Localized DNA Demethylation at Recombination Intermediates during Immunoglobulin Heavy Chain Gene Assembly

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (DH) and joining (JH) gene segments were methylated, DJH junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eμ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination

IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme

<p>Abstract</p> <p>Background</p> <p>SWI/SNF chromatin remodeling enzymes play a critical role in the development of T helper lymphocytes, including Th2 cells, and directly program chromatin structure at Th2 cytokine genes. Different versions of SWI/SNF complexes, including BAF and PBAF, have been described based on unique subunit composition. However, the relative role of BAF and PBAF in Th cell function and cytokine expression has not been reported.</p> <p>Results</p> <p>Here we examine the role of the PBAF SWI/SNF complex in Th cell development and gene expression using mice deficient for a PBAF-specific component, BAF180. We find that T cell development in the thymus and lymphoid periphery is largely normal when the BAF180 gene is deleted late in thymic development. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus.</p> <p>Conclusions</p> <p>These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription.</p

Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

<p>Abstract</p> <p>Background</p> <p>Non-coding DNA in and around the human Amyloid Precursor Protein (APP) gene that is central to Alzheimer’s disease (AD) shares little sequence similarity with that of <it>appb</it> in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish <it>appb</it> are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28–31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal <it>cis</it>-acting regulation, and use that information to identify a regulatory region of the human APP gene.</p> <p>Results</p> <p>Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of <it>appb</it>. A cluster of three E4BP4 sites at −31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at −31 kb specifically and efficiently bind the corresponding zebrafish proteins <it>in vitro</it>. These sites are statistically over-represented in both the zebrafish <it>appb</it> and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP) experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription factors.</p> <p>Conclusion</p> <p>The results suggest that the clock-regulated and immune system modulator transcription factor E4BP4/ NFIL3 likely regulates the expression of both <it>appb</it> in zebrafish and APP in humans. It suggests potential human APP gene regulatory pathways, not on the basis of comparing DNA primary sequences with zebrafish <it>appb</it> but on the model of conservation of transcription factors.</p

CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes.

CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease

Immunoglobulin heavy chain locus and B cell development.

<p>Schematic of the murine IgH locus showing variable (V_H, blue, n represents approximately 150 V_H gene segments), diversity (D_H, grey, n represents six to nine DSP gene segments), and joining (J_H, orange) gene segment <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Chevillard1" target="_blank">[11]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Johnston1" target="_blank">[12]</a>. Exons encoding the constant regions of IgM and IgD are indicated as Cμ and Cδ. A promoter 5′ of DQ52, the 3′-most D_H gene segment, is indicated by the yellow oval and the intronic enhancer Eμ by a teal oval. Top line shows the germline (GL) configuration with associated histone modifications in B lineage precursors <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Chakraborty1" target="_blank">[23]</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Chowdhury1" target="_blank">[25]</a>. Histone H3 and H4 acetylation are shown in orange and presence of heterochromatic H3K9 methylation by the red line. Vertical red arrows represent the tissue-specific DNase I hypersensitive sites in the germline state. Next two lines show sequential stages of VDJ recombination at the IgH locus. D_H to J_H rearrangement occurs first resulting in a DJ_H junction and, depending on which D_H rearranges, residual upstream unrearranged D_Hs may be present. V_H rearrangement occurs to the DJ_H junction to generate a VDJ_H junction; during this process unrearranged D_Hs are lost from the genome.</p

DNA methylation status of DNase 1 hypersensitive sites 5′ of DFL16.1.

<p>Three newly identified DNase 1 hypersensitive sites are located at approximately 6–6.5, 4–4.5, and 0.4–1.3 kb 5′ of DFL16.1. Genomic DNA from primary RAG-deficient pro-B cells and DP (CD4⁺ CD8⁺) thymocytes were used in bisulfite mapping experiments to examine CpG methylation. Five amplicons covering regions between 3 kb and 7 kb 5′ of DFL16.1 were modified, cloned, and sequenced. The distribution of CpG dinucleotides within each amplicon are noted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475.s003" target="_blank">Figure S3</a>. Filled and open circles represent methylated and unmethylated residues, respectively. Pie charts summarize the percentage of methylated alleles at each position.</p

DNA methylation state of unrearranged and DJ_H recombined alleles in mature B cells.

<p>(A) Mature B cells were purified from spleens of C57BL/6 mice, and the genomic DNA was subjected to bisulfite modification assays. 40% of these cells contain two VDJ_H recombined alleles and the remainder contains one VDJ_H and one DJ_H recombined allele. (B) Amplicons corresponding to unrearranged DFL16.1 gene segment and a region centered 1.3 kb 5′ to DFL16.1 were cloned and sequenced. For comparison, methylation of the same region in pro-B cells derived from RAG2-decificient bone marrow is shown in the bottom panel. Filled and open circles indicate methylated and unmethylated cytosines. Pie charts summarize the percentage of methylated cytosines at each position; data are derived from two independent spleen B cell preparations with two to four mice in each experiment. (C) DJ_H junctions were amplified from bisulfite modified DNA, followed by cloning and sequencing. Circles and squares represent cytosines from D_H and J_H1 gene segment, respectively. Filled and open circles, or squares, indicate methylated and unmethylated cytosines, respectively. Numbers within regions marked as DFL16.1, DSP, and J_H1 denote CpG dinucleotides corresponding to the configuration at the respective unrearranged gene segments. For example, of the five CpGs at unrearranged DFL16.1, only the first two are retained in DFL16.1/J_H1 junctions. Variations in the total number of cytosines are due to imprecise joining during VDJ recombination. Pie charts summarize the percentage of methylated cytosines. The asterisk indicates positions where less than 12 CpGs were observed due to reduced representation caused by junctional diversity.</p

DNA methylation status after the first step of IgH locus recombination.

<p>Pro-B cells were purified from the bone marrow of wild-type C57BL/6 mice. Data shown are derived from two independent preparations of pro- and pre-B cells obtained from six to eight mice in each experiment. This cell population contains a mix of germline and partially rearranged IgH alleles. After bisulfite modification, the genomic DNA was used to amplify unrearranged (A) and DJ_H rearranged junctions containing DFL16.1 and DSP gene segments (B). Cytosines derived from D_H and J_H gene segments are marked as circles and squares, respectively. Filled and open circles, or squares, indicate methylated and unmethylated cytosines, respectively. Numbers within regions marked as DFL16.1, DSP and J_H1 in (B) denote CpG dinucleotides corresponding to the configuration of these residues at the respective unrearranged gene segments. For example, of the five CpGs at unrearranged DFL16.1 only the first two are retained in DFL16.1/J_H1 junctions. The total numbers of CpG dinucleotides are reduced in junctional sequences because some residues are lost during VDJ recombination as described in the text. Additional heterogeneity is due to the imprecise nature of recombination. Pie charts summarize the percentage of methylated cytosines at each position, except where the number of alleles falls below 12 (indicated by asterisks). (C) Methylation state of recombined DJ_H alleles in purified pre-B cells. This population contains a mix of VDJ_H recombined and DJ_H recombined IgH alleles (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio-1001475-g005" target="_blank">Figure 5A</a>). Note that the number of circles and squares representing D_H- or J_H-associated CpGs differed in the DJ_H junctions compared to the corresponding unrearranged regions due to junctional variability.</p