Project description:Activation of splenic B cells induces formation of a 220kb DNA loop between Em and 3’RR enhancers in the immunoglobulin heavy chain locus (IgH). This DNA loop has been proposed to be necessary for the crucial immune diversification mechanism of IgH class switch recombination, but the factors that control its formation are unknown. We show that conditional deletion of transcription factor YY1 in primary splenic B cells results in a dramatic drop in formation of this DNA loop, as well as immunoglobulin class switch recombination. Reconstitution of YY1-deleted splenic B cells with various YY1 mutants showed that the C-terminal half of YY1 lacking the transactivation domain restored both Em-3’RR DNA loop formation as well as class switch recombination. RNA transcript analyses of YY1 conditional deleted splenic B cells suggest that YY1 does not regulate genes needed for DNA looping or CSR. Our results argue for a direct physical mechanism of YY1 mediating long-distance DNA loops and provide strong evidence of the importance of this DNA loop for class switching. Our results provide foundational mechanistic insight into a crucial immune function.

2016-08-06 | GSE85259 | GEO

YY1 controls Eμ-3'RR DNA loop formation and immunoglobulin heavy chain class switch recombination.

Project description: Not available

| S-EPMC5695717 | biostudies-literature

Germ line transcription of the immunoglobulin heavy chain locus directs production of mu chain without VDJ.

Project description:Immunoglobulin VDJ recombination is associated with transcriptional activation of the Ig variable region elements. We have previously described a novel Ig mu chain protein and mRNA produced by pre-B cell hybrids from normal and X-linked agammaglobulinemic bone marrow. We have now characterized the mRNA encoding this protein and find that it is composed of a 5' leader sequence spliced to C mu (LS-C mu), lacking the variable (V), diversity (D), and joining (J) gene sequences. The leader sequence is encoded by a novel exon 16 kb upstream of the JH locus. Transcription of the germ line heavy chain locus from this LS exon results in transcriptional activation of the JH locus, apparently the initial step in commitment to B lymphoid development. Polymerase chain reaction amplification of normal bone marrow shows that these germ line LS-C mu transcripts are a product of bone marrow pre-B cells. Production of LS-C mu commences a sequential process of transcriptional activation, with concordant translation of Ig rearrangement intermediates, in the process of creating a productive VDJ rearrangement.

| S-EPMC295921 | biostudies-other

E<sub>μ</sub> and 3'RR IgH enhancers show hierarchic unilateral dependence in mature B-cells.

Project description:Enhancer and super-enhancers are master regulators of cell fate. While they act at long-distances on adjacent genes, it is unclear whether they also act on one another. The immunoglobulin heavy chain (IgH) locus is unique in carrying two super-enhancers at both ends of the constant gene cluster: the 5'Eμ super-enhancer promotes VDJ recombination during the earliest steps of B-cell ontogeny while the 3' regulatory region (3'RR) is essential for late differentiation. Since they carry functional synergies in mature B-cells and physically interact during IgH locus DNA looping, we investigated if they were independent engines of locus remodelling or if their function was more intimately intermingled, their optimal activation then requiring physical contact with each other. Analysis of chromatin marks, enhancer RNA transcription and accessibility in Eμ- and 3'RR-deficient mice show, in mature activated B-cells, an unilateral dependence of this pair of enhancers: while the 3'RR acts in autonomy, Eμ in contrast likely falls under control of the 3'RR.

| S-EPMC5428668 | biostudies-literature

YY1 Controls Em-3â??RR DNA Loop Formation and Immunoglobulin Heavy Chain Class Switch Recombination.

Project description:Activation of splenic B cells induces formation of a 220kb DNA loop between Em and 3â??RR enhancers in the immunoglobulin heavy chain locus (IgH). This DNA loop has been proposed to be necessary for the crucial immune diversification mechanism of IgH class switch recombination, but the factors that control its formation are unknown. We show that conditional deletion of transcription factor YY1 in primary splenic B cells results in a dramatic drop in formation of this DNA loop, as well as immunoglobulin class switch recombination. Reconstitution of YY1-deleted splenic B cells with various YY1 mutants showed that the C-terminal half of YY1 lacking the transactivation domain restored both Em-3â??RR DNA loop formation as well as class switch recombination. RNA transcript analyses of YY1 conditional deleted splenic B cells suggest that YY1 does not regulate genes needed for DNA looping or CSR. Our results argue for a direct physical mechanism of YY1 mediating long-distance DNA loops and provide strong evidence of the importance of this DNA loop for class switching. Our results provide foundational mechanistic insight into a crucial immune function. Follicular B cells were isolated from the spleens of three C57Bl/6 yy1 fl/fl mice. For each spleen, half the cells received mock treatment and half received TATCRE. The 6 samples were then grown in RPMI medium along with LPS, Il4, OPI, and 20% FBS for 72 hours. The 6 groups of cells were lysed and RNA was isolated for library preparation. Expression differences between Mock and TATCRE treated cells were determined to understand the role of yy1 in B cell class switching.

2016-08-06 | E-GEOD-85259 | biostudies-arrayexpress

Heavy chain immunoglobulin sequencing of Lyme disease PBMCs

Project description:Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity world-wide. Here, we investigated B cell responses to Lyme disease through molecular identifier-enabled antibody heavy chain sequencing of bulk B cells from PBMCs. Single-cell immunoglobulin sequencing of paired heavy- and light-chain genes from this project will also be separately deposited. Additional information regarding patient characteristics and overlap with other data from the SLICE study is available upon request.

2018-12-21 | GSE113938 | GEO

Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions.

Project description:Activation-induced cytosine deaminase (AID) is essential for both somatic hypermutation (SHM) and class switch recombination (CSR), two processes involved in antibody diversification. Previously, various groups showed both in vitro and in vivo that AID initiates SHM and CSR by deaminating cytosines in DNA in a transcription-dependent manner. Although in vivo both DNA strands are equally targeted by AID, many in vitro and bacterial experiments found that AID almost exclusively targets the nontemplate strand of a transcribed substrate. Here, we report the detection of antisense transcripts in assembled Ig heavy chain (IgH) variable region exons and their immediate downstream region, as well as in switch regions, sequences that, respectively, are targets for SHM and CSR in vivo. In contrast, we did not detect antisense transcripts from the Cmu constant region exons, which lie between the IgH variable region exons and downstream S regions and which are not normally an AID target. Expression of the antisense variable region/flanking region and the S-region transcripts were found in all lymphocytes that transcribe these sequences in the sense direction. Steady-state levels of antisense transcripts appeared very low, and start sites potentially appeared heterogeneous. We discuss the potential implications of antisense IgH locus transcription for AID targeting or other processes.

| S-EPMC2268805 | biostudies-literature

Developmental regulation of DNA cytosine methylation at the immunoglobulin heavy chain constant locus.

Project description:DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.

| S-EPMC6380546 | biostudies-literature