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Mallard, William

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Mallard

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William

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Mallard, William
Author's Publications: search.filters.isAuthorOfPublication.d7a1588d-6ce9-4294-8d69-c68f6942032f

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Now showing 1 - 5 of 5
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    Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking
    (eLife Sciences Publications, Ltd, 2017) Kaewsapsak, Pornchai; Shechner, David Michael; Mallard, William; Rinn, John; Ting, Alice Y
    The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.
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    Multiple knockout mouse models reveal lincRNAs are required for life and brain development
    (eLife Sciences Publications, Ltd, 2013) Sauvageau, Martin; Goff, Loyal; Lodato, Simona; Bonev, Boyan; Groff, Abigail F.; Gerhardinger, Chiara; Sanchez-Gomez, Diana B; Hacisuleyman, Ezgi; Li, Eric; Spence, Matthew; Liapis, Stephen C; Mallard, William; Morse, Michael; Swerdel, Mavis R; D’Ecclessis, Michael F; Moore, Jennifer C; Lai, Venus; Gong, Guochun; Yancopoulos, George D; Frendewey, David; Kellis, Manolis; Hart, Ronald P; Valenzuela, David M; Arlotta, Paola; Rinn, John
    Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc–Brn1b and linc–Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr−/− neonates, whereas linc–Brn1b−/− mutants displayed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001
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    A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs
    (2016) Choi, Jiho; Lee, Soohyun; Clement, Kendell; Mallard, William; Tagliazucchi, Guidantonio Malagoli; Lim, Hotae; Choi, In Young; Ferrari, Francesco; Tsankov, Alex; Pop, Ramona; Lee, Gabsang; Rinn, John; Meissner, Alexander; Park, Peter; Hochedlinger, Konrad
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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.
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    Common variants in signaling transcription-factor-binding sites drive phenotypic variability in red blood cell traits
    (Springer Science and Business Media LLC, 2020-11-23) Choudhuri, Avik; Trompouki, Eirini; Abraham, Brian J.; Colli, Leandro M.; Kock, Kian Hong; Mallard, William; Yang, Min-Lee; Vinjamur, Divya S.; Ghamari, Alireza; Sporrij, Audrey; Hoi, Karen; Hummel, Barbara; Boatman, Sonja; Chan, Victoria; Tseng, Sierra; Nandakumar, Satish K.; Yang, Song; Lichtig, Asher; Superdock, Michael; Grimes, Seraj N.; Bowman, Teresa V.; Zhou, Yi; Takahashi, Shinichiro; Joehanes, Roby; Cantor, Alan; Bauer, Daniel; Ganesh, Santhi K.; Rinn, John; Albert, Paul S.; Bulyk, Martha; Chanock, Stephen J.; Young, Richard; Zon, Leonard
    Genome-wide association studies (GWAS) reveal genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell type-specific enhancers, but the molecular mechanism by which they cause phenotypic variation is understood in only a few instances. Here, we show that a striking proportion of enhancer-variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) that modulate levels of gene expression in response to extracellular signals. We find that the majority of the enhancer variants alter STF and not MTF motifs. Consequently, they perturb DNA-binding by various signaling factors including BMP/TGF-directed SMADs, WNT-induced TCFs, Hedgehog-responsive GLIs, Notch-dependent HES and affect downstream gene expression. Analysis of activity of SNPs in human CD34+ cells and eQTL analysis from the Framingham Heart Study (FHS) verifies that human alleles with disrupted STF binding lead to altered expression of genes that are upregulated during human erythroid differentiation. Our results propose that, of the RBC trait-associated variants that reside on TF binding sequences, the majority fall on DNA sequences recognized by STFs. This suggests that, in many cases, the phenotypic variation of RBC traits could be due to disruptions in STF motifs that lead to altered responsiveness to extracellular stimuli.