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Pre-B cell receptor signaling induces immunoglobulin κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions.

Stadhouders R, de Bruijn MJ, Rother MB, Yuvaraj S, Ribeiro de Almeida C, Kolovos P, Van Zelm MC, van Ijcken W, Grosveld F, Soler E, Hendriks RW - PLoS Biol. (2014)

Bottom Line: Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells.Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a.We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Erasmus MC Rotterdam, The Netherlands.

ABSTRACT
During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

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Long-range chromatin interactions of κ regulatory elements correlate with TF binding and histone modifications.(A-D) For fragments within the Vκ region, average 3C-seq interaction frequencies were calculated for fragments that did (+) or did not (−) contain binding sites for TFs or H3K4 histone modifications (as determined by previous ChIP-Seq studies; see Materials and Methods for references). Data for the three viewpoint and the five B-cell precursor fractions representing a pre-BCR signaling gradient are shown for Ctcf (A), Ikaros (B), E2a (C), and H3K4 di- and tri-methylation (Me2/3). Statistical significance was determined using a Mann–Whitney U test (*p<0.05; **p<0.01; ***p<0.001; n.s., not significant, p≥0.05).
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pbio-1001791-g006: Long-range chromatin interactions of κ regulatory elements correlate with TF binding and histone modifications.(A-D) For fragments within the Vκ region, average 3C-seq interaction frequencies were calculated for fragments that did (+) or did not (−) contain binding sites for TFs or H3K4 histone modifications (as determined by previous ChIP-Seq studies; see Materials and Methods for references). Data for the three viewpoint and the five B-cell precursor fractions representing a pre-BCR signaling gradient are shown for Ctcf (A), Ikaros (B), E2a (C), and H3K4 di- and tri-methylation (Me2/3). Statistical significance was determined using a Mann–Whitney U test (*p<0.05; **p<0.01; ***p<0.001; n.s., not significant, p≥0.05).

Mentions: Remarkably, we found similar striking correlations between the presence of in vivo binding sites for each of these TFs (as determined by ChIP experiments; see Materials and Methods for the relevant references) and long-range chromatin interactions with the κ regulatory elements (Figure 6A–C), even though Ctcf sites are mostly located in between Vκ genes [21] and Ikaros/E2a sites were frequently found close to Vκ gene promoter regions ([2]; Figure 7A). Even when pre-BCR signaling was absent (Rag1−/− pro B cells) or very low (Btk−/−Slp65−/− pre-B cells), the average interaction frequencies of the κ regulatory elements with fragments containing Ctcf, Ikaros, or E2a bindings sites were higher than those without binding sites. Irrespective of the presence or absence of bindings sites for these TFs, we found that upon pre-BCR signaling interaction frequencies with the Sis element increased and those with the iEκ did not change. In contrast, for the 3′Eκ we found that pre-BCR signaling specifically increased interaction frequencies with fragments occupied by Ctcf, Ikaros, or E2a.


Pre-B cell receptor signaling induces immunoglobulin κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions.

Stadhouders R, de Bruijn MJ, Rother MB, Yuvaraj S, Ribeiro de Almeida C, Kolovos P, Van Zelm MC, van Ijcken W, Grosveld F, Soler E, Hendriks RW - PLoS Biol. (2014)

Long-range chromatin interactions of κ regulatory elements correlate with TF binding and histone modifications.(A-D) For fragments within the Vκ region, average 3C-seq interaction frequencies were calculated for fragments that did (+) or did not (−) contain binding sites for TFs or H3K4 histone modifications (as determined by previous ChIP-Seq studies; see Materials and Methods for references). Data for the three viewpoint and the five B-cell precursor fractions representing a pre-BCR signaling gradient are shown for Ctcf (A), Ikaros (B), E2a (C), and H3K4 di- and tri-methylation (Me2/3). Statistical significance was determined using a Mann–Whitney U test (*p<0.05; **p<0.01; ***p<0.001; n.s., not significant, p≥0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928034&req=5

pbio-1001791-g006: Long-range chromatin interactions of κ regulatory elements correlate with TF binding and histone modifications.(A-D) For fragments within the Vκ region, average 3C-seq interaction frequencies were calculated for fragments that did (+) or did not (−) contain binding sites for TFs or H3K4 histone modifications (as determined by previous ChIP-Seq studies; see Materials and Methods for references). Data for the three viewpoint and the five B-cell precursor fractions representing a pre-BCR signaling gradient are shown for Ctcf (A), Ikaros (B), E2a (C), and H3K4 di- and tri-methylation (Me2/3). Statistical significance was determined using a Mann–Whitney U test (*p<0.05; **p<0.01; ***p<0.001; n.s., not significant, p≥0.05).
Mentions: Remarkably, we found similar striking correlations between the presence of in vivo binding sites for each of these TFs (as determined by ChIP experiments; see Materials and Methods for the relevant references) and long-range chromatin interactions with the κ regulatory elements (Figure 6A–C), even though Ctcf sites are mostly located in between Vκ genes [21] and Ikaros/E2a sites were frequently found close to Vκ gene promoter regions ([2]; Figure 7A). Even when pre-BCR signaling was absent (Rag1−/− pro B cells) or very low (Btk−/−Slp65−/− pre-B cells), the average interaction frequencies of the κ regulatory elements with fragments containing Ctcf, Ikaros, or E2a bindings sites were higher than those without binding sites. Irrespective of the presence or absence of bindings sites for these TFs, we found that upon pre-BCR signaling interaction frequencies with the Sis element increased and those with the iEκ did not change. In contrast, for the 3′Eκ we found that pre-BCR signaling specifically increased interaction frequencies with fragments occupied by Ctcf, Ikaros, or E2a.

Bottom Line: Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells.Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a.We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Erasmus MC Rotterdam, The Netherlands.

ABSTRACT
During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

Show MeSH
Related in: MedlinePlus