1. Volume 34, Issue 2, Pages 175-187 (February 2011)
The Distal VH Gene Cluster of the Igh Locus Contains Distinct Regulatory Elements with Pax5 Transcription Factor-Dependent Activity in Pro-B Cells 
Anja Ebert, Shane McManus, Hiromi Tagoh, Jasna Medvedovic, Giorgia Salvagiotto, Maria Novatchkova, Ido Tamir, Andreas Sommer, Markus Jaritz, Meinrad Busslinger 
Immunity 
Volume 34, Issue 2, Pages (February 2011)
DOI: /j.immuni
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2. Immunity 2011 34, 175-187DOI: (10.1016/j.immuni.2011.02.005)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
Active Chromatin Regions in the Distal VH Gene Cluster of the Igh Locus
(A) Schematic diagram of the Igh locus indicating the positions of three PAIR elements (indicated by arrows) together with the location of VH, DH, JH and CH gene segments and the Eμ enhancer. Different colors indicate members of distinct VH gene families, of which the VHJ558 (black) and VH3609 (pink) genes are most relevant for this study.
(B–D) ChIP-chip mapping of histone marks (H3K9ac, H3K4me2, H3K4me3) and transcription factor-binding sites for Pax5 and CTCF at VH3609 genes and PAIR elements. Short-term cultured Pax5+/+ (+/+) and Pax5−/− (–/–) pro-B cells on a Rag2-deficient background were used for ChIP-chip analysis with antibodies recognizing Pax5, CTCF, and the indicated histone modifications. Pax5-binding sites were additionally identified by Bio-ChIP-chip analysis of Pax5Bio/Bio Rag2−/− pro-B cells. The ChIP-chip results obtained with wild-type (Pax5+/+ and Pax5Bio/Bio) and Pax5 mutant (Pax5−/−) pro-B cells are shown in black (+/+) and red (–/–) colors, respectively. The logarithmic ratio (log2) of the hybridization intensities between precipitated and input DNA (bound/input) is shown as a vertical bar for each oligonucleotide on the microarray. Blue bars below the ChIP tracks identify significant Pax5 and CTCF peaks that reached values above the threshold of the peak-finder analysis. Gray shading highlights Pax5-binding sites in active chromatin regions. The results shown are representative of two ChIP-chip experiments, which were performed with independently prepared DNA probes from each pro-B cell type. The scale bar is shown in kilobases (kb). The mm8 sequence coordinates for the displayed regions of the Igh locus on chromosome 12 are 116,520,000–113,662,000 (A); 115,925,500–115,914,500 (B); 115,861,000–115,850,000 (C); and 115,715,700–115,703,000 (D). Figures S1I–S1K show the ChIP-chip data for three additional PAIR elements.
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4. Figure 2 Sequence Conservation of the PAIR Elements
A circular diagram of the mouse Igh locus indicates the positions of the VH, DH, JH, and CH gene segments, Eμ enhancer, Zfp386 gene, and 3′ regulatory region (3′ RR). The same color code described in Figure 1A is used for indicating members of the different VH gene families. The inner concentric tracks show the location of all VH3609 genes (pink), the binding sites for Pax5 (purple), and CTCF (blue), as well as the 14 PAIRs (red). All pairwise segmental similarities with a minimal length of 1500 bp and minimal identity of 75% are indicated as gray Bézier curves in the center. The red curves denote the repeat elements containing the PAIR sequences as shown and defined in Figure S2A.
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5. Figure 3 In Vivo Binding of Pax5, E2A, and CTCF to PAIR Elements
(A) In vivo footprinting of PAIR elements 4, 6, and 7. DMS footprinting was performed with bone marrow-derived macrophages (MØ), in vitro-cultured Rag2−/− and Pax5−/− pro-B cells. DMS-modified and piperidine-cleaved DNA was amplified by ligation-mediated PCR (LM-PCR) with PAIR-specific primers (Table S1). The G sequencing ladder of purified DNA (G) served as a control. G-residues with DMS protection (open circles) or enhanced DMS reactivity (closed circles) are shown to the right together with the extent of the binding sites for CTCF, E2A, and Pax5, which were mapped on the upper (CTCF and E2A) or lower (Pax5) strand, respectively.
(B) Summary of the DMS footprint results. The protected G-residues are shown above the CTCF, E2A, and Pax5 recognition sequences (bold face) of PAIR elements 4, 6, and 7.
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6. Figure 4 CTCF, Pax5, and E2A Bind to Conserved PAIR Sequences
(A) Sequence conservation. Nucleotides of PAIR2 with homologies to the consensus sequence of CTCF (Cuddapah et al., 2009), Pax5 (Cobaleda et al., 2007), or E2A (TRANSFAC Database) are indicated in red, and dots denote identical nucleotides in other PAIRs. An empty box indicates the absence of a G-residue in PAIR6, and arrowheads denote the insertion of an A-residue in PAIR8 and PAIR13.
(B and C) High-affinity binding of E2A (B) and Pax5 (C) to PAIR elements. A double-stranded oligonucleotide containing the E2A or Pax5 recognition sequence of PAIR7 was used as a probe for electrophoretic mobility shift assay (EMSA) with a nuclear extract of in vitro-cultured 70Z/3 pre-B cells (B) or wild-type pro-B cells (C), respectively. The presence of an E2A antibody (Ab) resulted in a supershift of the E2A-DNA complex (mark by an asterisk), whereas the addition of a Pax5 antibody prevented Pax5 binding. Double-stranded oligonucleotides containing the E2A- and Pax5-binding sites of other PAIRs were analyzed at 10-, 30-, and 100-fold molar excess for competition of protein binding. The PAIR7M oligonucleotide contained a 3-bp substitution in the Pax5-binding site (A). Recognition sequences of the human Cd19 promoter (Kozmik et al., 1992) or the mouse Igh Eμ (μE5) and Igk iEκ (κE2) enhancers (Murre et al., 1989) served as reference high-affinity binding sites for Pax5 and E2A, respectively. The E2A probe detected two non-specific (ns) proteins. All oligonucleotides used are listed in Table S2.
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7. Figure 5
Pax5-Dependent Expression of Antisense RNA from PAIRs in Pro-B Cells
(A) Mapping of the RIKEN cDNA transcript CJ to Igh sequences upstream of PAIR4.
(B) Polyadenylation of the spliced antisense transcripts originating from PAIR elements 4, 6, 7, and 12. PolyA+ RNA was isolated from Rag2−/− pro-B cells and analyzed by RT-PCR with primers designed in exons 1 and 2 of the antisense RNA transcribed from PAIR elements 4, 6, and 7 (Table S3). Unspliced (u) and spliced (s) transcripts are indicated. The reverse transcriptase (RT) was omitted in the control reaction (–). Sequencing of 122 PCR products obtained with the PAIR7 primers demonstrated that the PCR fragments amplified from the spliced antisense transcripts originated from either PAIR7 (57%) or PAIR12 (43%).
(C) Developmental regulation and Pax5 dependence of antisense RNA transcribed from the PAIR elements 4, 6, 7, and 12. Total RNA was isolated from short-term cultured Pax5−/− Rag2−/− and Rag2−/− pro-B cells as well as from sorted wild-type small pre-B cells (CD19+ CD25+c-Kit−IgM−) and immature B cells (CD19+IgM+CD25−c-Kit−) of the bone marrow and DP T cells (CD4+CD8+) of the thymus. The same PAIR-specific primers were used for semiquantitative RT-PCR analysis of 5-fold serially diluted cDNA obtained from total RNA of the different cell types. Hprt (encoding hypoxanthine guanine phosphoribosyl transferase) served as a loading control.
(D) Transient transfection assay. The luciferase reporter genes (schematically shown to the right) were transfected together with the control vector pRL-CMV into the mouse pro-B cell line 38B9 (Alt et al., 1984). After 24 hr, luciferase activities were measured, normalized, and displayed relative to the activity of the parental vector Luc-S or XPG. Three independent transfection experiments were used for determining the relative luciferase values and their standard deviations. Luc-S (Czerny and Busslinger, 1995) contains of a minimal β-globin promoter in contrast to the promoter-less luciferase construct XPG (Bert et al., 2000). An asterisk (P∗) denotes mutation of the Pax5-binding site (TGA to ACT; Figure 4A), and Ex1 refers to exon 1 of the antisense transcript.
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8. Figure 6
Loss of Pax5 Binding at PAIR Elements in Small Pre-B and Mature B Cells
(A) In vivo footprinting of PAIR elements 4, 6, and 7. DMS footprinting was performed with total thymocytes (T), in vitro-cultured wild-type pro-B cells and sorted bone marrow pre-B cells (CD19+CD25+c-Kit−IgM−), as described in the legends of Figure 3. The G sequencing ladder of naked DNA (G) served as a control. G-residues with DMS protection (open circles) or enhanced reactivity (closed circles) are shown to the right together with the extent of the binding sites for CTCF, E2A, and Pax5, which were mapped on the upper strand (CTCF and E2A) or lower strand (Pax5), respectively.
(B) Bio-ChIP analysis. Short-term cultured Pax5Bio/Bio Rag2−/− pro-B cells from the bone marrow and MACS-sorted mature B cells (B220+) from lymph nodes of Pax5Bio/Bio mice were used for streptavidin-mediated Bio-ChIP analysis. Input and precipitated DNA were quantified by real-time PCR with primer pairs derived from PAIR4 or PAIR7 (Figure S2A and Table S4), and the amount of precipitated DNA is shown as percentage relative to input DNA together with its standard deviation as determined in two independent experiments. The Pax5 target gene Blnk served as a positive control. Mock refers to the use of protein A-coupled Dynabeads instead of streptavidin beads for ChIP analysis. Cloning and sequencing of 83 PCR fragments obtained with the PAIR4 primers revealed the relative proportion of PAIR4 (48%), PAIR6 (40%), PAIR11 (8.4%), and other (3.6%) sequences in the PCR-amplified DNA from Pax5Bio/Bio Rag2−/− pro-B cells. Similarly, sequencing of 81 PCR fragments obtained with the PAIR7 primers determined the relative abundance of PAIR2 (18%), PAIR7 (31%), and PAIR12 (51%) sequences in the PCR-amplified DNA.
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9. Figure 7 Preferential Binding of CTCF to PAIR Elements in Pro-B Cells
Short-term cultured Rag2−/− pro-B cells from the bone marrow, mature (mat) B cells (depleted of Lin+ non-B cells) from lymph nodes and DP T cells (CD4+CD8+) from the thymus were used for ChIP analysis with a CTCF antibody combined with paired-end Solexa sequencing with a read length of 76 nucleotides. Rag2−/− pro-B cells were additionally analyzed by ChIP sequencing with a Rad21 antibody. The genome-wide analysis of CTCF-binding sites in the three different cell types is presented in Figure S4, whereas the location of the identified CTCF- and Rad21-binding sites within the Igh locus is shown in Figure S5A.
(A) Similar pattern and intensity of the CTCF and Rad21 peaks in the 3′ regulatory region (3′ RR) among all three cell types. The similar intensity of the CTCF peaks in the 3′ RR was used for normalizing the ChIP-sequencing data by keeping the same relative proportion of the scales among the different cell types for presenting the CTCF-binding sites in (B) and (C).
(B) Same CTCF-binding pattern in the proximal VH gene region between the three cell types. The intensity of the CTCF-binding sites is lower in mature B cells, as the proximal VH gene region is deleted on all Igh alleles that have undergone distal or central VH gene recombination.
(C) Preferential binding of CTCF and Rad21 to PAIR elements in pro-B cells. Orange boxes with vertical lines indicate the position of the PAIRs with their CTCF-binding motif.
Immunity  , DOI: ( /j.immuni )
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