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Karnik, Rahul

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Karnik

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Rahul

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Karnik, Rahul

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2014 - 20152

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Author's Publications: search.filters.isAuthorOfPublication.6957bc7b-02b7-452b-ad94-6d915f19f451

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    DNA methylation dynamics of the human preimplantation embryo
    (2014) Smith, Zachary; Chan, Michelle M.; Humm, Kathryn C.; Karnik, Rahul; Mekhoubad, Shila; Regev, Aviv; Eggan, Kevin; Meissner, Alexander
    In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell type-specific regulation directed by DNA binding factors1-3. This comparatively static landscape dramatically contrasts the events of fertilization, where the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, though methylation is maintained at several notable features4-7. While these dynamics have been extensively characterized in the mouse, only limited observations are available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved8-10. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell (ESC) derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of persistent maintenance are primarily restricted to gene bodies. While most features share similar dynamics to mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island (CGI) promoters that extend beyond known Imprint Control Regions (ICRs). Retrotransposon regulation is also highly diverse and transitions from maternally to embryonically expressed, species-specific elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation.
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    Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells
    (2015) Liao, Jing; Karnik, Rahul; Gu, Hongcang; Ziller, Michael; Clement, Kendell; Tsankov, Alexander M.; Akopian, Veronika; Gifford, Casey A.; Donaghey, Julie; Galonska, Christina; Pop, Ramona; Reyon, Deepak; Tsai, Shengdar Q.; Mallard, William; Joung, J. Keith; Rinn, John; Gnirke, Andreas; Meissner, Alexander
    DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate their roles and genomic targets. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets.