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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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Gene expression profiling strategy for the identification of genes regulated by Btk/Slp65-mediated pre-BCR signaling.(A) FACS sorting strategy for purification of pre-B cell fractions from the indicated mice on a VH81x transgenic Rag1−/− background. Lymphocytes were gated on the basis of forward/side scatter and B220+CD19+ pre-B cell fractions were sorted. Virtually all B220+CD19+ cells were cytoplasmic μ heavy chain positive [34], but showed genotype-dependent levels of expression of the CD2 differentiation marker, in agreement with previous findings [34]. (B) DNA microarray analysis of total mRNA from FACS-purified B220+CD19+ pre-B/pro-B cell fractions from the indicated mice. One-way ANOVA analysis (p = 0.01) identified 266 significantly different genes. MeV hierarchical clustering of gene expression differences are represented in the heatmap. (C) Validation of the expression of TFs implicated in Igκ gene rearrangement. Total mRNA isolated from FACS-sorted B220+CD19+ pre-B/pro-B cell fractions from the indicated mice was analyzed by quantitative RT-PCR for expression of TFs. Expression levels were normalized to those of Gapdh, whereby the values in WT pre-B cells were set to one. Bars represent mean values and error bars denote standard deviations for four independent mice per group.
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pbio-1001791-g001: Gene expression profiling strategy for the identification of genes regulated by Btk/Slp65-mediated pre-BCR signaling.(A) FACS sorting strategy for purification of pre-B cell fractions from the indicated mice on a VH81x transgenic Rag1−/− background. Lymphocytes were gated on the basis of forward/side scatter and B220+CD19+ pre-B cell fractions were sorted. Virtually all B220+CD19+ cells were cytoplasmic μ heavy chain positive [34], but showed genotype-dependent levels of expression of the CD2 differentiation marker, in agreement with previous findings [34]. (B) DNA microarray analysis of total mRNA from FACS-purified B220+CD19+ pre-B/pro-B cell fractions from the indicated mice. One-way ANOVA analysis (p = 0.01) identified 266 significantly different genes. MeV hierarchical clustering of gene expression differences are represented in the heatmap. (C) Validation of the expression of TFs implicated in Igκ gene rearrangement. Total mRNA isolated from FACS-sorted B220+CD19+ pre-B/pro-B cell fractions from the indicated mice was analyzed by quantitative RT-PCR for expression of TFs. Expression levels were normalized to those of Gapdh, whereby the values in WT pre-B cells were set to one. Bars represent mean values and error bars denote standard deviations for four independent mice per group.

Mentions: Whereas mice deficient for the pre-BCR signaling molecules Btk and Slp65 have a partial block at the pre-B cell stage [42],[43], in Btk/Slp65 double-deficient mice, only very few pre-B cells show progression to the immature B cell stage characterized by functional IgL chain gene recombination [44]. To enable analysis of the effects of pre-BCR signaling on (i) the expression of genes involved in Igκ gene rearrangement and on (ii) long-distance chromatin interactions in the Igκ locus in pre-B cells in the absence of Igκ gene recombination events, we bred Btk and Slp65 single- and double-deficient mice on the Rag1−/− background. In these mice, progression of B cell progenitors to the pre-B cell stage was conferred by the transgenic, functionally rearranged VH81x IgH μ chain, which ensures pre-BCR expression and cellular proliferation. The absence of functional Rag1 protein precludes IgL chain gene rearrangement and cells are completely arrested at the small pre-B cell stage (Figure 1A).


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)

Gene expression profiling strategy for the identification of genes regulated by Btk/Slp65-mediated pre-BCR signaling.(A) FACS sorting strategy for purification of pre-B cell fractions from the indicated mice on a VH81x transgenic Rag1−/− background. Lymphocytes were gated on the basis of forward/side scatter and B220+CD19+ pre-B cell fractions were sorted. Virtually all B220+CD19+ cells were cytoplasmic μ heavy chain positive [34], but showed genotype-dependent levels of expression of the CD2 differentiation marker, in agreement with previous findings [34]. (B) DNA microarray analysis of total mRNA from FACS-purified B220+CD19+ pre-B/pro-B cell fractions from the indicated mice. One-way ANOVA analysis (p = 0.01) identified 266 significantly different genes. MeV hierarchical clustering of gene expression differences are represented in the heatmap. (C) Validation of the expression of TFs implicated in Igκ gene rearrangement. Total mRNA isolated from FACS-sorted B220+CD19+ pre-B/pro-B cell fractions from the indicated mice was analyzed by quantitative RT-PCR for expression of TFs. Expression levels were normalized to those of Gapdh, whereby the values in WT pre-B cells were set to one. Bars represent mean values and error bars denote standard deviations for four independent mice per group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928034&req=5

pbio-1001791-g001: Gene expression profiling strategy for the identification of genes regulated by Btk/Slp65-mediated pre-BCR signaling.(A) FACS sorting strategy for purification of pre-B cell fractions from the indicated mice on a VH81x transgenic Rag1−/− background. Lymphocytes were gated on the basis of forward/side scatter and B220+CD19+ pre-B cell fractions were sorted. Virtually all B220+CD19+ cells were cytoplasmic μ heavy chain positive [34], but showed genotype-dependent levels of expression of the CD2 differentiation marker, in agreement with previous findings [34]. (B) DNA microarray analysis of total mRNA from FACS-purified B220+CD19+ pre-B/pro-B cell fractions from the indicated mice. One-way ANOVA analysis (p = 0.01) identified 266 significantly different genes. MeV hierarchical clustering of gene expression differences are represented in the heatmap. (C) Validation of the expression of TFs implicated in Igκ gene rearrangement. Total mRNA isolated from FACS-sorted B220+CD19+ pre-B/pro-B cell fractions from the indicated mice was analyzed by quantitative RT-PCR for expression of TFs. Expression levels were normalized to those of Gapdh, whereby the values in WT pre-B cells were set to one. Bars represent mean values and error bars denote standard deviations for four independent mice per group.
Mentions: Whereas mice deficient for the pre-BCR signaling molecules Btk and Slp65 have a partial block at the pre-B cell stage [42],[43], in Btk/Slp65 double-deficient mice, only very few pre-B cells show progression to the immature B cell stage characterized by functional IgL chain gene recombination [44]. To enable analysis of the effects of pre-BCR signaling on (i) the expression of genes involved in Igκ gene rearrangement and on (ii) long-distance chromatin interactions in the Igκ locus in pre-B cells in the absence of Igκ gene recombination events, we bred Btk and Slp65 single- and double-deficient mice on the Rag1−/− background. In these mice, progression of B cell progenitors to the pre-B cell stage was conferred by the transgenic, functionally rearranged VH81x IgH μ chain, which ensures pre-BCR expression and cellular proliferation. The absence of functional Rag1 protein precludes IgL chain gene rearrangement and cells are completely arrested at the small pre-B cell stage (Figure 1A).

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