Project description:We test the predicted interferon-inducible enhancer activity of a LINE within the first intron of the IFNAR1 gene (IFNAR1.L1M2a) by knocking out the predicted enhancer using CRISPR, and assessing chromatin and polymerase II occupancy in untreated and interferon beta-treated conditions compared to wildtype cells
Project description:We test the effect of CRISPR knockout of a predicted interferon inducible enhancer within the first intron of the IFNAR1 gene (IFNAR1.L1M2a) on the transcriptional response of GM12878 cells to treatment with interferon beta
Project description:To identify gene expression changes and pathways induced by interferon-? (IFN-?) in B cells, we studied the in vitro response of EBV-transformed B cells (lymphoblast cell lines-LCLs). LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-? exposure, with over 50 novel IFN-? response genes. Pathways analysis demonstrated that IFN-? affected LCLs in a similar manner to other cell types by activating known IFN-? canonical pathways. Additionally, IFN-? increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Several of the novel response genes were tested and validated as IFN-? response genes in human primary B cells. Total RNA from LCL samples treated for four hours with interferon beta (100u/ml) was compared to RNA from untreated LCLs (paired analysis). LCL samples were from patients with MS.
Project description:Noncoding variants play a central role in the genetics of complex traits, but we still lack a full description of the main molecular pathways through which they act. Here we used molecular data to quantify the contribution of cis-acting genetic effects at each major stage of gene regulation from chromatin to proteins, within a population sample of Yoruba lymphoblastoid cell lines (LCLs). We performed 4sU metabolic labeled transcripts in 65 YRI LCLs to identify genetic variants that affect transcription rates. As expected, we found an important contribution of genetic variation via chromatin, contributing â¼65% of eQTLs (expression Quantitative Trait Loci). The remaining eQTLs, which are not asso- ciated with chromatin-level variation, are highly enriched in transcribed regions, and hence may affect expression through co- or post-transcriptional processes. International HapMap lymphoblastoid cell lines (LCLs) derived from YRI (Yoruba in Ibadan, Nigeria); We adapted the 4sU labelling method from (PMID 21516085). Briefly, cell cultures were grown to log phase in volumes sufficient to yield about 300 ng of 4sU-labeled RNA. Cells were incubated with 4sU for the required length of time (0, 30, or 60 minutes), then washed, pelleted, and frozen. Total RNA was extracted, and 4sU-labeled RNA was separated from total RNA using a bead-based biotin-streptavidin purification protocol. We sequenced metabolic labeled transcripts in 65 YRI LCLs 30 minutes and 60 minutes after incubation.
Project description:Epstein-Barr virus (EBV) is a herpesvirus that establishes lifelong asymptomatic infection in up to 95% of the human population. In vitro EBV infection of resting B lymphocytes drives them to proliferate as lymphoblastoid cell lines (LCLs). EBV expresses limited genes, which include six nuclear proteins (EBNAs), three integral membrane proteins (LMPs), and more than 30 micro RNAs in LCLs. EBNA3C, one of the six nuclear proteins, is transcription factor and regulates host cell genes. The growth effects of EBNA3C appear to be primarily due to suppression of the CDKN2A gene products, p16INK4A and p14ARF. Conditional inactivation of EBNA3C results in increasing p16 expression and this is accompanied by the substantial reduction of the repressive H3K27me3 modification at the CDKN2A promoter and cell cycle arrest. To further elucidate the relationship between EBNA3C and H3K27me3 modification, we profiled H3K27me3 by ChIP-seq in CDKN2A knockout EBNA3CHT LCLs in the presence (4HT+) or absence (4HT-) of 4-hydroxytamoxifen, with a total of 4 biological replicates per condition.