A simple and robust plate-based single-cell ATAC-seq on various tissues and cell lines
ABSTRACT: The assay for transposase-accessible chromatin using sequencing (ATAC-seq) is widely used to identify regulatory regions throughout the genome. However, only a few studies have been done at the single cell level (scATAC-seq) due to technical difficulties. Here we developed a simple and robust plate-based scATAC-seq method, combining upfront bulk tagmentation with single-nuclei sorting, to investigate open chromatin regions. We applied this method on mouse splenocytes and unbiasedly revealed key regulatory regions and transcription factors that define each cell (sub)type.
Project description:B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre–BCR-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5′ to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which,at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination. ChIP-seq for 1 transcription factor and 2 histone modifications in flow purified mouse small pre-B cells. ATAC-seq and RNA-seq in WT and Brwd-Mut mouse flow purified small pre-B cells.
Project description:Activated T cells differentiate into functional subsets which require distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to provide substrate for the tricarboxylic acid cycle and epigenetic reactions and here we identify a key role for GLS in T cell activation and specification. Though GLS-deficiency diminished T cell activation, proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet and Interferon-γ expression and CD4 Th1 and CD8 CTL effector cell differentiation. These changes were mediated by differentially altered gene expression and chromatin accessibility, leading to increased sensitivity of Th1 cells to IL-2 mediated mTORC1 signaling. In vivo, GLS-null T cells failed to drive a Th17 mediated Graft-vs-Host Disease model. Transient inhibition of GLS, however, increased Th1 and CTL T cell numbers in viral and chimeric antigen receptor models. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.
Project description:Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer (10.7554/eLife.03848). Specifically, the 2.7 kb Mouse Smarcd3-F6 sequence (chr5: 24113559 -24116342 from mm9 assembly) was sub-cloned from a gateway entry vector into the Zebrafish Enhancer Detection (ZED). Tol2-mediated transgenesis was performed and stable lines were created. The Smarcd3-F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3-F6:EGFPhsc70 embryos were dissociated at 10 hours post fertilization for fluorescent activated cell sorting (FACS). 30,000-50,000 GFP positive and negative cells were collected from FACS for ATAC-seq. ATAC-seq libraries were created with Tn5 transposase and single-end sequenced on the Illumina HiSeq 2500 platform
Project description:Understanding the impact of cohesin mutations on HSPC chromatin structure We examined chromatin structure using ATAC-seq in CD34+ enriched-human HSPC that were transduced with either cohesin WT, mutant or empty vector controls