The long-term goal of this project is to develop a complete understanding of the spatial and temporal events involved in the regulation of gene expression in living ES cells as they transit from the pluripotent to the differentiated state. An understanding of the nuclear choreography of essential genes regulating the earliest stages of ES cell differentiation is integral to understanding the mechanisms that help shift the ES cell transcriptional program to cell-type specific gene expression and will provide the basis of identifying how alterations in these events alter development. Here, a series of Aims are proposed that will address questions relating to the impact of gene positioning and dynamics on transcriptional regulation using both live cell imaging and molecular approaches. Studies will focus on two transcriptional paradigms: (1) genes that go from the "on" to the "off" state, and (2) genes that switch to monoallelic expression upon commitment to differentiation. The nuclear position of a series of 4 genes encoding the transcription factors Oct4, Nanog, Sox2, and Klf4 will be examined, as transcriptional silencing of these genes serves as a trigger for the initiation of the differentiation process. Changes in the nuclear position/associations of one or more of these genes during the initiation of differentiation will be identified and characterized and the relationship to the transcriptional output of the genes will be examined at the single cell level. Based on these findings a live cell imaging system will be developed to allow the direct study of the spatial and temporal dynamics of the identified gene movements in living cells over time. This system will provide the visualization of the gene and its mRNA transcripts and will allow one to determine if changes in gene positioning/associations is the "cause" or "effect" of changes in the transcriptional output of these genes. Together, these proposed live cell imaging experiments will define the nature of allele dynamics and interactions, and provide insight into the functional consequences of such dynamics/interactions. In the final Aim the same differentiation paradigm will be used to identify genes that become monoallelically expressed upon differentiation. The active and inactive alleles will be characterized and a live cell imaging system will be developed to study the establishment of monoallelic gene expression. Together, the proposed studies will provide important insights into the underlying principles of nuclear organization and gene expression that will be critical to fully understanding the signals that regulate normal developmental progression and how they may be disrupted during disease progression and/or treatments.

Public Health Relevance

This study will identify the nuclear dynamics/associations of a series of genes that are central to the transition of pluripotent embryonic stem cells to the differentiated state. The findings of this study will provide important insights into the underlying principles of nuclear organization and gene expression that will be critical to fully understanding the signals that regulate normal developmental progression and how it may be altered during disease progression and/or treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042694-25
Application #
8641364
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Carter, Anthony D
Project Start
1990-04-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
25
Fiscal Year
2014
Total Cost
$746,284
Indirect Cost
$351,425
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724
Hogan, Megan S; Parfitt, David-Emlyn; Zepeda-Mendoza, Cinthya J et al. (2015) Transient pairing of homologous Oct4 alleles accompanies the onset of embryonic stem cell differentiation. Cell Stem Cell 16:275-88
Zepeda-Mendoza, Cinthya J; Mukhopadhyay, Swagatam; Wong, Emily S et al. (2015) Quantitative analysis of chromatin interaction changes upon a 4.3 Mb deletion at mouse 4E2. BMC Genomics 16:982
Bergmann, Jan H; Li, Jingjing; Eckersley-Maslin, Mélanie A et al. (2015) Regulation of the ESC transcriptome by nuclear long noncoding RNAs. Genome Res 25:1336-46
Eckersley-Maslin, Mélanie A; Thybert, David; Bergmann, Jan H et al. (2014) Random monoallelic gene expression increases upon embryonic stem cell differentiation. Dev Cell 28:351-65
Bergmann, Jan H; Spector, David L (2014) Long non-coding RNAs: modulators of nuclear structure and function. Curr Opin Cell Biol 26:10-8
Eckersley-Maslin, Mélanie A; Spector, David L (2014) Random monoallelic expression: regulating gene expression one allele at a time. Trends Genet 30:237-44
Shi, Junwei; Whyte, Warren A; Zepeda-Mendoza, Cinthya J et al. (2013) Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes Dev 27:2648-62
Bodnar, Megan S; Spector, David L (2013) Chromatin meets its organizers. Cell 153:1187-9
Hübner, Michael R; Eckersley-Maslin, Mélanie A; Spector, David L (2013) Chromatin organization and transcriptional regulation. Curr Opin Genet Dev 23:89-95
Dowen, Jill M; Bilodeau, Steve; Orlando, David A et al. (2013) Multiple structural maintenance of chromosome complexes at transcriptional regulatory elements. Stem Cell Reports 1:371-8

Showing the most recent 10 out of 69 publications