The overall goal of this component project is to identify and reverse engineer the cis-regulatory modules (CRMs) that direct the activation, maintenance and repression of gene expression in human pluripotent cells. The project innovates by applying high-throughput and quantitative genomic technologies to dissect key components of the pluripotency gene regulatory network (CRMs) at unprecedented resolution and scale. First, we will generate a genome-wide map of putative CRMs with specific activity in pluripotent cells, using computational analysis of new and existing TF localization and chromatin profiling data. We will then select genomic loci that harbor genes which correct regulation have previously been shown to be important for maintenance of pluripotency and self-renewal for in-depth validation and functional analysis. Second, we will select representative CRMs from these loci for systematic dissection of the relationship between their sequences and regulatory activities at single-nucleotide resolution. Third, we will classify pluripotency CRMs according to whether they are activated eariy or late in the reprogramming process, and then explore the mechanistic basis for these differences using systematic perturbation experiments. Finally, we will then use data from all of our experiments to construct quantitative models of pluripotency CRMs that integrate interactions between their DNA sequences, local chromatin structure and trans-regulatory factors.

Public Health Relevance

(See Instructions): The goal of this project is to uncover how the rules that govern gene expression in stem cells are encoded in human DNA. Insights into this question will help us understand the genetic basis for human development and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
1P01GM099117-01
Application #
8206145
Study Section
Special Emphasis Panel (ZGM1-GDB-8 (IP))
Project Start
Project End
Budget Start
2011-08-01
Budget End
2012-07-31
Support Year
1
Fiscal Year
2011
Total Cost
$472,938
Indirect Cost
Name
Harvard University
Department
Type
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Choi, Jiho; Clement, Kendell; Huebner, Aaron J et al. (2017) DUSP9 Modulates DNA Hypomethylation in Female Mouse Pluripotent Stem Cells. Cell Stem Cell 20:706-719.e7
Merkle, Florian T; Ghosh, Sulagna; Kamitaki, Nolan et al. (2017) Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 545:229-233
Smith, Zachary D; Shi, Jiantao; Gu, Hongcang et al. (2017) Epigenetic restriction of extraembryonic lineages mirrors the somatic transition to cancer. Nature 549:543-547
Melé, Marta; Mattioli, Kaia; Mallard, William et al. (2017) Chromatin environment, transcriptional regulation, and splicing distinguish lincRNAs and mRNAs. Genome Res 27:27-37
Lin, Shuibin; Choe, Junho; Du, Peng et al. (2016) The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol Cell 62:335-345
Groff, Abigail F; Sanchez-Gomez, Diana B; Soruco, Marcela M L et al. (2016) In Vivo Characterization of Linc-p21 Reveals Functional cis-Regulatory DNA Elements. Cell Rep 16:2178-2186
Santos, David P; Kiskinis, Evangelos; Eggan, Kevin et al. (2016) Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells. Curr Protoc Stem Cell Biol 38:5B.6.1-5B.6.60
Hacisuleyman, Ezgi; Shukla, Chinmay J; Weiner, Catherine L et al. (2016) Function and evolution of local repeats in the Firre locus. Nat Commun 7:11021
Liu, Lin L; Brumbaugh, Justin; Bar-Nur, Ori et al. (2016) Probabilistic Modeling of Reprogramming to Induced Pluripotent Stem Cells. Cell Rep 17:3395-3406
Tsankov, Alexander M; Akopian, Veronika; Pop, Ramona et al. (2015) A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells. Nat Biotechnol 33:1182-92

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