title: 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.
abstract: 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.
authors.affiliation: Immunology Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Inka
authors.initials: I
authors.lastname: Pawlitzky
authors.forename: Christina V
authors.initials: CV
authors.lastname: Angeles
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Siegel
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Stanton
authors.forename: Roy
authors.initials: R
authors.lastname: Riblet
authors.forename: Peter H
authors.initials: PH
authors.lastname: Brodeur
mesh_terms: D024506:5' Flanking Region / Q000276:immunology*; D000818:Animals; D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D003850:Deoxyribonuclease I / Q000378:metabolism*; D018985:Epitopes, B-Lymphocyte / Q000235:genetics* / Q000378:metabolism; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005819:Genetic Markers; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics / Q000276:immunology; D051757:PAX5 Transcription Factor / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D021381:Protein Transport / Q000235:genetics / Q000276:immunology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000276:immunology*; D013234:Stem Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D003850:Deoxyribonuclease I; D018985:Epitopes, B-Lymphocyte; D005819:Genetic Markers; D007143:Immunoglobulin Heavy Chains; D015514:Oncogene Proteins, Fusion; D051757:PAX5 Transcription Factor; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C489626:E2a-Hlf fusion protein, mouse; C497282:Pax5 protein, mouse