ATAC-sequencing in control and Stag2 knockdown cells
ABSTRACT: We performed ATAC-sequencing in LSK cells (Lin(neg)/c-Kit(+)/Sca-1(+)) from shRNA mice carrying an shRNA for either Renilla or Stag2. ATAC-sequencing control (Renilla) and Stag2 knockdown cells.
Project description:We performed RNA-sequencing in LSK cells (Lin(neg)/c-Kit(+)/Sca-1(+)) from shRNA mice carrying an shRNA for Renilla, Smc1a or Stag2. RNA-sequencing control (Renilla) and cohesin (Smc1a and Stag2) knockdown cells.
Project description:In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development. Mouse preimplantation embryos were obtained from crosses of C57BL/6N and DBA/2N. ATAC-seq was performed in these embryos at various stages in preimplantation development.
Project description:Methylation of histone 3 on lysine 79 (H3K79) is broadly associated with active gene expression in eukaryotes, and the H3K79 methyltransferase DOT1L is indispensable for specific leukemia subtypes like those with MLL-translocations. We found that suppression of the histone deacetylase SIRT1 rescued MLL-AF9 leukemia cells from their dependence on DOT1L. We show that upon DOT1L inhibition, SIRT1 is required for the acquisition of a repressive chromatin state consistent with facultative heterochromatin around MLL-AF9 target genes in leukemia and other genes possess an H3K79me2(hi), H3K9ac(hi), H3K9me2(low) histone modification profile in normal hematopoietic stem and progenitor cells. Examination of histone modifications and a chromatin modifier with and without drug treatment and RNA interference.
Project description:Runx1 is expressed in regenrating muscle, specifically in the muscle adult stem cells- the satellite cells. Its exact role and target genes were yet to be identified. We report here the genome wide open chromatin paatern, as established by ATAC-seq Examination ofgenome wide pattern naked and therefore trhansposase accecible genomic DNA in primary myoblasts (PM)
Project description:We report the IFN-induced dynamics in murine splenic B cells. Male C57BL/6 mice were injected subcutaneously with 10,000U IFNa and spleens were removed at 90min. B cells were negatively isolated using magnetic beads and profiled for the chromatin configuration by ATAC-seq. Profilings of chromatin configuration by ATAC-seq (0 and 90min, biological duplicate for each).
Project description:We performed RNA-sequencing in c-Kit+ cells that were infected with retroviruses expressing shRNAs for Renilla, Rad21, Smc1a, Smc3 or Stag2. These cells were grown in methylcellulose (M3434) for either one passage (P1) or replated for five passages (P5). RNA-sequencing control (Ren) and cohesin (Rad21, Smc1a, Smc3 and Stag2) knockdown cells.
Project description:Normal thymic T cell development is enabled by a stromal microenvironment most importantly composed of distinct epithelial cell populations in cortex and medulla. Their differentiation, growth and function require the expression of the transcription factor Foxn1. Direct targets of Foxn1 have, however, remained largely undefined. Utilizing newly created static and inducible genetic model systems, we now provide a genome wide map of Foxn1 target genes and the sequences bound by this master regulator. Foxn1 controls not only essential steps early in intrathymic lymphoid development including T cell lineage commitment but is also indispensable for later stages in T cell maturation such as the selection of CD4 and CD8 T cells. Thus, Foxn1 function critically choreographs both early and late events in thymic lympho-stromal cross-talk. Foxn1 ChIP-seq and RNA-seq in mouse models of hypofunctional or conditional knock-out of Foxn1 Brief sample descriptions are shown below: Foxn1 ChIP-seq (GSM1945905) - chromatin immunoprecipitated using an antibody against FOXN1-FLAG (wt*); 2 samples Input ChIP-seq (GSM1945906) - input chromatin; 2 samples 42cT (GSM1945907) - RNA-seq on wt*/-::nu/nu cTEC; 3 samples 42mT (GSM1945908) - RNA-seq on wt*/-::nu/nu mTEC; 2 samples 96cT (GSM1945909) - RNA-seq on wt*/wt*::nu/nu cTEC; 3 samples 96mT (GSM1945910) - RNA-seq on wt*/wt*::nu/nu mTEC; 2 samples NcT (GSM1945911) - RNA-seq on Dox-treated TetO- iFoxn1(del7,8) cTEC; 5 samples PcT (GSM1945912) - RNA-seq on Dox-treated TetO+ iFoxn1(del7,8) cTEC; 5 samples C57BL/6 mice (GSM1945913) - ATAC-seq on wild-type cTEC; 1 sample Please see each sample for more detailed information.
Project description:STAG2 is targeted by somatic aberrations in a subset (4%) of human PDAs. Transposon mediated disruption of STAG2 in a KRASG12D genetically engineered mouse model promotes the development of PDA and its progression to metastatic disease. There was a statistically significant loss of STAG2 protein expression in human tumor tissue (Wilcoxon-Rank test) with complete absence of STAG2 staining observed in 15 (4.3%) patients. In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (> 95% of nuclei positive) was associated with a median survival benefit of 6.41 months (p = 0.031). The survival benefit of adjuvant chemotherapy was only seen in patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; p = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in pancreatic cancer patients. Finally we show that RNAi mediated knockdown of STAG2 selectively sensitizes human PDA cell lines to platinum-based therapy. Overall design: We used DNA content flow sorting to identify and purify tumor nuclei of PDA samples from 50 patients. The genome of each sorted sample was profiled by oligonucleotide comparative genomic hybridization (aCGH) and targeted resequencing of STAG2. Transposon insertions within STAG2 in a KRASG12D driven genetically engineered mouse (GEM) model of PDA were screened by RT-PCR. We then used a tissue microarray (TMA) to survey STAG2 protein expression levels in 344 human PDA tumor samples and adjacent tissues. Univariate Kaplan Meier analysis and multivariate Cox Regression analysis was used to assess the association of STAG2 expression relative to overall survival and responses to adjuvant therapy. Finally RNAi based assays with PDA cell lines were used to assess the potential therapeutic consequence of STAG2 expression in responses to 18 therapeutic agents.
Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs