Chromatin Structural Analyses of the Mouse Igκ Gene Locus Reveal New Hypersensitive Sites Specifying a Transcriptional Silencer and Enhancer
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m204065200
ISSN1083-351X
AutoresZhimei Liu, Julia B. George-Raizen, Shuyu Li, Katherine C. Meyers, Mee Young Chang, William T. Garrard,
Tópico(s)RNA Research and Splicing
ResumoTo identify new regulatory elements within the mouse Igκ locus, we have mapped DNase I hypersensitive sites (HSs) in the chromatin of B cell lines arrested at different stages of differentiation. We have focused on two regions encompassing 50 kilobases suspected to contain new regulatory elements based on our previous high level expression results with yeast artificial chromosome-based mouse Igκ transgenes. This approach has revealed a cluster of HSs within the 18-kilobase intervening sequence, which we cloned and sequenced in its entirety, between the Vκ gene closest to the Jκ region. These HSs exhibit pro/pre-B cell-specific transcriptional silencing of a Vκ gene promoter in transient transfection assays. We also identified a plasmacytoma cell-specific HS in the far downstream region of the locus, which in analogous transient transfection assays proved to be a powerful transcriptional enhancer. Deletional analyses reveal that for each element multiple DNA segments cooperate to achieve either silencing or enhancement. The enhancer sequence is conserved in the human Igκ gene locus, including NF-κB and E-box sites that are important for the activity. In summary, our results pinpoint the locations of presumptive regulatory elements for future knockout studies to define their functional roles in the native locus. To identify new regulatory elements within the mouse Igκ locus, we have mapped DNase I hypersensitive sites (HSs) in the chromatin of B cell lines arrested at different stages of differentiation. We have focused on two regions encompassing 50 kilobases suspected to contain new regulatory elements based on our previous high level expression results with yeast artificial chromosome-based mouse Igκ transgenes. This approach has revealed a cluster of HSs within the 18-kilobase intervening sequence, which we cloned and sequenced in its entirety, between the Vκ gene closest to the Jκ region. These HSs exhibit pro/pre-B cell-specific transcriptional silencing of a Vκ gene promoter in transient transfection assays. We also identified a plasmacytoma cell-specific HS in the far downstream region of the locus, which in analogous transient transfection assays proved to be a powerful transcriptional enhancer. Deletional analyses reveal that for each element multiple DNA segments cooperate to achieve either silencing or enhancement. The enhancer sequence is conserved in the human Igκ gene locus, including NF-κB and E-box sites that are important for the activity. In summary, our results pinpoint the locations of presumptive regulatory elements for future knockout studies to define their functional roles in the native locus. kilobase(s) 3′ enhancer downstream enhancer intronic enhancer hypersensitive site intervening sequence luciferase reporter gene matrix association region minimal Vκ gene promoter intervening sequence silencer yeast artificial chromosome The mouse immunoglobulin (Ig) κ gene locus has provided a paradigm to investigate many challenging and biologically relevant problems, including site-specific recombination (1Sleckman B.P. Gorman J.R. Alt F.W. Annu. Rev. Immunol. 1996; 14: 459-481Crossref PubMed Scopus (259) Google Scholar, 2Lewis S.M. Wu G.E. Cell. 1997; 88: 159-162Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 3Papavasiliou F. Jankovic M. Gong S. Nussenzweig M.C. Curr. Opin. Immunol. 1997; 9: 233-238Crossref PubMed Scopus (33) Google Scholar, 4Grawunder U. West R.B. Lieber M.R. Curr. Opin. 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Although considerable insight has been revealed on the functional significance of the above elements, the results of several investigations strongly suggest that additional crucial regulatory elements within the Igκ locus remain to be discovered. For example, expression of rearranged Igκ transgenes containing both enhancers is influenced by the site integration and fails to exhibit copy-number dependence (16Blasquez V.C. Hale M.A. Trevorrow K.W. Garrard W.T. J. Biol. Chem. 1992; 267: 23888-23893Abstract Full Text PDF PubMed Google Scholar, 47Blasquez V.C., Xu, M. Moses S.C. Garrard W.T. J. Biol. Chem. 1989; 264: 21183-21189Abstract Full Text PDF PubMed Google Scholar, 48Xu M. Hammer R.E. Blasquez V.C. Jones S.A. Garrard W.T. J. Biol. Chem. 1989; 264: 21190-21195Abstract Full Text PDF PubMed Google Scholar). 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In an effort to identify new regulatory elements we have therefore focused on the aforementioned previously unstudied regions in the mouse Igκ locus suspected to possess transcriptional regulatory elements based on our transgenic mice studies (54Li S. Hammer R.E. George-Raizen J.B. Meyers K.C. Garrard W.T. J. Immunol. 2000; 164: 812-824Crossref PubMed Scopus (8) Google Scholar). Our approach takes advantage of the observation that when cis-acting elements are functional in a particular cell lineage, they often form nuclease hypersensitive sites (HSs) in chromatin (55Gross D.S. Garrard W.T. Annu. Rev. Biochem. 1988; 57: 157-197Crossref Scopus (953) Google Scholar). We report here the identification, sequencing, and initial functional characterization of several such HSs. Interestingly, one cluster of HSs specifies transcriptional silencing in pro/pre-B cells, whereas another acts as a powerful B cell-specific transcriptional enhancer. Long PCR Amplification of the Intervening Sequence ( IS )—Template DNA was prepared as total DNA from yeast cells bearing either YAC FAW.A3 or YAC FAW.A3 truncated at Vκ21G by chromosome fragmentation (24George J.B., Li, S. Garrard W.T. Capra J.D. Zanetti M. The Antibodies. 4. Gordon and Breach/Harwood Academic Publisher, San Diego1997: 41-62Google Scholar, 25George J.B., Li, S. Garrard W.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12421-12425Crossref PubMed Scopus (20) Google Scholar). To truncate FAW.A3, a genomic Vκ21 fragment was amplified by PCR (primers Vκ21L (5′-TGC TGC TGC TCT GGG TTC CAG GTG-3′) and Vκcdr2r (5′-GAT TCT AGG TTG GAT GCA GGA TAG-3′)). Amplification conditions were 1 min at 94 °C, 2 min at 55 °C, and 1 min at 72 °C for 30 cycles. The amplified sequence was introduced into the acentric fragmentation vector pBP81 (56Reeves R.H. Pavan W.J. Hieter P. Methods Enzymol. 1992; 216: 584-603Crossref PubMed Scopus (17) Google Scholar), linearized, and transformed into yeast harboring FAW.A3 using standard lithium acetate transformation (57Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 53: 163-168Crossref Google Scholar). Several transformants were analyzed using pulsed-field gel electrophoresis. Long PCR was performed using the Expand Long PCR System according to the manufacturer's recommendations (Roche Molecular Biochemicals). After equilibration in 1× PCR reaction buffer (excluding enzymes), 25 μl of master mix 1 (dNTPs plus primer) was added to gel block slices followed by incubation for 15 min at 65 °C and the subsequent addition of 25 μl of master mix 2 (including enzymes). Amplification conditions were 10 cycles of 94 °C for 30 s, 55 °C for 45 s, and 68 °C for 12 min. Twenty additional cycles were performed in which the extension cycle was increased by 10 s/cycle. Primers were Vκ21L (5′-TGC TGC TGC TCT GGG TTC CAG GTG-3′) and Jκ2R (5′-TTT GAG CTT GAG TAG ACA AAT ATC C-3′). 1–2% of the total products were separated by pulsed-field gel electrophoresis on 1% agarose gels using an auto algorithm for 5–50 kb on a CHEF mapper (Bio-Rad). Cell lines, except for S194, A20, and MPC-11, were maintained in RPMI 1640, 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mml-glutamine. β-Mercaptoethanol (50 μm) was added to pre-B cell cultures. S194 and A20 cells were cultured in Iscove's medium containing 5% fetal bovine serum, and MPC-11 cells were cultured in Dulbecco's modified Eagle's medium containing 20% horse serum. The engineered 103/BclII cell lines ΔN1 and ΔN7 (58Scherrer D.C. Brockman J.A. Bendall H.H. Zhang G.M. Ballard D.W. Oltz E.M. Immunity. 1996; 5: 563-574Abstract Full Text PDF PubMed Scopus (56) Google Scholar) were kind gifts of Eugene Oltz (Vanderbilt University). The pre-B cell 103Bcl2 lines were maintained at 34 °C with 5% CO2; S194, A20, and MPC-11cells were maintained at 37 °C and 10% CO2; all other lines were maintained at 37 °C and 5% CO2. Cells were permeabilized with hen egg white α-lysolecithin (Sigma) and treated with increasing concentrations of DNase I (Worthington Biochemicals) (0.25–8.0 units/ml) (17Roque M.A. Smith P.A. Blasquez V.C. Mol. Cell. Biol. 1996; 16: 3138-3155Crossref PubMed Google Scholar). After lysis, DNA was purified either using Qiagen genomic columns or by phenol:chloroform extraction (13Hale M.A. Garrard W.T. Mol. Immunol. 1998; 35: 609-620Crossref PubMed Scopus (12) Google Scholar), and samples were digested to completion with either BglII,HindIII, NcoI, or PstI as indicated below. For PstI digest mapping with probes A or B (see Fig.1), 10–15-μg samples were electrophoresed in 0.8% agarose (SeaKem GTG, FMC Bioproducts) in 0.5× Tris-acetate-buffered EDTA running buffer overnight at 1.1 V/cm. After blotting using standard neutral transfer to Nytran Plus membranes (59Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.38-9.40Google Scholar), 0.2-μm pore size (Schleicher and Schuell), prehybridized filters were hybridized overnight in 6× SSC (1× SSC = 0.15 m NaCl and 0.015m sodium citrate), 10× Denhardt's solution, 1% SDS, and 100 μg/ml herring testes DNA with a 1.8- or 0.8-kb DNA fragments, corresponding to probes A and B, respectively, labeled with [α-32P]dCTP using Rediprime II (AP Biotech). Wash conditions were 1× SSC, 0.5% SDS 3 times for 30 min at 65 °C or until sufficient background had been eliminated. For mapping with probes C-E, electrophoresis and blotting was as above, but pre-hybridization, hybridization, and washing were performed according to a modification of Church and Gilbert (13Hale M.A. Garrard W.T. Mol. Immunol. 1998; 35: 609-620Crossref PubMed Scopus (12) Google Scholar, 60Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7251) Google Scholar). Membranes were exposed to Eastman Kodak Co. XAR5 film with intensifying screens at −70 °C or to PhosphorImager screens (Molecular Dynamics). Probe A was a 1.8-kb ClaI/PstI fragment gel-isolated from the IS long PCR product. Primers for probe B PCR amplification were 5′-PstL (5′-TAA AAA TCC TGG TGC CAG GGG TG-3′) and 5′-PstR (5′-AGC TTA AGG ACG TCA CAT AGA CT-3′). PCR reactions were performed for 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C.BglII or NcoI digests were used for mapping with probe C, PstI digests were used for mapping with probe D, and HindIII digests were used for mapping with probe E (see Fig. 1). Probe C consisted of a 1.3-kbBglII/HindIII fragment isolated from the pRxR-1 recombinant plasmid (61Mu¨ller B. Stappert H. Reth M. Eur. J. Immunol. 1990; 20: 1409-1411Crossref PubMed Scopus (27) Google Scholar). Probe D consisted of a 1.5-kb SacI fragment isolated from the pRSB recombinant plasmid (61Mu¨ller B. Stappert H. Reth M. Eur. J. Immunol. 1990; 20: 1409-1411Crossref PubMed Scopus (27) Google Scholar). pRxR-1 and pRSB were the kind gifts of Michael Reth (Max-Plank-Institut fu¨r Immunobiologie, Freiburg, Germany). Probe E was a 510-bp fragment spanning exon 6 of the mouse ribose-5-phosphate isomerase gene (62Apel T.W. Scherer A. Adachi T. Auch D. Ayane M. Reth M. Gene. 1995; 156: 191-197Crossref PubMed Scopus (23) Google Scholar), prepared as above using the following PCR primers 5′-GCT TGC TTG GAC CTG CTG G-3′ and 5′-CGG CAG AGA AGA CAA AGG ATC C-3′. For functional tests, various fragments were inserted into the NheI or theSpeI site of the polylinker region of KpLUC or IM.KpLUC, respectively (63Fulton R. Van Ness B.G. Nucleic Acids Res. 1993; 21: 4941-4947Crossref PubMed Scopus (32) Google Scholar), designated here as PVκLuc and EiPVκLuc, respectively. A 3.6-kb fragment encompassing hypersensitive sites HS 3–6 was amplified from the IS long PCR product to add externalSpeI sites using PCR conditions similar to those described above for probe preparation. The primer pairs for fragments containing HS 3–6, HS 4–6, HS 5–6, HS 6, HS 3–4, HS 3, and HS 4–5 were, respectively: 5′-ACG CGT CGA CTA GTG TAC TCT GAA CCT TGT ATG GTG ATG-3′ and 5′-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3′; 5′-ACT CGT CGA CTA GTC TCT GGG CCT GCA CAG ATT CCA C-3′ and 5′-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3′; 5′-ACG CGT CGA CTA GTC TGC TAC ATA TGT GCG GGG GAG G-3′ and 5′-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3′; 5′-ACG CGT CGA CTA GTC CCA CCC TCA AGA CAG GCA CAG-3′ and 5′-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3′; 5′-ACG CGT CGA CTA GTG TAC TCT GAA CCT TGT ATG GTG ATG-3′ and 5′-ACG CGT CGA CTA GTC CTC CCC CGC ACA TAT GTA GCA G-3′; 5′-ACG CGT CGA CTA GTG TAC TCT GAA CCT TGT ATG GTG ATG-3′ and 5′-ACG CGT CGA CTA GTG GAA TCT GTG CAG GCC CAG AGA C-3′; 5′-ACG CAC GCG TCG ACT AGT CTC TGG GCC TGC ACA GAT TCC AC-3′ and 5′-ACG CAC GCG TCG ACT AGT GCC TGT CTT GAG GGT GGG ACT G-3′. The spacer DNA control was a 2.1-kb rat amylase cDNA fragment. PCR conditions were 1 min at 94 °C, 2 min at 55 °C, and 2 min at 72 °C for 30 cycles. For vector insertions we similarly amplified a 1-kb fragment encompassing HS 9 from recombinant plasmid pRxB5 (the kind gift of Michael Reth, Max-Plank-Institut fu¨r Immunobiologie, Freiburg, Germany) (61Mu¨ller B. Stappert H. Reth M. Eur. J. Immunol. 1990; 20: 1409-1411Crossref PubMed Scopus (27) Google Scholar) using the PCR primers L10F1 (5′-CCG CCG ACT AGT CGT TAG CCC CTG TCC TTG-3′) and L10R1 (5′-CCG CCG ACT AGT TGT GCA TAT GTG TGT GTA CAC ATG-3′). For testing smaller segments of the 1-kb sequences, we PCR-amplified the desired regions, again adding externalSpeI sites for vector insertions as above using the primers L10F2 (5′-CCG CCG ATC AGT GAA GCC AGG GAA ATG CCA C-3′), L10R2 (5′-CCG CCG ATC AGT CTA GCT TTA CAG CTT GTC-3′), L10R3 (5′-CCG CCG ATC AGT GCT TAA GCA GCA GAC AGT G-3′), L10R4 (5′-CCG CCG ATC AGT GTG CCC TGC ACC TTC AGG-3′), and L10R5 (5′-CCG CCG ATC AGT GTG GCA TTT CCC TGG CTT C-3′). PCR conditions were 1 min at 94 °C, 30 s at 58 °C, and 75 s at 72 °C for 35 cycles. Finally, to replace the Vκ promoter with a 420-bp BglII/HindIII fragment containing the SV40 early enhancer/promoter from the pRL-SV40 vector (Promega), the Vκ promoter was deleted from PVκLuc by excision withNheI and HindIII and from PVκLuc containing the 3.6-kb silencer by HindIII digestion. Then the sticky ends of these two vectors were filled in with Klenow and dephosphorylated. The sticky ends of the SV40 early enhancer/promoter 420-bp fragment were also filled in with Klenow and ligated to the treated vectors to construct PSV40Luc and 3.6kbPSV40Luc. Cell lines were transiently transfected either in triplicate in the same batch or in duplicate in separate batches using either optimized DEAE dextran concentrations (100–250 μg/ml; 500 μg/ml for S194 cells) as previously described (58Scherrer D.C. Brockman J.A. Bendall H.H. Zhang G.M. Ballard D.W. Oltz E.M. Immunity. 1996; 5: 563-574Abstract Full Text PDF PubMed Scopus (56) Google Scholar), LipofectAMINE-Plus, or LipofectAMINE 2000 (Invitrogen). Typically, 106–107 cells and 1–2 μg of DNA were used per transfection, adjusted for insert sizes to provide equimolar comparisons, along with 20–50 ng of pRL-CMVRenilla luciferase reporter (Promega Corp). Pre-B and more mature cell lines were harvested 24 and 48 h post-transfection, respectively. The 24-h time chosen for pre-B cells allowed for optimum reproducibility of ±10 μg/ml lipopolysaccharide (LPS) comparisons (data not shown). Cell extracts were assayed for luciferase activity using Dual-Luciferase™ reporter assay systems (Promega) following the manufacturers' instructions. The Renilla luciferase activity was used for normalization of transfection efficiencies, except for the pre-B cell samples in Fig. 5, A andB, where extract protein levels were used. Data from a minimum of triplicate experiments are represented with error bars, whereas duplicate experiments are represented as means. Data were internally consistent between triplicates with the same batch of cells. Two-step PCR mutagenesis was used to create mutations of NFκB and E-box sites as described elsewhere (64Schanke J.T. Van Ness B.G. J. Immunol. 1994; 153: 4565-4572PubMed Google Scholar) in a 600-bp fragment amplified with L10F1 and L10R3 primers as described above. Sense and antisense primers for NFκB site mutation were 5′-GAA GTC AAA TTG GTT TCC ACT GTG CCA C-3′ and 5′-GAA ACC AAT TTG ACT TCA TTA CCT CAT G-3′; sense and antisense primers for first E-box mutation were 5′-CCT GCATTT TTG CAG TGC AGA TGG AC-3′ and 5′-CAC TGC AAA AAT GCA GGG CTG GAC TC-3′; sense and antisense primers for second E-box mutation were 5′-CAG TGC ATT TTG ACT TGG CAA AAG AAG-3′ and 5′-CAA GTCAAA ATG CAC TGC ACA GGT G-3′ (mutated bases are underlined). To locate new candidate regulatory elements within the mouse Igκ gene locus, we assayed for the presence of DNase I-hypersensitive sites in the chromatin of B cell lines representing different stages of lymphocyte differentiation. Such an approach has been proven to be successful previously and has contributed to the discovery and functional analyses of several enhancer elements in the mouse light and heavy chain Ig gene loci (16Blasquez V.C. Hale M.A. Trevorrow K.W. Garrard W.T. J. Biol. Chem. 1992; 267: 23888-23893Abstract Full Text PDF PubMed Google Scholar,17Roque M.A. Smith P.A. Blasquez V.C. Mol. Cell. Biol. 1996; 16: 3138-3155Crossref PubMed Google Scholar, 19O'Brien D.P. Oltz E.M. Van Ness B.G. Mol. Cell. Biol. 1997; 17: 3477-3787Crossref PubMed Google Scholar, 65Parslow T.G. Granner D.K. Nature. 1982; 299: 449-451Crossref PubMed Scopus (66) Google Scholar, 66Hagman J. Rudin C.M. Haasch D. Chaplin D. Storb U. 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