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 #
5P01GM099117-04
Application #
8717680
Study Section
Special Emphasis Panel (ZGM1-GDB-8)
Project Start
Project End
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
4
Fiscal Year
2014
Total Cost
$562,487
Indirect Cost
$115,911
Name
Harvard University
Department
Type
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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
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
Lin, Shuibin; Choe, Junho; Du, Peng et al. (2016) The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol Cell 62:335-45
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 16:2178-86
Merkle, Florian T; Neuhausser, Werner M; Santos, David et al. (2015) Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep 11:875-83
Galonska, Christina; Ziller, Michael J; Karnik, Rahul et al. (2015) Ground State Conditions Induce Rapid Reorganization of Core Pluripotency Factor Binding before Global Epigenetic Reprogramming. Cell Stem Cell 17:462-70
Meissner, Alexander (2015) (Epi)genomics approaches and their applications. Methods 72:1-2
Molyneaux, Bradley J; Goff, Loyal A; Brettler, Andrea C et al. (2015) DeCoN: genome-wide analysis of in vivo transcriptional dynamics during pyramidal neuron fate selection in neocortex. Neuron 85:275-88
Ziller, Michael J; Edri, Reuven; Yaffe, Yakey et al. (2015) Dissecting neural differentiation regulatory networks through epigenetic footprinting. Nature 518:355-9
Liao, Jing; Karnik, Rahul; Gu, Hongcang et al. (2015) Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nat Genet 47:469-78

Showing the most recent 10 out of 50 publications