Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 23, 15030-15035, November 12, 2002 Immunology Identification of E2A target genes in B lymphocyte development by using a gene t agging-based chromatin immunoprecipitation system Stephen Greenbaum and Yuan Zhuang* Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 2 7710 Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 16, 2002 (received for review May 17, 2002) Abstract The transcription factors encoded by the E2A gene are known to be essential for B lymphocyte development, and ectopic expression or gene inactivation studies ha ve revealed several potential lineage-specific E2A target genes. However, it rem ains unknown whether these target genes are directly regulated by E2A at the tra nscriptional level. We therefore generated mice carrying an affinity-tagged E2A knock-in allele to provide a system for the direct elucidation of E2A target gen es based on E2A binding to target regulatory regions. Abelson-transformed pre-B cell lines derived from these mice were used in chromatin immunoprecipitation ex periments to identify regulatory sequences bound by E2A in the context of an ear ly B lymphocyte environment. Significant E2A binding was detected at the promote rs and enhancers of several essential B-lineage genes, including the Ig intronic and 3' enhancers, 5 and VpreB surrogate light chain promoters, the EBF locus pr omoter region, and the mb-1 (Ig) promoter. Low levels of E2A binding were observ ed at several other lymphoid-restricted regulatory regions including the Ig heav y chain (IgH) intronic enhancer, the IgH 3' enhancers hs3b/hs4, the RAG-2 enhanc er, and the 5' regions of the B29 and TdT loci. An E2A target gene, the predicte d butyrophilin-like gene NG9 (BTL-II), was also identified by using a chromatin immunoprecipitation-based cloning strategy. In summary, our studies have provide d evidence that E2A is directly involved in the transcriptional regulation of a number of early B-lineage genes. Introduction Top Abstract Introduction Materials and Methods Results Discussion References The development of lymphocytes from hematopoietic stem cells involves a series o f highly regulated differentiation events that depend on the collaborative effor ts of a number of transcription factors. These transcription factors coordinatel y regulate lymphopoiesis through the initiation, maintenance, and restriction of lineage-specific gene expression programs. The transcription factors encoded by the E2A gene are known to play critical roles in the regulation of lymphocyte d evelopment. E2A proteins are highly expressed in developing lymphoid cells (1, 2 ), and gene targeting studies have shown that E2A proteins are required for the initiation of B cell development in the bone marrow. Mice deficient in E2A demon strate a complete and persistent block at the earliest stage of B cell developme nt before the initiation of Ig heavy chain gene rearrangements, as well as a dra matic reduction in thymocyte number (3, 4). The mammalian E2A gene encodes two major products, E12 and E47, which are member s of the basic helix-loop-helix (bHLH) family of transcription factors (5, 6). b HLH proteins are characterized by a conserved HLH dimerization domain and an adj acent basic region that mediates DNA binding (7, 8). E12 and E47 are generated b y alternative splicing of adjacent exons encoding their bHLH domains and bind th e consensus E-box sequence CANNTG as homodimers or as heterodimers with other bH LH transcription factors (5-7, 9, 10). As E-box binding factors, E12 and E47 are members of a subfamily of bHLH transcription factors denoted the E proteins (9) . The E protein family also includes the transcription factors E2-2 and HeLa E-b ox binding protein (HEB), which play important roles in lymphocyte development a s well (11-14). E2A proteins were initially characterized for their binding activity at the Ig h eavy and light chain enhancers, which were subsequently shown to contain several functionally relevant E2A binding sites (10, 15-18). Ectopic expression of E2A in non-B cells was also found to induce germ-line Ig transcription and rearrange ment, suggesting that E2A might play a key role in Ig gene regulation (19-21). M ore recent studies demonstrated that transfection of the kidney cell line BOSC23 with E2A and the RAG recombination machinery was sufficient to drive diverse re combination events at the Ig heavy and light chain loci (22, 23). Overexpression studies have also implicated E2A in the regulation of other genes involved in t he initiation and maintenance of the B cell developmental program. Ectopic expre ssion of E12 in a macrophage line was shown to result in the induction of the B- lineage transcription factors EBF and Pax5/BSAP, as well as IL-7R and RAG-1 (24) . Other potential E2A targets include several components of the pre-B cell and B cell receptor (BCR) complexes, including the surrogate light chains 5 and VpreB and the BCR signaling molecules mb-1 (Ig) and B29 (Ig) (25, 26). However, it re mains unclear whether the lineage and stage-specific expression of these genes i s directly regulated by E2A at the transcriptional level or, alternatively, is a ctivated by other factors downstream of E2A. Chromatin immunoprecipitation (ChIP) strategies have recently been used for more direct assessments of target gene regulation based on transcription factor bind ing to suspected regulatory regions. For example, ChIP has been successfully use d in the verification and cloning of several E2F target genes in human cell line s (27). In yeast, ChIP-based analysis of transcription factor binding to relevan t target sequences has been facilitated by the introduction of affinity tags to endogenous proteins by homologous recombination (28). We have therefore combined an in vivo murine gene-tagging approach with a ChIP assay to investigate both s uspected and novel E2A target genes in lymphoid cells. Mice carrying a dual affi nity-tagged E2A knock-in allele have been generated and used to derive pre-B cel l lines that express a functional E2A fusion protein. This fusion protein provid es a means for highly efficient immunoprecipitation of E2A-bound DNA fragments, which have been screened for the presence of several suspected E2A target sequen ces. We have used this approach to identify a subset of B-lineage genes whose re gulatory regions are bound by E2A under physiological conditions, providing evid ence for direct transcriptional regulation of these genes by E2A. The pre-B cell lines expressing affinity-tagged E2A have also been used in ChIP-based cloning of a previously uncharacterized E2A target gene. Materials and Methods Top Abstract Introduction Materials and Methods Results Discussion References E2AFH Knock-In Mice. The E2AFH gene-targeting construct was generated by using an E2A genomic sequenc e isolated from the 129/sv strain. A 2-kb genomic fragment of homologous sequenc e was used in subcloning to create an in-frame fusion of the E2A carboxyl termin us with oligonucleotides encoding the following amino acid sequence: Ala-Gly-Asp -Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Ala-Gly-His-His-His-His-His-His-Stop. A phosphoglyc erate kinase promoter-driven neomycin resistance cassette (PGK-Neo) was inserted into a unique XbaI site downstream of the E2A poly(A) signal and between the ho mologous segments for positive selection of clones undergoing homologous recombi nation. A PGK promoter-driven thymidine kinase (PGK-TK) cassette was placed outs ide of the homologous targeting sequences to allow for negative selection agains t random nonhomologous recombination events. The final targeting construct was l inearized and transfected into embryonic stem cells by electroporation. Transfec ted clones were cultured under double selection with gancyclovir and G418, and c orrect targeting of 27 of 95 clones was determined by PCR screening. Germ-line t ransmission was obtained from one of two injected clones, and mice carrying the E2AFH allele were maintained in a specific pathogen-free environment at Duke Uni versity's animal facility. Derivation of Abelson Pre-B Cell Lines. Abelson murine leukemia virus (AMLV) was prepared as supernatant from ABO10 cell s, pretreated with polybrene, and added to E2AFH/FH or E2Aloxp/loxp bone marrow cells cultured in RPMI 1640 media supplemented with 10% FBS, penicillin/streptom ycin, -ME, and gentamycin. After 3 wk, bulk cultures were subcloned to yield the Abelson lines E2AFH1B, E2AFH4, and E2Aloxp 1AB1. Chromatin Extracts and Immunoprecipitations. Cells were fixed and washed for preparation of chromatin extracts essentially as reported by Fernandez et al. (29). Fixed cells were then sonicated (Fisher Scie ntific 550 tapered microtip probe, setting 4) for 20-25 cycles of 25 s on a cold block with 15 s cooling between each burst, to obtain 0.5- to 1.0-kb DNA fragme nts. Sonicates were centrifuged at 14,000 × g for 5 min, and supernatants were harvested and stored at 80°C. ChIP procedures were adapted from those described by Fernandez et al. (29). Chromatin extracts (1-2 mg) from the E2AFH1B pre-B ce ll line or the untagged control cell line AMLV-3B (derived from C57BL/6 bone mar row) were diluted 1:10 in ChIP buffer (140 mM NaCl/100 µg/ml BSA/100 &micr o;g/ml yeast tRNA/1% Triton X-100/1 mM PMSF) and incubated with 50 µl of a nti-FLAG agarose for 2 h at 4°C, rotating slowly. The bound agarose beads were harvested by centrifugation (14,000 × g for 15 s) and washed three times in 1 m l of IP buffer, twice in 1 ml of IP buffer containing 500 mM NaCl, twice in 1 ml of wash buffer (10 mM Tris·HCl, pH 8.0/250 mM LiCl/1 mM EDTA), and three times in 1 ml of TE buffer (10 mM Tris·HCl, pH 8.0/1 mM EDTA). Bound DNA was eluted, incubated at 65°C overnight to reverse cross-links, and then RNase-treated, de proteinized, and precipitated as described by Fernandez et al. (29). Processed D NA fragments were resuspended in 100 µl of distilled water. Cloning of imm unoprecipitated DNA fragments was performed essentially as described by Weinmann et al. (27). PCR Screening of Suspected Target Genes. A series of 4-fold dilutions of input chromatin and immunoprecipitated DNA from E2AFH and control cell lines was PCR-amplified for 29-32 cycles (94°C, 1 min; 5 8°C, 1 min; 72°C, 1min; with 2 min final extension at 72°C) in 20 µl PC R buffer containing 3 mM MgCl2 and Platinum Taq polymerase (Invitrogen). The ent ire PCR samples were then resolved on 1.5% agarose gels and visualized by ethidi um bromide staining. Relative band intensity was used in qualitative comparisons of target sequence enrichment from ChIPs of E2AFH vs. untagged control cell lin es. All oligonucleotides used in PCR screening of immunoprecipitated DNA were 23 -mers (IDT DNA) designed to amplify 150- to 200-bp regions surrounding potential E2A-binding sites within the regulatory regions of suspected target genes (Tabl e 1, Regulatory region and Clone). In some cases, multiple primer sets were used to cover larger genomic sequences and potential regulatory regions; representat ive results for each of these regions are presented in Fig. 4. View this table: [in this window] [in a new window] Table 1. Oligonucleotide primer pairs used for ChIP-PCR screening of putati ve E2A target genes (Regulatory region), ChIP-PCR enrichment confirmation of clo nes isolated as E2A-bound sequences (Clone), and RT-PCR analysis of Abelson cell line transcripts (Transcript) Results Top Abstract Introduction Materials and Methods Results Discussion References Generation of Affinity-Tagged E2AFH Mice. We wanted to use a ChIP strategy to elucidate target genes that are directly reg ulated by E2A during lymphocyte development. Because the efficiency of immunopre cipitation-based studies can be limited by antibody specificity or affinity, we used an in vivo gene-tagging approach in generating an affinity-tagged E2A knock -in mouse model to facilitate the isolation of endogenous E2A-bound DNA sequence s. Oligonucleotides encoding dual affinity tags were cloned into the 3' exon of the genomic E2A locus such that both E12 and E47 would be expressed as fusion pr oteins carrying carboxyl-terminal FLAG epitope and hexahistidine sequences (Fig. 1A). Proper targeting of the locus with the knock-in allele was confirmed by So uthern blotting (Fig. 1B) and PCR genotyping (Fig. 1C). Bands representing the e xpected wild-type and E2AFH alleles were detected, and normal distributions of e ach genotype were observed, indicating that the knock-in allele did not have an effect on neonatal survival. View larger version (25K): [in this window] [in a new window] Fig. 1. Generation of dual affinity-tagged E2A knock-in allele. (A) Wild-ty pe E2A locus and E2A-FLAGHis knock-in allele containing in-frame fusion of 3' E2 A exon sequence with FLAG and hexahistidine affinity tags. (B) Southern blot gen otyping confirmation of BamHI-digested tail DNA from E2AFH/+ intercross pups. A 1.3-kb PCR-generated genomic E2A probe was used to detect the wild-type and knoc k-in alleles. (C) PCR genotyping of intercross pups by using YZ-104 (E2A antisen se), YZ-29 (Neo sense), and YZ-164 (E2A sense) primers (31). E2AFH Fusion Protein Supports Normal Lymphocyte Development. B lymphocyte development is highly dependent on E2A protein dosage, such that ev en a 50% reduction in E2A protein activity translates into altered B cell develo pment in the bone marrow (4, 13, 30). We therefore wanted to investigate B-lymph opoiesis in E2AFH mice as a sensitive indicator of E2A fusion protein expression and function. Fluorescence-activated cell sorter (FACS; Becton Dickinson) analy sis of bone marrow and splenocytes from wild-type, E2AFH/+ heterozygous, and E2A FH/FH homozygous mice showed that lymphocyte development was normal in the knock -in mice. The E2AFH allele led to no significant alterations in the relative per centages or numbers of pro- and pre-B cell populations (Fig. 2A) and mature IgM+ B cell populations in the bone marrow (Fig. 2B). Normal B and T lymphocyte prof iles were also observed in the spleen (Fig. 2 C and D) and thymus, and other hem atopoietic populations in the bone marrow and spleen were also normal (data not shown). The fusion protein was therefore expressed from the targeted locus and w as functional in supporting normal lymphocyte development. View larger version (44K): [in this window] [in a new window] Fig. 2. Normal lymphocyte development in E2AFH mice. Bone marrow (A and B) and splenocytes (C and D) from 3-wk-old wild-type (E2AWT/WT), heterozygous (E2AF H/WT), and homozygous (E2AFH/FH) littermates were analyzed by FACS for expressio n of lymphocyte lineage surface markers by using the following fluorescent antib ody conjugates: IgM-FITC, B220-APC, CD19-PE, CD4-FITC, and CD8-APC (Caltag, Sout h San Francisco, CA); and CD43-PE (PharMingen). (A) Bone marrow staining for B22 0 and CD43. (B) Bone marrow staining for B220 and surface IgM. (C) Splenocyte st aining for B220 and surface IgM. (D) Splenocyte staining for CD4 and CD8. Abelson-Transformed Pre-B Cell Lines Express Functional E2AFH Fusion Protein. To establish a culture system for studying E2A target sequences in the context o f an early B cell environment, bone marrow was isolated from E2AFH/FH mice and t ransformed with AMLV. The Abelson virus encodes the v-Abl oncogene and selective ly targets developing pre-B cells for transformation. Primary transformants were established from E2AFH/FH bone marrow cultures, and two subcloned lines, E2AFH1 B and E2AFH4, were then derived for subsequent studies. These lines, as well as an untagged control Abelson line (E2Aloxp 1AB1) generated from the bone marrow o f mice carrying a loxp-flanked E2A knock-in allele (E2Aloxp/loxp), displayed a t ypical B220+CD19+ pre-B cell phenotype (Fig. 3A; ref. 31). Anti-FLAG immunopreci pitations of E2A from nuclear extracts from E2AFH1B or E2AloxP control cells wer e then performed to verify the expression and functional utility of the E2AFH fu sion proteins. An anti-E2A Western blot of the immunoprecipitated extract confir med that the fusion proteins were expressed and that affinity purifications coul d be performed by using the FLAG epitope tag (Fig. 3B). A band corresponding to the molecular mass of the immunoprecipitated E2A fusion protein (74 kDa) was als o detected by colloidal staining of an SDS/PAGE gel (Fig. 3C). Electrophoretic m obility-shift analysis of nuclear extracts from the three Abelson clones (E2AFH1 B, E2AFH4, and E2AloxP) indicated that the E2A fusion protein bound to a µ E5 E-box oligonucleotide probe as expected and that the E2AFH-DNA complex could be mobility-shifted with anti-FLAG antibody (Fig. 3D). View larger version (45K): [in this window] [in a new window] Fig. 3. Characterization of Abelson-transformed pre-B cell lines. (A) FACS analysis for expression of B-lineage markers CD19 and B220 on E2AFH clone 1B, E2 AFH clone 4, and E2Aloxp control clone 1AB1. (B) Anti-E2A (G193-86, PharMingen) Western blot of elution fractions from anti-FLAG agarose affinity-purification o f primary E2Aloxp and E2AFH/FH transformant nuclear extracts (*, E2A monomer). ( C) Colloidal Coomassie staining of FLAG peptide elution fractions from affinity- purification of subcloned Abelson lines. E2A monomer (*) is indicated, as well a s a specific higher-molecular-weight band representing a commonly observed alter native isoform or modification of E2A. (D) Electrophoretic mobility-shift analys is on Abelson pre-B nuclear extracts for binding to radiolabeled µE5 oligo nucleotide (10). E2A in µE5-binding complexes unshifted (*) or supershifte d (**) with 1 µl of anti-FLAG antibody. E2A Is Bound to a Subset of Lineage-Restricted Regulatory Regions. Phenotypic and biochemical analysis of the E2AFH Abelson lines demonstrated that the E2A fusion protein was expressed, displayed normal DNA-binding characterist ics in vitro, and could be immunoprecipitated with anti-FLAG antibody. The affin ity-tagged protein should facilitate efficient immunoprecipitation of E2A-bound DNA sequences from cross-linked chromatin fragments. Chromatin extracts were the refore prepared from the E2AFH1B or untagged control cell lines, and E2A-bound D NA fragments were obtained by anti-FLAG immunoprecipitation. These sequences wer e then screened for enrichment of promoter and enhancer regions within several p otential lymphoid-restricted E2A target genes. Unfractionated input chromatin an d immunoprecipitated DNA from the E2AFH1B cell line and an untagged control cell line (AMLV-3B) were PCR-amplified by using primers surrounding the E-box sites within the regulatory elements of these genes (Table 1, Regulatory Region). Comp arisons of the relative signals observed after amplification of immunoprecipitat ed DNA from tagged vs. control chromatin extracts allowed for qualitative determ inations of whether each potential target sequence was enriched due to E2A bindi ng. Significant enrichment was observed for several suspected E2A targets, including the Ig intronic and 3' enhancers, the mb-1 (Ig) promoter, the 5 and VpreB promo ters, and the 5' region of the EBF locus (Fig. 4A). Selective enrichment of thes e regulatory regions indicated E2A protein binding and suggested that E2A is lik ely to play a direct role in the transcriptional regulation of these genes durin g B cell development. Interestingly, several other potential E2A target sequence s appeared to be only slightly enriched in the ChIP-PCR screen, including the Ig H intronic and hs3b/hs4 3' enhancers, the RAG-2 enhancer, and the 5' regions of the B29 and TdT loci (Fig. 4B). Meanwhile, no enrichment was observed for severa l other potential E2A targets, including the IgH 3' enhancer hs3a and the promot er sequences for Pax5, RAG-1, RAG-2, Oct-2, CD5, and CD19 (Fig. 4C). The observa tion that only a subset of putative E2A targets was actually enriched provided s upport for both the selectivity and relevance of the assay because enrichment re quired more than just an accessible locus containing potential E2A binding sites . View larger version (91K): [in this window] [in a new window] Fig. 4. ChIP-PCR screen for E2A binding to regulatory regions of potential target genes. A series of 4-fold dilutions of input chromatin and immunoprecipit ated DNA from E2AFH1B or untagged control pre-B cell lines were PCR-amplified by using primers specific for suspected E2A target regions (see Table 1, Regulator y region). Sequence enrichment in immunoprecipitated DNA from E2AFH1B vs. contro l chromatin indicated E2A binding within genomic region. Each experiment was rep eated at least three times, and results shown were reproducible for all targets. (A) Significant enrichment of the Ig enhancers, 5 and VpreB promoters, 5' EBF l ocus, and mb-1 promoter was observed. (B) Low levels of enrichment were seen for the IgH intronic and hs3b/hs4 enhancers, 5' B29 and TdT loci, and RAG-2 enhance r. (C) No enrichment was indicated for the Pax5, CD19, Oct-2, RAG-1, RAG-2, and CD5 promoters and the IgH hs3a enhancer. Identification of Novel E2A Target Genes by ChIP-Based Cloning. Comparative PCR analysis provided a means for qualitative assessment of enrichme nt (and thus E2A binding) at a suspected target sequence. However, the regulator y regions identified as E2A targets by ChIP-PCR screening probably represent onl y a small subset of all genes subject to transcriptional regulation by E2A durin g B cell development. A more general approach was required to broaden the scope of the study to include the identification of novel E2A target genes. ChIP-based cloning strategies have recently been used in identifying novel target genes re gulated by ubiquitous transcription factors such as E2F (27). We therefore used our E2AFH pre-B cell ChIP system for the cloning and characterization of novel E 2A target sequences. Immunoprecipitated DNA fragments from E2AFH1B chromatin ext racts were blunt-ended by polymerase treatment and ligated into pBluescript vect or. The transformed clones were then screened by restriction digest, and 13 of 2 4 clones were found to contain inserts representing immunoprecipitated DNA fragm ents. These inserts were sequenced by using vector-specific primers, and the seq uences obtained were then identified by BLAST or ENSEMBLE database searches for mouse genome matches. Ten of 13 sequences had single murine genome matches, wher eas the remaining 3 yielded either no match or multiple genomic matches (Table 2 ). Sequence analysis of the 10 genomic clones indicated that 9 of 10 inserts con tained at least one E box representing a potential E2A binding site. Importantly , eight of these nine sequences were subsequently found to be enriched by ChIP-P CR screening, providing strong confirmation that the majority of DNA fragments i solated by ChIP-based cloning represented actual E2A-bound sequences (Table 1, C lone; Table 2). Interestingly, seven of the eight sequences found to be enriched by ChIP-PCR were also located within predicted or known gene loci (Table 2). View this table: [in this window] [in a new window] Table 2. Summary of inserts isolated as E2A-bound sequences from ChIP-based cloning of novel E2A target genes The ChIP-1 insert yielded a perfect match to an intronic sequence of the putativ e mouse butyrophilin-like gene NG9 (BTL-II), located within the extended MHC II locus (GenBank accession no. AF050157.1). The 428-bp sequence also contained two E-box sites, including one canonical E2A binding site, CAGGTG (Fig. 5A). The ge nomic region represented by the ChIP-1 clone was therefore selected for further analysis. Primers (I) specific for the sequences flanking the prominent ChIP-1 E box were used to confirm E2A-dependent enrichment of the region by PCR (Fig. 5 A and B). Because the clone represented intronic sequence between central exons of the gene, two additional primer sets (II, III) were also designed to screen f or enrichment of the putative promoter region of NG9. A fourth primer pair (IV) covering what was speculated to be a nonregulatory region upstream of the first NG9 exon was used in ChIP-PCR screening as well. Interestingly, the genomic regi ons corresponding to both the ChIP-1 clone and the putative NG9 promoter region were moderately enriched in immunoprecipitated DNA from E2AFH1B chromatin (Fig. 5C). No significant enrichment of the nonregulatory region represented by primer pair IV was observed, supporting the specificity of the enrichment seen with th e other primers. View larger version (72K): [in this window] [in a new window] Fig. 5. E2A target gene isolated by ChIP-based cloning. Immunoprecipitated DNA fragments from E2AFH1B chromatin extract were cloned, sequenced, and identif ied by mouse genome database search (see Table 2). (A) ChIP-1 clone insert with an exact match to an intronic region within the putative butyrophilin-like gene NG9. Clone-specific primers (I; in italics) surrounding the consensus E box (CAG GTG) were used to confirm E2A binding by ChIP-PCR. (B) Primers to the ChIP-1 ins ert (I), the predicted promoter region (II and III) of the NG9 locus, and a regi on upstream of the transcription start site (IV). (C) ChIP-PCR screen for E2A bi nding at the ChIP-1 insert genomic sequence and NG9 5' regulatory regions. (D) S emiquantitative RT-PCR analysis of four 3-fold dilutions of E2AFH1B cDNA for the predicted NG9 transcript, along with relevant B-lineage genes (Ig, 5, Iµ) and a loading control (EF1) (see Table 1, Clone). Thirty cycles (21 cycles for EF1) of PCR amplification (94°C, 30 s; 57°C, 30 s; 72°C, 30 s, with 1 min ext ension at 72°C) were performed in PCR buffer containing 3 mM MgCl2 and Platinum Taq polymerase (Invitrogen). The NG9 gene contains several conserved domains characteristic of the butyrophil in gene family, including two Ig variable-like (IgV) domains and two Ig constant region-like (IgC) domains (Fig. 5A) (32). No functional information on the gene product is available, however, and little is known about its expression in lymp hoid cells. Because E2A was shown to bind to putative regulatory regions of NG9 and typically functions as a positive regulator of transcription, we wanted to e valuate NG9 transcription in the cells from which the ChIP clone was isolated. c DNA from E2AFH1B cells was therefore PCR-amplified by using primers specific for the 5' and 3' regions of the predicted NG9 transcript, as well as primers to se veral other B-lineage genes (Table 1, Clone). NG9 transcripts were detectable in E2AFH1B cells along with Ig, 5, and Iµ transcripts, further supporting a role for E2A in the regulation of NG9 expression (Fig. 5D). Discussion Top Abstract Introduction Materials and Methods Results Discussion References To our knowledge, there have been no previous studies in which an in vivo murine gene-tagging approach has been used to facilitate analysis of target genes regu lated by an endogenously expressed transcription factor. The gene-tagging system provides a distinctive alternative to the use of ectopically expressed proteins and also bypasses the challenges of low antibody affinity and specificity that can sometimes interfere with traditional protein isolation approaches. Similarly , to our knowledge there have been no previous studies to directly characterize E2A binding to a number of lymphoid lineage-restricted regulatory regions at the chromatin level. Here, we have demonstrated E2A binding to regulatory regions w ithin a select subset of genes required for B lymphocyte development. Previous e ctopic expression and promoter-analysis studies have implicated E2A in the regul ation of many of these target genes, including the Ig heavy and light chains, s urrogate light chains 5 and VpreB, and the transcription factor EBF (22, 24, 26) . Significant enrichment of these sequences by E2A-ChIP provides more conclusive evidence for direct transcriptional regulation by E2A. Binding of E2A to 5' reg ulatory regions within the EBF locus is also in agreement with recent studies by Smith et al. (33), who have characterized a potential E2A binding site within t he newly identified EBF promoter region. We were also intrigued to see strong en richment of the mb-1 (Ig) promoter region, because this observation provided dir ect evidence for the regulation of yet another component of the B cell receptor complex by E2A. Although mb-1 has been implicated as a potential E2A target in g enetic studies, it has generally been considered a primary target for other tran scription factors, such as Pax5/BSAP, Ets, and Oct-2 (34, 35). However, our data on E2A binding to the mb-1 promoter supports unpublished work by Hagman et al. demonstrating occupation of an mb-1 promoter E-box site by DNA footprinting anal ysis (J. Hagman, personal communication). These studies provide further insight into the collaborative regulation of gene expression by E2A and other transcript ion factors during B-lymphopoiesis. We consistently observed that certain regulatory regions (Ig enhancers, mb-1 pro moter, 5' EBF locus, and 5 and VpreB promoters) are significantly enriched after E2A-DNA immunoprecipitations, whereas other putative targets (IgH intronic and hs3b/hs4 enhancers, 5' TdT and B29 loci, and RAG-2 enhancer) show only very low levels of enrichment. We have also evaluated a group of lymphoid lineage-restric ted genes that show no detectable enrichment in our ChIP-PCR assay, including th e IgH 3' enhancer hs3a and the promoters for Pax5, RAG-1, RAG-2, CD19, Oct-2, an d CD5. The lack of enrichment of these sequences further substantiates the speci ficity of the ChIP-PCR system as a means for elucidation of direct vs. indirect E2A target genes. Although the transcription of these genes has been shown to be affected by E2A activity, previous studies have provided no significant evidenc e for direct transcriptional regulation by E2A. Our data suggests that Pax5/BSAP , Oct-2, CD19, and RAG-1 may be targets for transcriptional regulation by other genes downstream of E2A. This possibility is supported by previous observations that ectopic expression of EBF in a macrophage line leads to activation of a sub set of E2A-responsive genes including Pax5 (24). E2A-mediated activation of the EBF locus may therefore play a role in establishing the hierarchy of transcripti on factors at the earliest stages of B-lymphopoiesis, with EBF subsequently cont ributing to the induction of Pax5 and other downstream genes. Perhaps the most surprising data obtained from the ChIP-PCR screen were the mini mal enrichment observed for the IgH enhancers. The presence of abundant Iµ transcripts in the pre-B cell lines indicates that the IgH locus is accessible and transcriptionally active. The IgH enhancers also contain well-characterized E-box sites, which are required for normal Ig gene transcription and rearrangeme nt (15, 18). However, previous transfection and DNase hypersensitivity studies h ave shown that only one of the 3' IgH enhancers, hs4, is thought to be active at the early stages of B cell development (36, 37). Interestingly, hs4 was the mos t prominently enriched of the 3' enhancers, with its most proximal enhancer, hs3 b, showing slightly lower enrichment. No detectable enrichment of IgH enhancer h s3a was observed, which is in agreement with previous data suggesting that this enhancer is only functional in activated mature B cells (reviewed in ref. 37). H ere, we must also emphasize that a lack of significant E2A binding at any given regulatory region in the pre-B cell lines does not exclude the possibility of di rect transcriptional regulation by E2A. Alternatively, certain regulatory region s may be bound by E2A only at restricted developmental stages during B cell deve lopment and/or activation. This caveat may be particularly relevant in the analy sis of genes involved in Ig rearrangement, such as RAG-1 and RAG-2, TdT, and the Ig genes themselves. The tight and highly ordered developmental regulation of t hese loci during B-lymphopoiesis may involve significant E2A binding only during specific phases of Ig gene rearrangement, assembly, and expression. Future stud ies on E2A-bound regulatory regions in primary cells at different stages of deve lopment should provide further insight on these dynamic regulatory processes. A ChIP-based cloning strategy was used to isolate several novel E2A-bound sequen ces, including an intronic region within the novel E2A target gene NG9 (also kno wn as BTL-II for butyrophilin-like MHC class II-associated). E2A binding at E-bo x-containing genomic regions across this locus was subsequently investigated by sequence analysis and ChIP-PCR screening. The NG9 locus lies downstream from the E locus and upstream from a cluster of other butyrophilin-like genes within the extended MHC II locus (32). As with other butyrophilin genes, each conserved do main of NG9 is encoded by a separate exon, providing support for the notion that the butyrophilin gene clusters arose through exon shuffling and duplication. Ho wever, NG9 lacks both the transmembrane domain and the conserved carboxyl-termin al B30.2 domain of the traditional butyrophilins. Interestingly, sequencing data from multiple cell lines indicates that the NG9/BTL-II locus is also highly pol ymorphic with respect to HLA haplotype (32). These polymorphisms translate into at least five different NG9 alleles and may have been generated and maintained d uring the diversification of the MHC/HLA loci. Although NG9 transcripts have pre viously been detected in skeletal muscle and a number of gut tissues, to our kno wledge no previous work has evaluated potential regulatory regions and character ized NG9 transcription specifically in lymphoid cells (32). A number of other po tential target sequences were also isolated by ChIP-based cloning, most of which appear to be bona fide E2A-bound targets based on their enrichment and their lo cation within known or predicted gene loci. The ChIP-PCR screening strategy used in characterizing E2A binding at the NG9 locus should also prove quite useful i n evaluating these and other large genomic regions for transcription factor bind ing in vivo. In future studies, we will be combining the ChIP-based cloning syst em with gene array analysis on E2A-deficient cell lines to characterize addition al E2A target genes. This powerful two-tiered approach will allow for the elucid ation of target genes based on both E2A binding and the requirement for E2A in t he normal expression of these genes. Acknowledgements We are grateful to Cheryl Bock at the Duke University Transgenic Mouse Facility for her work in generating the E2AFH mice. 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