptional dysregulation in mature naïve T cells approximates the situation in RTEs , one way to explain why mature T cells are less affected could be that the deregulated genes and their associated pathways are most critical during late-thymic to early post-thymic maturation , after which they become dispensable . In support of this concept , it has been reported that IKK2 is required transiently in RTEs but not in mature T cells for normal IL-7Rα upregulation and homeostasis ( Silva et al . , 2014 ) , although we have no evidence that this pathway is affected in blt/blt T lymphocytes . Alternatively , stochastic variations in gene expression or upregulation of compensatory mechanisms may have resulted in comparatively ‘fitter’ cells being preferentially selected into the mature naïve T cell pool . Zfp335 is broadly expressed in hematopoietic cells ( Heng et al . , 2008 ) and in a variety of non-lymphoid tissues such as brain , kidney , heart , and lung ( Wu et al . , 2009 ) . Given its widespread expression and the early embryonic lethality resulting from its complete ablation ( Yang et al . , 2012 ) , it is highly likely that Zfp335 serves wider developmental roles . The poor contribution of Zfp335-overexpressing cells to hematopoietic lineages in BM chimeric mice also hints at a wider role . It may therefore seem remarkable that a mutation in such a ubiquitously expressed gene could produce a defect that appears to be selective for this particular stage of T cell development . It is possible that the loss of Zfp335 binding to the target sites described in our study may be unique to T lymphocytes , resulting in T cell-specific dysregulation; however , this hypothesis is not favored by preliminary evidence showing that target genes such as Ankle2 were also downregulated in B cells ( data not shown ) . As discussed earlier , our data that only a small number of target genes are significantly deregulated suggests that other aspects of the Zfp335-dependent transcriptional program may remain sufficiently intact to allow most developmental processes , except for T cell maturation , to proceed relatively normally . For future studies , it will be important to test whether a null allele has additional consequences for immune regulation beyond the selective T cell maturation effects revealed by the bloto mutation . Genome-wide , we estimate that there are more than 2000 promoter regions containing Zfp335 motif sites . Our study detected approximately 150 ChIP-seq peaks in thymocytes , consistent with the generally accepted principle that additional molecular requirements beyond DNA sequence preference determine the stability of transcription factor binding to a given site . At this early stage , it is not known what these requirements are for Zfp335 , but they are likely to involve local chromatin context and interactions with co-binding partners which remain to be defined . It is also unclear whether Zfp335 displays cell type-specific binding patterns , which may allow Zfp335 to fulfill various developmental roles by regulating different gene expression programs . Finally , although it has been proposed that Zfp335 activates target genes by recruiting H3K4 methyltransferases ( Yang et al . , 2012 ) , it is not clear if this is the sole mechanism , or that Zfp335 functions exclusively as a transcriptional activator . Consistent with the view that Zfp335 drives transcriptional activation , we observed that most target genes with deregulated expression in blt/blt thymocytes tended to be downregulated . However , we have noted exceptions in which some target genes—including Zfp335 itself—were modestly upregulated , suggesting the possibility that Zfp335 may also act as a repressor . Further investigations will be needed to identify the interaction partners with which Zfp335 cooperates to control gene expression . To conclude , our findings regarding Zfp335R1092W add to other recent work ( Hsu et al . , 2011; Landry et al . , 2011; Silva et al . , 2014 ) to highlight unique gene expression requirements in late stage thymocytes and recent thymic emigrants for the formation of a normal sized naïve T cell compartment . In this regard , the function of Zfp335 is similar to its action in the brain where it is needed for the formation of a normal sized forebrain structure ( Yang et al . , 2012 ) . In future work , it will be important to build from these findings to understand how this broad set of gene expression changes are integrated to promote formation of a normal sized compartment of cells . It will also be important to understand whether Zfp335 acts constitutively during late stages of T cell development or whether its function is regulated by external inputs and thus serves as a checkpoint that can influence the size and properties of the naïve T cell compartment .
The Zfp335bloto strain was established through ethylnitrosourea ( ENU ) -mediated mutagenesis of C57BL/6 ( B6 ) mice at the Australian National University using methods previously described ( Randall et al . , 2009 ) . Putative mutants were identified as having blood CD4+ and CD8+ T cell frequencies more than one standard deviation below the mean . CD45 . 1+ congenic mice were from the National Cancer Institute ( 01B96; B6-LY5 . 2/Cr ) . CD45 . 1+CD45 . 2+ mice were generated by crossing B6 and Boy/J ( Jackson Laboratory , 002014; B6 . SJL-PtprcaPepcb/BoyJ ) mice . OTII TCR transgenic mice ( Tg[TcraTcrb]426-6Cbn ) were from an internal colony . Rag1-GFP transgenic mice were provided by N Sakaguchi ( Kumamoto University , Kumamoto , Japan ) ( Kuwata et al . , 1999 ) . Lck-BCL2 transgenic mice were generated by S Korsmeyer ( Dana-Farber Cancer Institute , Boston , MA ) ( Sentman et al . , 1991 ) and provided by A Winoto ( University of California Berkeley , Berkeley , CA ) . RIP-mOVA transgenic mice ( Tg[Ins2-OVA]59Wehi ) were provided by S Sanjabi ( University of California San Francisco ) . Mice were housed in specific pathogen-free conditions , and all experiments were done according to the Institutional Animal Care and Use Committee guidelines of the University of California San Francisco . Affected bloto mice were crossed onto the CBA/J background to generate heterozygous F1 mice . F1 mice were intercrossed to yield F2 progeny homozygous for the bloto mutation and carrying a mix of C57BL/6 and CBA/J single nucleotide polymorphisms ( SNPs ) . SNP mapping using an Amplifluor assay ( EMD Millipore , Billerica , MA ) with Platinum Taq ( Life Technologies , Carlsbad , CA ) was carried out on genomic DNA isolated from affected and unaffected mice . Exome enrichment was performed using the SeqCap EZ Mouse Exome kit ( Roche Nimblegen , Basel , Switzerland ) , followed by 75 bp paired-end sequencing on the Illumina Genome Analyzer IIx platform ( Illumina , San Diego , CA ) . Computational analysis to detect novel single-nucleotide variants was done as previously described ( Andrews et al . , 2012 ) . The affected exon was PCR-amplified from genomic DNA and Sanger sequencing ( TACGen , Richmond , CA ) was carried out to confirm the mutation . Zfp335bloto mice were genotyped by allele-specific PCR using the following primers: WT-F: 5ʹ-AGAACAAGAAGGATCTGAGGC-3ʹ; bloto-F: 5ʹ-AAGAACAAGAAGGATCTGAGGT-3ʹ; common-R: 5ʹ-GGCTCGGGCTGTAGAAGT-3ʹ . WT and bloto allele-specific primers were run in separate reactions with GoTaq Hot Start polymerase ( Promega , Madison , WI ) . Full-length Zfp335 was cloned from cDNA into an MSCV retroviral vector containing a Thy1 . 1 ( CD90 . 1 ) reporter downstream of an internal ribosome entry site ( IRES ) . The bloto mutation ( c . 3274C > T ) was introduced by site-directed mutagenesis . For use in transfections , WT and bloto Zfp335 inserts were subcloned into a pcDNA3 . 1 vector ( Life Technologies ) with a FLAG epitope tag at the N-terminus . Ankle2 , Cnpy2 , Nme6 , Sep15 , Tbck , and Wdr47 were PCR amplified from mouse cDNA and cloned into the MSCV-IRES-Thy1 . 1 vector . All constructs were verified by sequencing . Reference sequences used in this study are as follows—protein: Zfp335 ( NP_950192 . 2 ) ; mRNA: Zfp335 ( NM_199027 . 2 ) , Ankle2 ( NM_001253814 . 1 ) , Cnpy2 ( NM_019953 . 1 ) , Nme6 ( NM_018757 . 1 ) , Sep15 ( NM_053102 . 2 ) , Tbck ( NM_001163455 . 1 ) , Wdr47 ( NM_181400 . 3 ) . Cells were isolated from thymus , spleen , and lymph nodes by mechanical disaggregation through a 40-μm nylon sieve and stained as described ( Schmidt et al . , 2013 ) . Antibodies were as follows: anti-CD4 ( GK1 . 5 , RM4-5 ) , CD8 ( 53–6 . 7 ) ( Biolegend , San Diego , CA; Tonbo Biosciences , San Diego , CA ) ; CD62L ( MEL-14 ) , CD44 ( IM7 ) , CD69 ( H1 . 2F3 ) , CD24 ( M1/69 ) , CD45 . 1 ( A20 ) , CD45 . 2 ( 104 ) , TCRβ ( H57-597 ) , CD19 ( 6D5 ) , TCRγδ ( GL3 ) , Thy1 . 1 ( OX-7 ) , CD25 ( PC61 ) ( Biolegend ) ; NK1 . 1 ( PK136 ) , Vα2 ( B20 . 1 ) , CD5 ( 53-7 . 3 ) ( BD Biosciences , San Jose , CA ) ; IL-7Rα ( A7R34 ) , Foxp3 ( FJK-16 s ) , BrdU ( BU20A ) ( eBioscience , San Diego , CA ) ; mCD1d/PBS-57 tetramer ( NIH Tetramer Core Facility ) . 4ʹ , 6-diamidino-2-phenylindole ( DAPI ) was used for dead cell exclusion . For all intracellular staining , cells were stained for surface antigens before fixation . Foxp3 was stained using the Foxp3 Staining Buffer Set ( eBioscience ) . BrdU staining was performed as per manufacturer's guidelines ( BD Biosciences ) . For cell cycle analysis by DNA content , cells were fixed with BD Cytofix/Cytoperm Buffer and stained with 5 μM DAPI in Perm/Wash buffer ( BD Biosciences ) . Annexin V staining was performed using the Annexin V-PE Apoptosis Detection kit ( BD Biosciences ) according to manufacturer's instructions . Samples were acquired on an LSRII cytometer ( BD Biosciences ) and analyzed with FlowJo software ( TreeStar , Ashland , OR ) . For microarray analysis , hematopoietic chimeras were generated with a mix of CD45 . 1+CD45 . 2+ Rag1-GFP WT and CD45 . 2+ Rag1-GFP blt/blt bone marrow . Thymocytes were isolated in MACS buffer ( PBS +2% FBS , 2 mM EDTA ) , incubated with anti-CD8 microbeads ( Miltenyi Biotec , Bergisch Gladbach , Germany ) , and MACS-depleted to enrich for CD4SP thymocytes , after which cells were stained with anti-CD45 . 1 , CD4 , CD8 , CD69 , and CD62L . Mature CD4SP thymocytes ( GFPhiCD4+CD8−CD62LhiCD69lo ) were sorted into WT ( CD45 . 1+ ) and blt/blt ( CD45 . 1− ) subsets . For RTE sorting , spleen , and lymph node cells were pooled and erythrocytes were lysed in a solution of Tris-buffered NH4Cl . Cells were labeled with anti-CD45 . 1 , CD4 , CD8 , CD62L , and CD44 . RTEs were sorted as CD4+CD62LhiCD44lo Rag1-GFPhi CD45 . 1+ ( WT ) , and CD45 . 1− ( blt/blt ) subsets . Dead cells were excluded with DAPI . Samples were sorted on a FACSAria with ≥98% purity . Mature CD4SP thymocytes were sorted and allowed to adhere to poly-L-lysine-coated glass slides ( P0425; Sigma–Aldrich , St . Louis , MO ) . Cells were then fixed with 4% PFA in PBS and permeabilized with 0 . 1% Triton X-100 for 10 min on ice , followed by blocking ( 5% normal goat serum , 2% BSA in PBS ) and staining ( 2% goat serum , 0 . 1% BSA , 0 . 1% Tween-20 in PBS ) . For Zfp335 detection , slides were incubated at RT with primary antibody ( A300-797A , A300-798A; Bethyl Laboratories , Montgomery , TX ) for at least 3 hr , followed by biotin goat anti-rabbit ( BD Biosciences ) and lastly SA-Cy3 ( Jackson Immunoresearch , West Grove , PA ) . Slides were counterstained with 1 μM DAPI and mounted with Fluoromount-G ( Southern Biotech , Birmingham , AL ) . Confocal imaging was performed on a Leica SP5 inverted microscope with a 63× oil immersion objective . Images were processed with Leica LAS software . Mixed bone marrow chimeras were generated by intravenously transferring 3 × 106 to 5 × 106 cells from the following mixes into lethally irradiated ( 2 × 450 rads , 3 hr apart ) CD45 . 1+ congenic mice: blt/blt CD45 . 2+: WT CD45 . 1+CD45 . 2+; blt/+ CD45 . 2+: WT CD45 . 1+CD45 . 2+ , at a 50:50 ratio . For OTII/ RIP-mOVA experiments , bone marrow from OTII blt/+ or OTII blt/blt mice was incubated with biotin-anti-CD3 , CD4 , and CD8 followed by anti-biotin microbeads ( Miltenyi Biotec ) and T cells were depleted by MACS prior to transfer into lethally irradiated WT B6 or RIP-mOVA recipients . Retroviral transduction of bone marrow from blt/blt or Rag1-GFP blt/blt mice was performed as described ( Green et al . , 2011 ) using the Platinum-E packaging cell line . Chimeric mice were analyzed at least 8 weeks after reconstitution . For peripheral T cell transfers , spleen and lymph node cells were harvested from WT ( control ) or blt/blt mice and RBC-lysed . For thymocyte transfers , bulk thymocytes were prepared from blt/+ ( control ) or blt/blt mice . To distinguish between the two populations , either control or blt/blt cells were labeled with 1 μM CFSE ( Life Technologies ) for 10 min at 37°C before they were mixed and intravenously injected into lymphoreplete Boy/J ( CD45 . 1+ ) mice . Recipients were analyzed at days 1 and 7 post-transfer and the percentage of blt/blt naïve T cells in the CD45 . 2+ donor population was determined . For Rag1-GFP T cell transfers , spleen and lymph node cells were harvested from Rag1-GFP blt/+ or Rag1-GFP blt/blt mice and RBC-lysed . Non-transgenic CD45 . 2+ blt/+ cells were used as a mixing control . Either control or Rag1-GFP cells were labeled with 10 μM CellTrace Violet ( Life Technologies ) for 20 min at 37°C before mixing and intravenous transfer into Boy/J recipients . At days 1 , 3 , and 5 post-transfer , Rag1-GFP blt/+ or Rag1-GFP blt/blt naïve T cells were assessed as a proportion of the total CD45 . 2+ donor population . For BrdU experiments , mice received two intraperitoneal ( i . p . ) injections of 1 mg BrdU spaced 2 hr apart and were euthanized 4 hr after the first dose , or were fed 1 mg/ml BrdU in drinking water for longer-term labeling . To block thymic egress and induce mature SP thymocyte accumulation , mice were injected i . p . twice with FTY720 ( Cayman Chemical , Ann Arbor , MI ) dissolved in saline at a dose of 1 mg/kg body weight on days 0 and 2 , and sacrificed on day 4 . FACS-purified CD4+ naïve T cells were labeled with 5 μM CFSE for 10 min at 37°C , quenched with fetal bovine serum ( FBS ) and washed in 10% FBS . Labeled cells were stimulated with plate-bound anti-mouse CD3 ( clone 2C11; 1 μg/ml ) and anti-mouse CD28 ( clone 37 . 51; 1 μg/ml ) for 3 days . For in vitro cell viability assays , semi-mature and mature CD4SP thymocytes were sorted from chimeras reconstituted with a mix of either blt/+ and WT or blt/blt and WT BM . Sorted cells were cultured in a 96-well plate at a density of 6–10 × 104 cells per well . All cells were cultured at 37°C in 5% CO2 , using RPMI media supplemented with 10% FBS , L-glutamine , β-mercaptoethanol , penicillin , and streptomycin . Thymocytes were lysed in RIPA buffer containing protease inhibitor cocktail ( EMD Millipore ) . Samples were resolved on NuPAGE Bis-Tris gels ( Life Technologies ) and transferred to Immobilon-FL membranes ( EMD Millipore ) . Primary antibodies used: anti-Zfp335 ( A300-797A , A300-798A; Bethyl Laboratories ) , anti-actin ( A2066 , Sigma–Aldrich ) , and anti-FLAG M2 ( F1804 , Sigma–Aldrich ) . Ankle2 was detected using rabbit antiserum raised against human ANKLE2 , provided by I Mattaj ( EMBL , Heidelberg , Germany ) ( Asencio et al . , 2012 ) . Secondary antibodies used: goat anti-mouse IRDye 800CW , donkey anti-rabbit IRDye 700DX ( Rockland Immunochemicals , Gilbertsville , PA ) . Blots were scanned using the Odyssey Infrared Imaging System ( LI-COR Biosciences , Lincoln , NE ) . HEK293T cells were transfected with FLAG-tagged WT or bloto Zfp335 ( cloned as described ) and nuclear extracts prepared using a modified Dignam protocol . Relative protein amounts were determined by Western blot using anti-Zfp335 ( A300-797A ) . Gel mobility shift assays were performed as described ( Lo et al . , 1991 ) . Briefly , nuclear extracts were mixed with 10 mM HEPES pH 7 . 9 , 0 . 1 mM EDTA , 10% glycerol , 0 . 5 mM DTT , 0 . 1 mM PMSF , 0 . 08 mg/ml BSA , 0 . 04 mg/ml poly dI/dC , 0 . 4 mM ZnCl2 , and 1ʹ mM biotin-labeled probe and incubated at room temperature for 20 min . For experiments using competitor oligos , 20- ( Figure 7F ) or 50-fold ( Figure 7G ) molar excess of unlabeled probe was pre-incubated with nuclear extracts on ice for 30 min before adding biotinylated probe . Samples were loaded on a 4% polyacrylamide gel with 3 . 5% glycerol in 0 . 25X TBE and run at constant voltage in 0 . 25X TBE running buffer , then transferred to Biodyne B nylon membranes ( Pall Corporation , Port Washington , NY ) in 0 . 5X TBE at 380 mA for 1 hr . Signal was detected with LightShift Chemiluminescent Nucleic Acid Detection Module kit ( Thermo Scientific , Waltham , MA ) . All probes were derived from synthetic double-stranded 5ʹ-biotinylated oligonucleotides , listed in Supplementary file 5 ( Integrated DNA Technologies , San Jose , CA ) . Total RNA was isolated using the RNeasy Micro Kit ( Qiagen , Venlo , The Netherlands ) ; cDNA was synthesized using MMLV reverse transcriptase and random primers ( Life Technologies ) according to manufacturer's instructions . Real-time PCR was carried out using a StepOnePlus real-time PCR system ( Applied Biosystems , Foster City , CA ) with SYBR Green PCR Master Mix ( Applied Biosystems ) and the appropriate primer pairs ( Supplementary file 6; Integrated DNA Technologies ) . Relative mRNA abundance of target genes was determined by subtracting the threshold cycle for the internal reference ( Hprt1 ) from that of the target . Primer pairs were tested for linear amplification over two orders of magnitude . For microarray analysis , cDNA was prepared using the Ovation Pico WTA System V2 and labeled using the Encore Biotin Module ( NuGEN , San Carlos , CA ) . Labeled cDNA libraries were hybridized to Affymetrix Mouse 1 . 0 ST arrays ( Affymetrix , Santa Clara , CA ) and scanned with the Affymetrix GeneChip Scanner 3000 7G System . Raw data were normalized by RMA and probesets mapped to unique Entrez Gene IDs using a custom Brainarray CDF . The limma R package was used for analyzing differential gene expression . GENE-E software ( Broad Institute ) was used for heat map generation and hierarchical clustering . Total thymocytes ( 20 × 106 ) or CD4SP thymocytes ( 4–10 × 106 ) were harvested from WT and blt/blt mice , cross-linked in 1% formaldehyde for 10 min at room temperature , quenched with 125 mM glycine and washed twice in ice-cold PBS . Cells were lysed in buffer containing 20 mM Tris–HCl ( pH 8 . 0 ) , 85 mM KCl , 0 . 5% Nonidet-P40 , followed by nuclear lysis buffer ( 50 mM Tris–HCl pH 8 . 0 , 10 mM EDTA , 1% SDS ) . Chromatin was sonicated with a Bioruptor ( Diagenode , Liège , Belgium ) , cleared by centrifugation and diluted in buffer containing 20 mM Tris–HCl ( pH 8 . 0 ) , 1 . 1 mM EDTA , 140 mM NaCl , 0 . 01% SDS , and 1 . 1% Triton X-100 . Diluted chromatin was incubated overnight at 4°C with antibody bound to Protein A Dynabeads ( Life Technologies ) . All buffers up to this point were supplemented with protease inhibitors . Beads were washed in low salt buffer ( 10 mM Tris–HCl pH 8 . 0 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 ) , high salt buffer ( 20 mM Tris–HCl pH 8 . 0 , 500 mM NaCl , 2 mM EDTA , 1% Triton X-100 , 0 . 1% SDS ) , LiCl buffer ( 10 mM Tris–HCl pH 8 . 0 , 250 mM LiCl , 1 mM EDTA , 0 . 5% sodium deoxycholate , 0 . 5% Nonidet-P40 ) , and TE buffer . Protein/DNA complexes were eluted ( 50 mM Tris–HCl pH 8 . 0 , 10 mM EDTA , 1% SDS ) at 65°C for 30 min with shaking on a Thermomixer ( Eppendorf ) . Eluted complexes were reverse-crosslinked overnight at 65°C and then treated with RNase A ( Sigma-Aldrich ) and proteinase K ( Life Technologies ) . ChIP DNA was purified using the QIAquick PCR purification kit ( Qiagen ) and quantitative PCR was performed using SYBR Green PCR Master Mix ( Applied Biosystems ) and primer pairs listed in Supplementary file 7 . For Zfp335 ChIP , a polyclonal antibody specific for a C-terminal epitope ( A300-798A; Bethyl Laboratories ) was used . ChIP was performed with total thymocytes ( 60 × 106 cells ) as described above . Two antibodies were used: one raised against a C-terminal epitope ( A300-798A; Bethyl Laboratories ) and the other against an N-terminal epitope ( A300-797A; Bethyl Laboratories ) of ZNF335 . 7–10 ng ChIP DNA and 10 ng input DNA were used for library preparation , performed according to Illumina's TruSeq protocol with some modifications . DNA clean-up , removal of adapter dimers , and size selection were done using Agencourt AMPure XP beads ( Beckman Coulter , Brea , CA ) . Libraries were checked for quality using the High Sensitivity DNA Bioanalyzer kit ( Agilent Technologies , Santa Clara , CA ) , quantified with the Qubit dsDNA HS Assay kit ( Life Technologies ) , and sequenced as 50 bp single-end reads on the Illumina HiSeq 2000 platform . Illumina adapter sequences were removed using the cutadapt tool . Trimmed reads were aligned to the mm9 reference genome using bwa , allowing for a maximum of two mismatches . Reads aligned with a MAPQ score of less than 20 were filtered out using samtools . Basic peak calling was performed with MACS2 ( parameters: -g mm --bw = 300 –q 0 . 05 ) . Peaks were annotated with their nearest RefSeq TSS using HOMER . Genomic feature annotation summary statistics were generated using the Galaxy/Cistrome CEAS module ( version 1 . 0 . 0 ) , and the full set of target genes ( n = 177 ) defined as having a TSS within 1 kb of high-confidence peaks were identified using the BETA-minus module ( version 1 . 0 . 0 ) . To call differential binding events , samtools rmdup was first used to remove duplicate reads , after which regions of differential enrichment were identified using the MACS2 callpeak and bdgdiff modules . Normalized pileup tracks were generated from nonredundant reads using MACS2 callpeak--SPMR ( fragment pileup per million reads ) and converted to the bigWig format for visualization on the UCSC Genome Browser . The C-terminal antibody ( A300-798A ) was found to give better enrichment so we based our analyses of general Zfp335 binding properties on the ChIP-C WT data set , unless otherwise stated . H3K4me3 , H3K27ac , and H3K27me3 ChIP-seq data for mouse thymus were downloaded from ENCODE/LICR ( ENCODE Project Consortium , 2012 ) , and aggregate density profiles were computed using bwtool . Gene ontology enrichment analysis of Zfp335 binding regions was performed using the Genomic Regions Enrichment of Annotations Tool ( GREAT ) . Each ChIP-seq peak was associated with the two nearest genes within 10 kb . 400 bp sequences centered on peak summits were extracted from the 119 top-scoring WT Zfp335 peak regions , repeat-masked and used for de novo motif discovery with MEME ( parameters: min . width = 6 , max . width = 30 , zero or one instances of a given motif per sequence ) . The top-scoring motif obtained with MEME was replicated using HOMER's motif finding function . HOMER was used to detect Zfp335 motif occurrences and locations within defined genomic regions . HOMER was used to generate a histogram of motif density ( bin size = 50 bp ) for a region from −500 bp to +500 bp of Zfp335 ChIP-seq peaks . DNase I-seq signal and phyloP conservation scores for motifs occurring within Zfp335 peaks or ±2 kb of RefSeq TSS were aggregated using bwtool . The DNase I digital genomic footprinting ( DGF ) signal track for mouse thymus was obtained from ENCODE/UW ( ENCODE Project Consortium , 2012 ) ; sequence conservation tracks were downloaded from UCSC Genome Browser ( Raney et al . , 2014 ) . Software tools used:R 3 . 0 . 2affy R package ( Gautier et al . , 2004 ) limma R package ( Smyth , 2004 ) Brainarray custom CDF ( mogene10st_Mm_ENTREZG_17 . 1 . 0 ) ( Dai et al . , 2005 ) cutadapt 1 . 4 . 1 ( Martin , 2011 ) bwa 0 . 7 . 7 ( Li and Durbin , 2009 ) samtools 0 . 1 . 18 ( Li et al . , 2009 ) bedtools 2 . 17 . 0 ( Quinlan and Hall , 2010 ) bwtool ( Pohl and Beato , 2014 ) MACS 2 . 0 . 10 ( Zhang et al . , 2008 ) MEME 4 . 9 . 1 ( Bailey et al . , 2009 ) HOMER 4 . 5 ( Heinz et al . , 2010 ) GREAT 2 . 0 . 2 ( McLean et al . , 2010 ) GSEA 2 . 0 . 14 ( Subramanian et al . , 2005 ) Galaxy/Cistrome ( Liu et al . , 2011 ) UCSC Genome Browser ( http://genome . ucsc . edu ) ( Kent et al . , 2002 , 2010; Rosenbloom et al . , 2012 ) A homology model was generated by SWISS-MODEL utilizing using the known structure of a designed DNA-binding zinc finger protein ( Protein Data Bank ID: 1MEYC ) as template . Figures were created using MacPyMOL ( version 1 . 5 . 0 . 5 ) . Data were analyzed with Prism 5 ( GraphPad Software ) . The two-tailed non-parametric Mann–Whitney test was used for comparison of two unpaired groups for all data sets unless otherwise indicated . Microarray and ChIP-seq data sets generated in this study were deposited to NCBI's Gene Expression Omnibus under SuperSeries GSE58293 .
The generation of naïve T lymphocytes is critical for immune function yet the mechanisms governing their maturation remain incompletely understood . We have identified a mouse mutant , bloto , that harbors a hypomorphic mutation in the zinc finger protein Zfp335 . Zfp335bloto/bloto mice exhibit a naïve T cell deficiency due to an intrinsic developmental defect that begins to manifest in the thymus and continues into the periphery , affecting T cells that have recently undergone thymic egress . The effects of Zfp335bloto are multigenic and cannot be attributed to altered thymic selection , proliferation or Bcl2-dependent survival . Zfp335 binds to promoter regions via a consensus motif , and its target genes are enriched in categories related to protein metabolism , mitochondrial function , and transcriptional regulation . Restoring the expression of one target , Ankle2 , partially rescues T cell maturation . These findings identify Zfp335 as a transcription factor and essential regulator of late-stage intrathymic and post-thymic T cell maturation .
To defend our bodies against a variety of foreign microbes , our immune system makes cells called T cells that can identify these invaders and help to destroy them . There are several types of T cells that play different roles in the immune response: some activate other immune cells , while others destroy cells that have been infected by viruses or other pathogens . T cells develop in a specialized organ called the thymus , where they go through a rigorous selection process before being released as mature T cells into the rest of the body . This selection process includes eliminating individual T cells that are found to be sub-standard , perhaps because they might mistake our own cells for enemy cells . However , many of the details of the later stages of T cell development are not fully understood . Han et al . have now found that a protein called Zfp335 that is involved in the production of mature T cells . Mice carrying a mutation in the gene that makes this protein have fewer mature T cells than normal mice . Han et al . also reveal that Zfp335 is a transcription factor that can control whether or not other genes are expressed as proteins , and further show that one of these proteins , Ankle2 , has an important role in the production of mature T cells . A next step in the work is to define exactly how Zfp335 controls the expression of these genes . It will also be important to determine whether mutations in Zfp335 contribute to human T-cell immunodeficiency .