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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: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 Vkappa Gene Rearrangement in Pre-B Cells and Pro-B Cells
| PRJNA483209 | ENA
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]
| PRJNA483208 | ENA
Project description:Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 μM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.
| S-EPMC8388921 | biostudies-literature
Project description:Nutrition represents one of the greatest environmental determinants of an individual's health. While nutrient quantity and quality impart direct effects, the interaction of nutrition with genetic and epigenetic modifications is often overlooked despite being shown to influence biological variation in mammals. Dissecting complex traits, such as those that are diet or nutrition related, to determine the genetic and epigenetic contributions toward a phenotype can be a formidable process. Epigenetic modifications add another layer of complexity as they do not change the DNA sequence itself but can affect transcription and are important mediators of gene expression and ensuing phenotypic variation. Altered carbohydrate metabolism and rates of fat and protein deposition resulting from diet-induced hypo- or hyper-methylation highlight the capability of nutritional epigenetics to influence livestock commodity quality and quantity. This interaction can yield either products tailored to consumer preference, such as marbling in meat cuts, or potentially increasing productivity and yield both in terms of carcass yield and/or offspring performance. Understanding how these and other desirable phenotypes result from epigenetic mechanisms will facilitate their inducible potential in livestock systems. Here, we discuss the establishment of the epigenome, examples of nutritional mediated alterations of epigenetics and epigenetic effects on livestock production.
| S-EPMC5075561 | biostudies-other
Project description:"Epigenetic plasticity" refers to the capability of mammalian cells to alter their differentiation status via chromatin remodeling-associated alterations in gene expression. While epigenetic plasticity has been best associated with lineage commitment of embryonic stem cells, recent studies have demonstrated chromatin remodeling even in terminally differentiated normal cells, and advanced-stage melanoma and breast cancer cells, in context-dependent responses to alterations in their microenvironment. In the current study, we extend this attribute of epigenetic plasticity to aggressive ovarian cancer cells, by using an integrative approach to associate cellular phenotypes with chromatin modifications ("ChIP-chip") and mRNA and microRNA expression. While we identified numerous gene promoters possessing the well-known "bivalent mark" of H3K27me3/H3K4me2, we also report 14 distinct, lesser-known bi-, tri-, and tetravalent combinations of activating and repressive chromatin modifications, in platinum-resistant CP70 ovarian cancer cells. The vast majority (>90%) of all the histone marks studied localized to regions within 2000 bp of transcription start sites, supporting a role in gene regulation. Upon a simple alteration in the microenvironment, transition from two- to three-dimensional culture, an increase (17% to 38%) in repressive-only marked promoters was observed, concomitant with a decrease (31% to 21%) in multivalent (i.e., juxtaposed permissive and repressive histone marked) promoters. Like embryonic/tissue stem and other (non-ovarian) carcinoma cells, ovarian cancer cell epigenetic plasticity reflects an inherent transcriptional flexibility for context-responsive alterations in phenotype. It is possible that this plasticity could be therapeutically exploited for the management of this lethal gynecologic malignancy.
| S-EPMC3052886 | biostudies-other
Project description:Maternal care alters epigenetic programming of glucocorticoid receptor (GR) gene expression in the hippocampus, and increased postnatal maternal licking/grooming (LG) behavior enhances nerve growth factor-inducible protein A (NGFI-A) transcription factor binding to the exon 1(7) GR promoter within the hippocampus of the offspring. We tested the hypothesis that NGFI-A binding to the exon 1(7) GR promoter sequence marks this sequence for histone acetylation and DNA demethylation and that such epigenetic alterations subsequently influence NGFI-A binding and GR transcription. We report that (1) NGFI-A binding to its consensus sequence is inhibited by DNA methylation, (2) NGFI-A induces the activity of exon 1(7) GR promoter in a transient reporter assay, (3) DNA methylation inhibits exon 1(7) GR promoter activity, and (4) whereas NGFI-A interaction with the methylated exon 1(7) GR promoter is reduced, NGFI-A overexpression induces histone acetylation, DNA demethylation, and activation of the exon 1(7) GR promoter in transient transfection assays. Site-directed mutagenesis assays demonstrate that NGFI-A binding to the exon 1(7) GR promoter is required for such epigenetic reprogramming. In vivo, enhanced maternal LG is associated with increased NGFI-A binding to the exon 1(7) GR promoter in the hippocampus of pups, and NGFI-A-bound exon 1(7) GR promoter is unmethylated compared with unbound exon 1(7) GR promoter. Knockdown experiments of NGFI-A in hippocampal primary cell culture show that NGFI-A is required for serotonin-induced DNA demethylation and increased exon 1(7) GR promoter expression. The data are consistent with the hypothesis that NGFI-A participates in epigenetic programming of GR expression.
| S-EPMC2951014 | biostudies-literature