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S region sequence, RNA polymerase II, and histone modifications create chromatin accessibility during class switch recombination.

Wang L, Wuerffel R, Feldman S, Khamlichi AA, Kenter AL - J. Exp. Med. (2009)

Bottom Line: We show that S and C(H) regions are dynamically modified with histone marks that are associated with active and repressed chromatin states, respectively.We propose that RNAP II enrichment facilitates recruitment of histone modifiers to generate accessibility.Thus, the histone methylation pattern produced by transcription localizes accessible chromatin to S regions, thereby focusing AID attack.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Immunoglobulin class switch recombination is governed by long-range interactions between enhancers and germline transcript promoters to activate transcription and modulate chromatin accessibility to activation-induced cytidine deaminase (AID). However, mechanisms leading to the differential targeting of AID to switch (S) regions but not to constant (C(H)) regions remain unclear. We show that S and C(H) regions are dynamically modified with histone marks that are associated with active and repressed chromatin states, respectively. Chromatin accessibility is superimposable with the activating histone modifications, which extend throughout S regions irrespective of length. High density elongating RNA polymerase II (RNAP II) is detected in S regions, suggesting that the transcription machinery has paused and stalling is abolished by deletion of the S region. We propose that RNAP II enrichment facilitates recruitment of histone modifiers to generate accessibility. Thus, the histone methylation pattern produced by transcription localizes accessible chromatin to S regions, thereby focusing AID attack.

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RNAP II p-ser5 is enriched in the Sμ and Sγ3 regions of activated B cells. B cells were stimulated with LPS for 48 h and analyzed in ChIP assays using antisera against total RNAP II or RNAP II CTD p-ser5, as indicated. Primer pairs used in the ChIP analyses are named below the histograms, and their positions in the μ and γ3 I-S-CH loci are shown as arrows above the schematics. In the locus diagrams, the vertical lines indicate Hind III (H) sites. (A) B cells were unstimulated or activated, and B cell nuclei were analyzed in ChIP assays at the μ I-S-CH locus by qPCR. Two to seven ChIP samples from each culture condition were derived from two independent experiments and were analyzed in duplicate and averaged, and SEMs are shown. (B) LPS-stimulated B cells were analyzed in ChIP assays at the γ3 I-S-CH locus. Five ChIP samples from three independent experiments were analyzed. Samples from the RNAP II p-ser5 (H14) ChIP were concentrated sixfold and were analyzed in duplicate and averaged, and SEMs are shown. ND, not done.
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fig6: RNAP II p-ser5 is enriched in the Sμ and Sγ3 regions of activated B cells. B cells were stimulated with LPS for 48 h and analyzed in ChIP assays using antisera against total RNAP II or RNAP II CTD p-ser5, as indicated. Primer pairs used in the ChIP analyses are named below the histograms, and their positions in the μ and γ3 I-S-CH loci are shown as arrows above the schematics. In the locus diagrams, the vertical lines indicate Hind III (H) sites. (A) B cells were unstimulated or activated, and B cell nuclei were analyzed in ChIP assays at the μ I-S-CH locus by qPCR. Two to seven ChIP samples from each culture condition were derived from two independent experiments and were analyzed in duplicate and averaged, and SEMs are shown. (B) LPS-stimulated B cells were analyzed in ChIP assays at the γ3 I-S-CH locus. Five ChIP samples from three independent experiments were analyzed. Samples from the RNAP II p-ser5 (H14) ChIP were concentrated sixfold and were analyzed in duplicate and averaged, and SEMs are shown. ND, not done.

Mentions: In yeast, the H3K4 histone methyltransferase (HMT) binds to initiating RNAP II p-ser 5 and introduces H3K4me3 into promoter-proximal regions (Hampsey and Reinberg, 2003). Genome-wide studies in humans, mice, and yeast confirm the distribution of H3K4me3 to the promoter-proximal areas, suggesting that this mark is targeted to chromatin via a conserved mechanism involving the RNAP II p-ser5 isoform (Bernstein et al., 2005; Pokholok et al., 2005). These observations imply that the extended distribution of H3K4me3 in S regions might derive from high occupancy RNAP II p-ser5. ChIP has been used to determine RNAP II association at specific promoters and transcribed regions and on a genome-wide scale (Muse et al., 2007; Guenther et al., 2007; Zeitlinger et al., 2007). A ChIP signal exclusively at the promoter indicates that RNAP II is poised at this site, whereas if the ChIP signal is found at short distances downstream of the transcription start sites (TSSs) then RNAP II is paused in early elongation (Saunders et al., 2006; for review see Wade and Struhl, 2008). When the transition from transcription initiation to elongation is rapid, RNAP II occupancy will be roughly equivalent across the gene. Alternatively, if the transition is slow then RNAP II levels will be higher at the promoter than in downstream coding regions. To examine this issue in S regions, ChIP assays focused on μ and γ3 I-S-CH regions were performed using an antibody against total RNAP II or RNAP II p-ser 5 (H14) detected during the early stages of elongation in B cells. The Sμ tandem repeats are located between Sμ-U and Sμ-D.2 sites, whereas Sμ-D is located ∼600 bp downstream and outside the repetitive zone (Fig. 2). There are three documented TSSs for μ GLT, one immediately upstream of the Iμ exon and two that are located 5′ of Sμ (Fig. 6 A; Alt et al., 1982; Nelson et al., 1983; Kuzin et al., 2000). Before B cell activation, relatively low levels of RNAP II were found throughout the μ I-S-CH locus, consistent with the constitutive expression of the μ GLT (Fig. 6 A) and suggesting rapid transition of RNAP II from transcription initiation to elongation, as has been previously described for some genes (Zeitlinger et al., 2007). After B cell activation, RNAP II occupancy increased across the Iμ-Sμ-Cμ transcription unit, with the greatest gains at the 3′ end of the Sμ tandem repeats, whereas the sites adjacent to Sμ were relatively depleted of RNAP II (Fig. 6 A). The high occupancy of RNAP II p-ser5 at the 3′ end of Sμ occurs at least 3.2 kb downstream of the TSSs and, therefore, may occur through a unique mechanism that is distinct from the pause associated with the transition from promoter-proximal transcription initiation to elongation. Increased RNAP II occupancy at Sμ (Fig. 6 A) with little concomitant change in μ GLT expression (Wang et al., 2006) could occur from increased transcription initiation that rapidly moves off the promoter but stalls in the S region and never completes the μ GLT. Alternatively, transcription initiation at alternative start sites (Fig. 6 A) might occur, thereby increasing transcription but not changing the level of the classical μ GLT. Finally, antisense RNA transcription initiating within the S region (Perlot et al., 2008) might lead to accumulation of RNAP II in Sμ DNA.


S region sequence, RNA polymerase II, and histone modifications create chromatin accessibility during class switch recombination.

Wang L, Wuerffel R, Feldman S, Khamlichi AA, Kenter AL - J. Exp. Med. (2009)

RNAP II p-ser5 is enriched in the Sμ and Sγ3 regions of activated B cells. B cells were stimulated with LPS for 48 h and analyzed in ChIP assays using antisera against total RNAP II or RNAP II CTD p-ser5, as indicated. Primer pairs used in the ChIP analyses are named below the histograms, and their positions in the μ and γ3 I-S-CH loci are shown as arrows above the schematics. In the locus diagrams, the vertical lines indicate Hind III (H) sites. (A) B cells were unstimulated or activated, and B cell nuclei were analyzed in ChIP assays at the μ I-S-CH locus by qPCR. Two to seven ChIP samples from each culture condition were derived from two independent experiments and were analyzed in duplicate and averaged, and SEMs are shown. (B) LPS-stimulated B cells were analyzed in ChIP assays at the γ3 I-S-CH locus. Five ChIP samples from three independent experiments were analyzed. Samples from the RNAP II p-ser5 (H14) ChIP were concentrated sixfold and were analyzed in duplicate and averaged, and SEMs are shown. ND, not done.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig6: RNAP II p-ser5 is enriched in the Sμ and Sγ3 regions of activated B cells. B cells were stimulated with LPS for 48 h and analyzed in ChIP assays using antisera against total RNAP II or RNAP II CTD p-ser5, as indicated. Primer pairs used in the ChIP analyses are named below the histograms, and their positions in the μ and γ3 I-S-CH loci are shown as arrows above the schematics. In the locus diagrams, the vertical lines indicate Hind III (H) sites. (A) B cells were unstimulated or activated, and B cell nuclei were analyzed in ChIP assays at the μ I-S-CH locus by qPCR. Two to seven ChIP samples from each culture condition were derived from two independent experiments and were analyzed in duplicate and averaged, and SEMs are shown. (B) LPS-stimulated B cells were analyzed in ChIP assays at the γ3 I-S-CH locus. Five ChIP samples from three independent experiments were analyzed. Samples from the RNAP II p-ser5 (H14) ChIP were concentrated sixfold and were analyzed in duplicate and averaged, and SEMs are shown. ND, not done.
Mentions: In yeast, the H3K4 histone methyltransferase (HMT) binds to initiating RNAP II p-ser 5 and introduces H3K4me3 into promoter-proximal regions (Hampsey and Reinberg, 2003). Genome-wide studies in humans, mice, and yeast confirm the distribution of H3K4me3 to the promoter-proximal areas, suggesting that this mark is targeted to chromatin via a conserved mechanism involving the RNAP II p-ser5 isoform (Bernstein et al., 2005; Pokholok et al., 2005). These observations imply that the extended distribution of H3K4me3 in S regions might derive from high occupancy RNAP II p-ser5. ChIP has been used to determine RNAP II association at specific promoters and transcribed regions and on a genome-wide scale (Muse et al., 2007; Guenther et al., 2007; Zeitlinger et al., 2007). A ChIP signal exclusively at the promoter indicates that RNAP II is poised at this site, whereas if the ChIP signal is found at short distances downstream of the transcription start sites (TSSs) then RNAP II is paused in early elongation (Saunders et al., 2006; for review see Wade and Struhl, 2008). When the transition from transcription initiation to elongation is rapid, RNAP II occupancy will be roughly equivalent across the gene. Alternatively, if the transition is slow then RNAP II levels will be higher at the promoter than in downstream coding regions. To examine this issue in S regions, ChIP assays focused on μ and γ3 I-S-CH regions were performed using an antibody against total RNAP II or RNAP II p-ser 5 (H14) detected during the early stages of elongation in B cells. The Sμ tandem repeats are located between Sμ-U and Sμ-D.2 sites, whereas Sμ-D is located ∼600 bp downstream and outside the repetitive zone (Fig. 2). There are three documented TSSs for μ GLT, one immediately upstream of the Iμ exon and two that are located 5′ of Sμ (Fig. 6 A; Alt et al., 1982; Nelson et al., 1983; Kuzin et al., 2000). Before B cell activation, relatively low levels of RNAP II were found throughout the μ I-S-CH locus, consistent with the constitutive expression of the μ GLT (Fig. 6 A) and suggesting rapid transition of RNAP II from transcription initiation to elongation, as has been previously described for some genes (Zeitlinger et al., 2007). After B cell activation, RNAP II occupancy increased across the Iμ-Sμ-Cμ transcription unit, with the greatest gains at the 3′ end of the Sμ tandem repeats, whereas the sites adjacent to Sμ were relatively depleted of RNAP II (Fig. 6 A). The high occupancy of RNAP II p-ser5 at the 3′ end of Sμ occurs at least 3.2 kb downstream of the TSSs and, therefore, may occur through a unique mechanism that is distinct from the pause associated with the transition from promoter-proximal transcription initiation to elongation. Increased RNAP II occupancy at Sμ (Fig. 6 A) with little concomitant change in μ GLT expression (Wang et al., 2006) could occur from increased transcription initiation that rapidly moves off the promoter but stalls in the S region and never completes the μ GLT. Alternatively, transcription initiation at alternative start sites (Fig. 6 A) might occur, thereby increasing transcription but not changing the level of the classical μ GLT. Finally, antisense RNA transcription initiating within the S region (Perlot et al., 2008) might lead to accumulation of RNAP II in Sμ DNA.

Bottom Line: We show that S and C(H) regions are dynamically modified with histone marks that are associated with active and repressed chromatin states, respectively.We propose that RNAP II enrichment facilitates recruitment of histone modifiers to generate accessibility.Thus, the histone methylation pattern produced by transcription localizes accessible chromatin to S regions, thereby focusing AID attack.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Immunoglobulin class switch recombination is governed by long-range interactions between enhancers and germline transcript promoters to activate transcription and modulate chromatin accessibility to activation-induced cytidine deaminase (AID). However, mechanisms leading to the differential targeting of AID to switch (S) regions but not to constant (C(H)) regions remain unclear. We show that S and C(H) regions are dynamically modified with histone marks that are associated with active and repressed chromatin states, respectively. Chromatin accessibility is superimposable with the activating histone modifications, which extend throughout S regions irrespective of length. High density elongating RNA polymerase II (RNAP II) is detected in S regions, suggesting that the transcription machinery has paused and stalling is abolished by deletion of the S region. We propose that RNAP II enrichment facilitates recruitment of histone modifiers to generate accessibility. Thus, the histone methylation pattern produced by transcription localizes accessible chromatin to S regions, thereby focusing AID attack.

Show MeSH
Related in: MedlinePlus