Volume 27, Issue 5, Pages 842-850 (September 2007) Repeat Organization and Epigenetic Regulation of the DH-Cμ Domain of the Immunoglobulin Heavy-Chain Gene Locus  Tirtha Chakraborty, Dipanjan Chowdhury, Amanda Keyes, Anant Jani, Ramesh Subrahmanyam, Irina Ivanova, Ranjan Sen  Molecular Cell  Volume 27, Issue 5, Pages 842-850 (September 2007) DOI: 10.1016/j.molcel.2007.07.010 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Histone Modification State in the DH-Cμ Domain of the Immunoglobulin Heavy-Chain Gene Locus The top line shows a schematic of the DH-Cμ region of the immunoglobulin locus in C57/BL6 mice. The DH segments are represented as brown boxes and parentheses with subscript 6 signifying the presence of six DSP2 gene segments in this strain. The intronic enhancer Eμ is shown as an oval. Approximate locations of primer pairs used in chromatin immunoprecipitation studies (ChIP) are indicated by short black lines below the schematic. Numbers in brackets represent positions of each amplicon (in kb) relative to the closest DH gene segment; negative numbers refer to positions 5′ of the DH gene segment. ChIP assays used antibodies directed against the indicated modifications (H3K9ac, [A] and [B]) and (H3K9me2, [C] and [D]). (A) and (C) show data using cultured cell lines: RAG−/−, pro-B cell line from RAG2-deficient mice; 2017, Moloney virus transformed pro-T cell line; and MEL, mouse erythroleukemia cells. (B) and (D) utilized primary CD19+ pro-B cells and CD4−CD8− thymocytes isolated from RAG2-deficient mice. Equal amounts of coimmunoprecipitated DNA were analyzed by quantitative (real-time) PCR using indicated primer pairs. The y axis represents the abundance of each amplicon in the immunoprecipitate relative to input, calculated as described in the Experimental Procedures section. Data shown are the average of three independent chromatin preparations from each cell type, or cell line, using at least two different antibody preparations. Error bars represent standard deviation of the results. Molecular Cell 2007 27, 842-850DOI: (10.1016/j.molcel.2007.07.010) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Sterile Transcription in the DH-Cμ Region Total RNA from RAG−/− and 2017 cell lines (A) or primary CD19+ pro-B cells and thymocytes from RAG-deficient mice (B) was reverse transcribed with random hexamer primers and assayed by quantitative PCR using Taqman probes as described in the Experimental Procedures Section. No RT controls were done with each sample (Figures S4 and S5). Direction of DH-associated transcripts was determined using strand-specific oligonucleotides to prime reverse transcription reactions with RNA obtained from RAG-deficient cell line (C) or primary RAG−/− CD19+ bone marrow cells (D). cDNA amplification and quantitation were carried out as described above. The proportions of transcripts in the sense (blue) and antisense (red) directions were calculated as described in the Experimental Procedures section and expressed as a percentage of the average total transcript shown in (A) and (B). Data shown were obtained from three preparations of RNA analyzed in triplicate with each primer pair. Error bars represent the standard deviation between experiments. Molecular Cell 2007 27, 842-850DOI: (10.1016/j.molcel.2007.07.010) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Effect of Trichostatin A Treatment on DH Region Histone Modifications and Transcription RAG−/− pro-B cells and 2017 pro-T cells treated with trichostatin A (TSA) for 22 hr, or untreated parallel cultures, were used for ChIP with anti-H3K9ac (A) or anti-H4ac (B) antibodies. Primer nomenclature is as described in the Figure 1 legend. The data presented are normalized to the relative abundance for each amplicon in the immunoprecipitate in the absence of TSA, which is assigned the value 1 (indicated by the dotted red line). Values greater than 1 signify increased proportion of the amplicon in the immunoprecipitate from TSA-treated samples; conversely, values less than 1 indicate decreased proportion of the amplicon in TSA-treated samples. The absolute values from which the normalized numbers were obtained are shown in Figures S6 and S7. Amplicons from β-globin and β2-microglobulin (β2m) genes served as controls for inactive and active loci, respectively. (C) Total RNA isolated from TSA-treated and untreated RAG−/− cells was converted to cDNA using random hexamers and RT, followed by real-time PCR analysis with the indicated primer pairs. Data shown are the average of three independent experiments. Error bars represent the standard deviation between experiments. Molecular Cell 2007 27, 842-850DOI: (10.1016/j.molcel.2007.07.010) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Transcription Analysis of DJH Recombined Alleles Transcription analyses were carried out on DJH recombined cell lines (solid bars) and in RAG parental cells (hatched bars). The recombination status of selected cell lines is as follows: 2C10, DFL16.1 to JH1, second allele missing; 2B9, DSP2.2(2) to JH1, second allele missing; and 3F3, DSP2.11 to JH3, second allele germline. Levels and directionality of transcripts upstream of the DJH recombination site are shown in (A), (C), and (E). Specific primer pairs were designed for each cell line and used to amplify cDNA generated using random hexamers or strand-specific oligonucleotides. The proportion of sense versus antisense transcripts was obtained as described in Figure 3. Sense-directed transcripts are in blue, and antisense-directed transcripts are in red. The data presented are derived from at least three preparations of RNA from each cell line, with error bars representing standard deviations between experiments. Flags represent regions with proposed bidirectional promoter activity as described in the text. ChIP assays were carried out using anti-Pol II antibody with each cell line as indicated ([B], [D], and [F]). Coprecipitated DNA was analyzed using real-time PCR with the indicated primer sets. The data are presented as relative abundance of each amplicon (y axis) in the immunoprecipitate relative to input and represent the average of three independent experiments. Error bars indicate the standard deviation between experiments. Molecular Cell 2007 27, 842-850DOI: (10.1016/j.molcel.2007.07.010) Copyright © 2007 Elsevier Inc. Terms and Conditions