DNMT3A haploinsufficiency causes dichotomous DNA methylation defects at enhancers in mature human immune cells.

DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.

Physiological role of the 3'IgH CBEs super-anchor in antibody class switching.

IgH class switch recombination (CSR) replaces Cµ constant region (CH) exons with one of six downstream CHs by joining transcription-targeted double-strand breaks (DSBs) in the Cµ switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3'IgH regulatory region (3'IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are 10 consecutive CTCF-binding elements (CBEs) downstream of the 3'IgHRR, termed the "3'IgH CBEs." Prior studies showed that deletion of eight 3'IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3'IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except that of Sγ1. Moreover, deletion of all 3'IgH CBEs rendered the 6-kb region just downstream highly transcribed and caused sequences within to be aligned with Sµ, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3'IgH CBEs as critical insulators for focusing loop extrusion-mediated 3'IgHRR transcriptional and CSR activities on upstream CH locus targets.

Cutting Edge: CRISPR-Based Transcriptional Regulators Reveal Transcription-Dependent Establishment of Epigenetic Memory of Foxp3 in Regulatory T Cells.

Transcription factor Foxp3 specifies and maintains regulatory T cell (Treg) identity. During Treg differentiation, a CpG-rich Foxp3 intronic enhancer, conserved noncoding sequence 2 (CNS2), is activated via DNA demethylation to establish epigenetic memory of Foxp3 expression to protect Treg identity. However, it is unclear how this epigenetic memory of Foxp3 expression is established, as CNS2 is thought to be demethylated independently of Foxp3 expression. In this article, we uncover an unexpected causal relationship between Foxp3-transcriptional activation and CNS2 demethylation in mice. CRISPR/dCas9-mediated Foxp3-transcriptional activation elicits CNS2 demethylation. Sustaining Foxp3-transcriptional activation in induced Tregs also promotes CNS2 demethylation, enhancing Treg lineage stability and suppressive function. Importantly, CRISPR-mediated silencing of Foxp3 transcription, but not protein expression, abolishes CNS2 demethylation. The novel finding that Foxp3-transcriptional activation promotes CNS2 demethylation may facilitate the development of Treg-based therapies and represent a general mechanism for the establishment of epigenetic memory of immune gene expression.

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells.

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2.

Identification of a novel cis-regulatory element essential for immune tolerance.

Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.

CTCF-binding elements 1 and 2 in the Igh intergenic control region cooperatively regulate V(D)J recombination.

Ig heavy chain (IgH) variable region exons are assembled from V, D, and J gene segments during early B-lymphocyte differentiation. A several megabase region at the "distal" end of the mouse IgH locus (Igh) contains hundreds of V(H)s, separated by an intergenic region from Igh Ds, J(H)s, and constant region exons. Diverse primary Igh repertoires are generated by joining Vs, Ds, and Js in different combinations, with a given B cell productively assembling only one combination. The intergenic control region 1 (IGCR1) in the V(H)-to-D intergenic region regulates Igh V(D)J recombination in the contexts of developmental order, lineage specificity, and feedback from productive rearrangements. IGCR1 also diversifies IgH repertoires by balancing proximal and distal V(H) use. IGCR1 functions in all these regulatory contexts by suppressing predominant rearrangement of D-proximal V(H)s. Such IGCR1 functions were neutralized by simultaneous mutation of two CCCTC-binding factor (CTCF)-binding elements (CBE1 and CBE2) within it. However, it was unknown whether only one CBE mediates IGCR1 functions or whether both function in this context. To address these questions, we generated mice in which either IGCR1 CBE1 or CBE2 was replaced with scrambled sequences that do not bind CTCF. We found that inactivation of CBE1 or CBE2 individually led to only partial impairment of various IGCR1 functions relative to the far greater effects of inactivating both binding elements simultaneously, demonstrating that they function cooperatively to achieve full IGCR1 regulatory activity. Based on these and other findings, we propose an orientation-specific looping model for synergistic CBE1 and CBE2 functions.

NLRC5 cooperates with the RFX transcription factor complex to induce MHC class I gene expression.

Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression.

Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus.

Regulatory elements located within an ∼28-kb region 3' of the Igh gene cluster (3' regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5-7 region (hs5-7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the D(H) region. To test the function of hs5-7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5-7 knockout (KO) (hs5-7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5-7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal V(H)7183 gene families. Hs5-7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5-7, a high-density CTCF-binding region at the 3' end of the Igh locus, impacts usage of V(H) regions as far as 500 kb away.

The IgH 3' regulatory region and its implication in lymphomagenesis.

The 3' regulatory region (3'RR) located downstream of the IgH gene is the master element that controls class switch recombination and sustains high-level transcription at the plasma-cell stage. This latter role suggests that the 3'RR may be involved in oncogene deregulation during the frequent IgH translocation events associated with B-cell malignancies. A convincing demonstration of the essential contribution of 3'RR in lymphomagenesis has been provided by transgenic animal models. The mouse 3'RR shares a strong structural homology with the regulatory regions located downstream of each human Cα gene. Mouse models exploring the role of the 3'RR in B-cell physiology and in malignancies should provide useful indications about the pathophysiology of human cell lymphocyte proliferation.

The crucial role of GATA-1 in CCR3 gene transcription: modulated balance by multiple GATA elements in the CCR3 regulatory region.

GATA-1, a zinc finger-containing transcription factor, regulates not only the differentiation of eosinophils but also the expression of many eosinophil-specific genes. In the current study, we dissected CCR3 gene expression at the molecular level using several cell types that express varying levels of GATA-1 and CCR3. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1. A reporter plasmid assay showed that constructs harboring exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of endogenous GATA-1. Introduction of a dominant-negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1 bound to virtually all seven putative GATA elements present in exon 1-intron 1, the first GATA site in exon 1 exhibited the highest binding affinity for GATA-1 and was solely responsible for GATA-1-mediated transactivation. The fourth and fifth GATA sites in exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1-mediated transcription of eosinophil-specific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore, mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the CCR3 gene.

Transcription-dependent mobilization of nucleosomes at accessible TCR gene segments in vivo.

Accessibility of chromosomal recombination signal sequences to the RAG protein complex is known to be essential for V(D)J recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis-acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible recombination signal sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at Dbeta, Jbeta, and Jalpha segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to recombination signal sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at Jalpha61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified.

Allele *1 of HS1.2 enhancer associates with selective IgA deficiency and IgM concentration.

Selective IgA deficiency (IGAD) is the most common primary immunodeficiency, yet its pathogenesis is elusive. The IG (heavy) H chain human 3' Regulatory Region harbors three enhancers and has an important role in Ig synthesis. HS1.2 is the only polymorphic enhancer of the 3' RRs. We therefore evaluated HS1.2 allelic frequencies in 88 IGAD patients and 101 controls. Our data show that IGAD patients have a highly significant increase of homozygousity of the allele *1 (39% in the IGAD patients and 15% in controls), with an increase of 2.6-fold. Allele *4 has a similar trend of allele *2, both showing a significant decrease of frequency in IGAD. No relationship was observed between allele *1 frequencies and serum levels of IgG. However, allele *1 was associated in IGAD patients with relatively low IgM levels (within the 30th lowest percentile of patients). The HS1.2 polymorphism influences Ig seric production, but not IgG switch, in fact 30th lowest or highest percentile of IgG in patients did not associate to different frequencies of HS1.2 alleles. The control on normal healthy subjects did not correlate high or low levels of IgM or IgG with HS1.2 allelic frequence variation. Overall our candidate gene approach confirms that the study of polymorphisms in human diseases is a valid tool to investigate the function of these Regulatory Regions that confers multiple immune features.

Integration of distinct intracellular signaling pathways at distal regulatory elements directs T-bet expression in human CD4+ T cells.

T-bet is a key regulator controlling Th1 cell development. This factor is not expressed in naive CD4(+) T cells, and the mechanisms controlling expression of T-bet are incompletely understood. In this study, we defined regulatory elements at the human T-bet locus and determined how signals originating at the TCR and at cytokine receptors are integrated to induce chromatin modifications and expression of this gene during human Th1 cell differentiation. We found that T cell activation induced two strong DNase I-hypersensitive sites (HS) and rapid histone acetylation at these elements in CD4(+) T cells. Histone acetylation and T-bet expression were strongly inhibited by cyclosporine A, and we detected binding of NF-AT to a HS in vivo. IL-12 and IFN-gamma signaling alone were not sufficient to induce T-bet expression in naive CD4(+) T cells, but enhanced T-bet expression in TCR/CD28-stimulated cells. We detected a third HS 12 kb upstream of the mRNA start site only in developing Th1 cells, which was bound by IL-12-induced STAT4. Our data suggest that T-bet locus remodeling and gene expression are initiated by TCR-induced NF-AT recruitment and amplified by IL-12-mediated STAT4 binding to distinct distal regulatory elements during human Th1 cell differentiation.

TLR7 and TLR8 ligands and antiphospholipid antibodies show synergistic effects on the induction of IL-1beta and caspase-1 in monocytes and dendritic cells.

TLRs represent the first line of defense against invading pathogens in the innate immune system. Certain cytokines are important mediators and essentially necessary to assure an appropriately regulated immune response. Recent data gave initial evidence that IL-1beta is one of the most relevant members of these regulating cytokines. We investigated the induction of IL-1beta production in monocytes and pDCs stimulated with ligands for TLR7 and TLR8 and with antiphospholipid antibodies (aPL). Using human monocytes and pDCs for stimulation with specific TLR7 and TLR8 ligands such as resiquimod (R848) and single stranded RNA (RNA42) as well as with a human monoclonal aPL HL5B resulted in a specific upregulation of IL-1beta mRNA and protein in these cells. Determination of expression-levels using real-time RT-PCR showed significantly augmented TLR-dependent IL-1beta and caspase-1 expression. This increase could be substantially enhanced by adding the monoclonal aPL HL5B. To demonstrate the direct dependency between TLR stimulation and IL-1beta production, specific TLR inhibitors were applied and the IL-1beta and caspase-1 secretion could be explicitly decreased. The respective protein levels were determined using Western Blot, FACS analysis or ELISA assays. In conclusion we demonstrated that the downstream signaling pathway of TLR7 and TLR8 in monocytes and pDCs after stimulation with specific ligands included not only the secretion of cytokines such as TNFalpha and IL-1beta but as well the activation of necessary regulating proteins like caspase-1. APL seem to enforce this process hinting that endogenous stimulation of TLRs in the Antiphospholipid Syndrome (APS) patients resulted in enhanced secretion of proinflammatory cytokines.

NFATc1 regulates PD-1 expression upon T cell activation.

PD-1 is a transmembrane protein involved in the regulation of immunological tolerance. Multiple studies have reported an association between high levels of PD-1 expressed on T cell surfaces and exhaustion in lymphocyte populations when challenged by chronic viral infections, such as HIV. By using model systems consisting of murine EL4 cells, which constitutively express PD-1, and primary murine CD8 T cells that express PD-1 upon T cell stimulation, we have identified two tissue-specific hypersensitive sites at the 5' CR of the PD-1 locus. Gene reporter assays in CD8 T cells have shown that one of these sites has robust transcriptional activity in response to cell stimulation. Cell treatment with the calcineurin inhibitor cyclosporine A or a NFAT-specific inhibitor led to a sharp reduction in PD-1 expression in the constitutive and inducible systems. Furthermore, analysis of this region by chromatin immunoprecipitation assay revealed NFATc1 binding associated with gene activation in EL4 and primary CD8 T cells. Mutation of the NFATc1 binding site in PD-1 reporter constructs resulted in a complete loss of promoter activity. Together, these results demonstrate that PD-1 gene regulation occurs in part via the recruitment of NFATc1 to a novel regulatory element at the pdcd1 locus and provides the molecular mechanism responsible for the induction of PD-1 in response to T cell stimulation.

Prolactin can modulate CD4+ T-cell response through receptor-mediated alterations in the expression of T-bet.

Low-dose prolactin induces proinflammatory responses and antibody production, whereas high-dose prolactin suppresses these responses. Mechanisms for these opposing effects remain incompletely defined. We have previously demonstrated that T-bet, a key transcription factor directing T helper type 1 inflammatory responses, is regulated by female steroid hormones in human mucosal epithelial cells via Stat1 and 5 pathways. T-bet was also modulated in a CD4+ T cell line by prolactin exposure. Prolactin rapidly induced T-bet transcription through phosphorylation of JAK2 and Stat5, but not Stat1. Phosphorylated Stat5 then bound to the T-bet regulatory region. These effects were weaker with high-dose prolactin exposures. Upon long-term prolactin exposure, low-dose prolactin induced T-bet expression, whereas high-dose prolactin tended to suppress it. Prolactin induced the suppressors of cytokine signaling (SOCS) 1 and 3 in a dose-dependent manner. With high-dose exposure, this was associated with an inhibition of the phosphorylation of T-bet regulatory region-bound Stat5. Further, the dose-dependent prolactin effects on T-bet expression were confirmed in murine primary CD4+ T cells. These data suggest that the divergent immune effects of low- and high-dose prolactin may involve modulation of T-bet and alterations in the balance of the prolactin/JAK2/Stat5 and the prolactin/SOCS1 and 3 pathways.

Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus.

Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3' regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus.

1,25-dihydroxyvitamin D3 induces CCR10 expression in terminally differentiating human B cells.

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD-CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD-CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD-CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.

Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus.

Immunoglobulin variable region exons are assembled from discontinuous variable (V), diversity (D), and joining (J) segments by the process of V(D)J recombination. V(D)J rearrangements of the immunoglobulin heavy chain (IgH) locus are tightly controlled in a tissue-specific, ordered and allele-specific manner by regulating accessibility of V, D, and J segments to the recombination activating gene proteins which are the specific components of the V(D)J recombinase. In this review we discuss recent advances and established models brought forward to explain the mechanisms underlying accessibility control of V(D)J recombination, including research on germline transcripts, spatial organization, and chromatin modifications of the immunoglobulin heavy chain (IgH) locus. Furthermore, we review the functions of well-described and potential new cis-regulatory elements with regard to processes such as V(D)J recombination, allelic exclusion, and IgH class switch recombination.

Identification of a candidate regulatory element within the 5' flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A.

The Igh locus is controlled by cis-acting elements, including Emu and the 3' IgH regulatory region which flank the C region genes within the well-studied 3' part of the locus. Although the presence of additional control elements has been postulated to regulate rearrangements of the VH gene array that extends to the 5' end of the locus, the 5' border of Igh and its flanking region have not been characterized. To facilitate the analysis of this unexplored region and to identify potential novel control elements, we physically mapped the most D-distal VH segments and scanned 46 kb of the immediate 5' flanking region for DNase I hypersensitive sites. Our studies revealed a cluster of hypersensitive sites 30 kb upstream of the most 5' VH gene. Detection of one site, HS1, is restricted to pro-B cell lines and HS1 is accessible to restriction enzyme digestion exclusively in normal pro-B cells, the stage defined by actively rearranging Igh-V loci. Sequence motifs within HS1 for PU.1, Pax5, and E2A bind these proteins in vitro and these factors are recruited to HS1 sequence only in pro-B cells. Transient transfection assays indicate that the Pax5 binding site is required for the repression of transcriptional activity of HS1-containing constructs. Thus, our characterization of the region 5' of the VH gene cluster demonstrated the presence of a single cluster of DNase I hypersensitive sites within the 5' flanking region, and identified a candidate Igh regulatory region defined by pro-B cell-specific hypersensitivity and interaction with factors implicated in regulating VDJ recombination.