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Single cell paired heavy- and light-chain immunoglobulin sequencing of sorted Lyme disease plasmablasts

ABSTRACT: Single cell paired heavy- and light-chain immunoglobulin sequencing of sorted Lyme disease plasmablasts

PROVIDER: PRJNA470930 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7145740_1.fastq.gz	Fastqsanger.gz
	SRR7145740_2.fastq.gz	Fastqsanger.gz
	SRR7145750_1.fastq.gz	Fastqsanger.gz
	SRR7145750_2.fastq.gz	Fastqsanger.gz
	SRR7145760_1.fastq.gz	Fastqsanger.gz

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Single cell paired heavy- and light-chain immunoglobulin sequencing of sorted Lyme disease plasmablasts

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 barcode-enabled single cell sequencing of activated B cells (plasmablasts) sorted from PBMCs. Bulk immunglobulin heavy-chain sequencing from this project has also been separately deposited. Additional information regarding patient characteristics and overlap with other data from the SLICE study is available upon request.

2018-12-21 | GSE114309 | GEO

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YY1 Controls Em-3’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 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.

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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.

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Allele specific analysis of the immunoglobulin heavy chain locus by simultaneous analysis of the productive and the non-productive allele through paired-end 4C sequencing analysis in mature B cells

Project description:In developing B cells the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different, repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is however unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells - but not in T cells – the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element. Allele specific analysis of the nuclear organization of the IgH locus in resting and activated B cells and T cells and fetal brain cells

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