48 research outputs found
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Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling
Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.Stem Cell and Regenerative Biolog
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Epigenomics and Chromatin Dynamics
A report of the 'Joint Keystone Symposium on Epigenomics and Chromatin Dynamics', Keystone, Colorado, 17-22 January 2012. This year's Joint Keystone Symposium on Epigenomics and Chromatin Dynamics was one of the largest Keystone meetings to date, reflecting the excitement and many developments in this area. Richard Young opened the meeting by giving a historic overview before sharing more detailed insights from his recent work in describing the role of the lysine demethylase Lsd1 in mouse embryonic stem (ES) cell differentiation. He also set the broader stage and highlighted the excitement concerning recent advances in epigenetic drugs such as the new bromodomain inhibitors.Stem Cell and Regenerative Biolog
A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development
While gene expression dynamics have been extensively cataloged during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs. Our findings indicate that HSCs manifest distinct alternative splicing patterns in key hematopoietic regulators. Detailed analysis of the splicing dynamics and function of one such regulator, HMGA2, identified an alternative isoform that escapes miRNA-mediated targeting. We further identified the splicing kinase CLK3 that, by regulating HMGA2 splicing, preserves HMGA2 function in the setting of an increase in let-7 miRNA levels, delineating how CLK3 and HMGA2 form a functional axis that influences HSC properties during development. Collectively, our study highlights molecular mechanisms by which alternative splicing and miRNA-mediated post-transcriptional regulation impact the molecular identity and stage-specific developmental features of human HSCs. Human hematopoietic stem cells (HSCs) display substantial transcriptional diversity during development. Here, we investigated the contribution of alternative splicing to such diversity by analyzing the dynamics of a key hematopoietic regulator, HMGA2. Next, we showed that CLK3, by regulating the splicing pattern of HMGA2, reinforces an HSC-specific program
Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
Biological and medical implications of discordance in C phosphate G methylation
Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2017.Cataloged from PDF version of thesis.Includes bibliographical references (pages 121-160).DNA methylation is an important epigenetic mark that is linked to the regulation of gene expression. It is a critical part of controlling cellular identity and is essential for normal development. DNA methylation is generally studied by comparing methylation levels at individual cytosines or computing region-level averages to identify differential methylation. Here we extended this classic viewpoint by capitalizing on a unique feature of next-generation sequencing, which provides the methylation status of CpGs that are located on the same sequencing read and hence originate from the same DNA molecule. When comparing methylation states of CpGs on the same read, we observed different levels of discordant methylation, defined as molecules where the methylation of neighboring cytosines are not correlated. We quantified the proportion of discordantly methylated reads (PDR) in normal and cancer samples, and found that global PDR levels were elevated in cancer, suggesting widespread epigenetic deregulation. While we have not yet established the mechanistic contribution of this feature, we find that discordant methylation is linked to higher genetic diversity, greater cell-to-cell transcriptional heterogeneity, and adverse clinical outcome in chronic lymphocytic leukemia (CLL). Our analytic approach introduces a novel perspective on utilizing epigenomic sequencing data, which we anticipate will be a valuable tool in understanding the regulation of DNA methylation and its contribution to cellular identity.by Mark Kendell Clement.Ph. D. in Medical Engineering and Medical Physic
CRISPR-SURF: discovering regulatory elements by deconvolution of CRISPR tiling screen data
Tiling screens that use CRISPR-Cas technologies provide a powerful approach for the mapping of regulatory elements to phenotypes of interest1–6. Here we present CRISPR screening uncharacterized region function (CRISPR-SURF), a deconvolution framework that can be used to identify functional regulatory regions in the genome from data generated by CRISPR-Cas nuclease, CRISPR interference (CRISPRi), or CRISPR activation (CRISPRa) tiling screens. CRISPR-SURF can be run as a stand-alone command line utility (https://github.com/pinellolab/CRISPR-SURF) or as a web application (http://crisprsurf.pinellolab.org/)National Human Genome Research Institute (U.S.) (Career Development Award R00 HG008399)National Human Genome Research Institute (U.S.). Centers of Excellence in Genomic Science (Grant RM1HG009490)National Institutes of Health (U.S.) (Grant R35 GM118158)National Institutes of Health (U.S.) (Grant RM1 HG009490)National Human Genome Research Institute (U.S.) (Grant 1K99HG009917-01
Generation of mouse models carrying B cell restricted single or multiplexed loss-of-function mutations through CRISPR-Cas9 gene editing
Summary: Here, we present a protocol to generate B cell restricted mouse models of loss-of-function genetic drivers typical of lymphoproliferative disorders, using stem cell engineering of murine strains carrying B cell restricted Cas9 expression. We describe steps for preparing lentivirus expressing sgRNA-mCherry, isolating hematopoietic stem and progenitor cells, and in vitro transduction. We then detail the transplantation of engineered cells into recipient mice and verification of gene edits. These mouse models represent versatile platforms to model complex disease traits typical of lymphoproliferative disorders.For complete details on the use and execution of this protocol, please refer to ten Hacken et al.,1 ten Hacken et al.,2 and ten Hacken et al.3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics