Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vkappa Gene Rearrangement in Pre-B Cells and Pro-B Cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA483209 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7028350.fastq.gz | Fastqsanger.gz | |||
| SRR7028351.fastq.gz | Fastqsanger.gz | |||
| SRR7028352.fastq.gz | Fastqsanger.gz | |||
| SRR7028353.fastq.gz | Fastqsanger.gz | |||
| SRR7028348.fastq.gz | Fastqsanger.gz |
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Project description:Deep sequencing has the potential to provide great insight into the composition of the antibody repertoire and is now widely used. To date there has not been a study directly comparing relative rearrangement frequencies obtained with unbiased amplification from genomic DNA (gDNA) and cDNA from the same set of cells to elucidate any differences. Here we use a recently developed method for unbiased amplification of gDNA and directly compare the data to the unbiased RNA IgK repertoire from the same pre-B cell pool. We find that ~20% of Vk genes have relative rearrangement frequencies > 2-fold up or down in the RNA vs DNA libraries, including many members of the Vk4, Vk3 and Vk6 families. Regression analysis indicates that Ikaros and E2A binding are associated with strong promoters. Within the pre-B repertoire, we found that the vastly unequal Vk gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many Vk genes in pre-B cells are most predictive of rearrangement levels. Comparison of IgK rearrangements that occur in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias towards usage of Jk-distal Vk genes, particularly Vk 10-96 and surrounding genes. Regression analysis indicates that PU.1 binding is the highest predictor of high Vk gene rearrangement frequency in pro-B cells. Lastly, repertoires of iEk -/- pre-B cells reveal that the intronic enhancer actively influences Vk gene usage and displays a germline transcriptional sphere of influence extending to Vk 6-15, overlapping with the deficit in Jk proximal Vk gene usage. These represent previously unknown roles for iEk in addition to its major function in stimulating overall IgK rearrangement.
2018-10-01 | GSE113306 | GEO
Project description:There has been no study to date that has examined genomic DNA (gDNA) and RNA Igkappa repertoires directly from the same set of cells. In this study we performed deep sequencing on Igkappa repertoire library preparations from both gDNA and RNA from the same sorted cells. This was done for both pre-B cells, where most Igkappa rearrangement takes places as well as pro-B cells where some B cells undergo early Igkappa rearrangement. In addition, we perfored gDNA repertoire analysis on iEkappa-/- pre-B cells. We find that some Vk genes display skewed DNA or RNA representiation relative to the other. We also find that pro-B cell Igkappa rearrangement is skewed to Jkappa-distal Vkappa genes. Lastly, we find that iEkappa-/- pre-B cells are not capable of rearranging to the most proximal Vkappa family genes.
2018-10-01 | GSE117800 | GEO
Project description:Epigenetic enhancer marks and transcription factor binding influence Vk gene rearrangement frequencies in pre-B cells and pro-B cells
| PRJNA450755 | ENA
Project description:Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vk Gene Rearrangement in Pre-B Cells and Pro-B Cells [VDJ-seq]
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Project description:To date there has not been a study directly comparing relative Igκ rearrangement frequencies obtained from genomic DNA (gDNA) and cDNA and since each approach has potential biases, this is an important issue to clarify. Here we used deep sequencing to compare the unbiased gDNA and RNA Igκ repertoire from the same pre-B cell pool. We find that ~20% of Vκ genes have rearrangement frequencies ≥2-fold up or down in RNA vs. DNA libraries, including many members of the Vκ3, Vκ4, and Vκ6 families. Regression analysis indicates Ikaros and E2A binding are associated with strong promoters. Within the pre-B cell repertoire, we observed that individual Vκ genes rearranged at very different frequencies, and also displayed very different Jκ usage. Regression analysis revealed that the greatly unequal Vκ gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many Vκ genes in pre-B cells are most predictive of rearrangement levels. Since H3K4me1 is associated with long range chromatin interactions which are created during locus contraction, our data provides mechanistic insight into unequal rearrangement levels. Comparison of Igκ rearrangements occurring in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias toward usage of Jκ-distal Vκ genes, particularly Vκ10-96 and Vκ1-135. Regression analysis indicates that PU.1 binding is the highest predictor of Vκ gene rearrangement frequency in pro-B cells. Lastly, the repertoires of iEκ-/- pre-B cells reveal that iEκ actively influences Vκ gene usage, particularly Vκ3 family genes, overlapping with a zone of iEκ-regulated germline transcription. These represent new roles for iEκ in addition to its critical function in promoting overall Igκ rearrangement. Together, this study provides insight into many aspects of Igκ repertoire formation.
| S-EPMC6146092 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2018-10-01 | GSE117804 | GEO
Project description:LGR6 marks nephron progenitor cells
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Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina HiSeq2000.
2013-08-05 | E-GEOD-47766 | biostudies-arrayexpress
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. RNA was extracted from C57BL/6 RAG-/- pro-B cells using TrizolM-BM-. (Life Technologies Corp., Carlsbad CA) and genomic DNA was eliminated using the genomic DNA wipeout buffer in the QuantiTect Reverse transcription kit (QIAGEN). A final purification of the RNA was performed with the RNeasy kit from QIAGEN. For each sample, 100 ng of total RNA was used to make RNASeq libraries using the NuGEN Encore Complete DR kits following manufacturer's recommended protocols. Sequencing libraries were gel purified to ensure insert sizes were larger than 100 bp in length and sequenced on an Ilumina HiSeq2000 for 100 bases plus 7 bases for indexing.
2013-08-05 | E-GEOD-47885 | biostudies-arrayexpress
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5’-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus.
2013-08-05 | GSE47766 | GEO