Enhancer, promoters, and insulators are regulatory sequences essential for the establishment of specific patterns of gene expression during cell differentiation It is becoming increasingly evident that these sequences, often located far from each other in the genome, interact in the nuclear space over long distances to regulate the establishment of distinct transcriptional profiles characteristic of specific cellular fates. The requirement for loal interactions at specific loci, for example between enhancers and promoters, has been studied in detail. Nevertheless, the extent and function of long-range inter- and intra-chromosomal interactions mediated by these sequences at the level of the whole genome and its role in establishing and/or maintaining specific gene expression profiles is largely unexplored. During the last funding period we have characterized the protein composition of Drosophila insulators and determined the genome-wide localization patterns of known insulator proteins. We have also used 5C/HiC technology to gather data that give us a glimpse into the complexity of the intra-chromosomal interactions mediated by insulators. We are now in an ideal position to carry out the experiments required to understand the role of the three-dimensional arrangement of the chromatin fiber in the nuclear space in the regulation of gene expression. Here we propose to use HiC and ChIA-PET to establish detailed maps of intra- and inter-chromosomal interactions in the Drosophila genome. Preliminary experiments suggest that transcription and insulators collaborate in the establishment of the 3D organization of the genome and we will carry out functional experiments to test this hypothesis. Point to point interactions between enhancer, promoters and insulators will be analyzed by ChIA-PET in order to understand the mechanisms by which insulators control gene expression. The 3D arrangement of the chromatin during S and M phases will be analyzed by HiC and results will be compared to those obtained with interphase cells to understand the possible role of insulators in the control of genome architecture throughout the cell cycle. Finally, covalent modifications responsible for the regulation of insulator activity will be analyzed and their role in mediating specific inter- and intra-chromosomal interactions will be determined. In the end, we hope to define the role of insulators in the regulation of the three- dimensional organization of the genome and its role in different aspects of nuclear biology. This three-dimensional organization may serve as a fingerprint of cell identity that synthesizes the epigenetic status of the cell, and may help to identify the state of differentiated, trans- differentiated or reprogrammed cells for use in human cell therapy.

Public Health Relevance

This study will determine the contribution of the three-dimensional organization of the genetic material in the nucleus to the establishment of distinct patterns of gene expression required during cell differentiation. This organization may represent a fingerprint indicative of the differentiation status of stem or reprogrammed cells used for cell therapy of various human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM035463-26A1
Application #
8515726
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
1986-04-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
26
Fiscal Year
2013
Total Cost
$577,836
Indirect Cost
$204,736
Name
Emory University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Ong, Chin-Tong; Corces, Victor G (2014) CTCF: an architectural protein bridging genome topology and function. Nat Rev Genet 15:234-46
Van Bortle, Kevin; Corces, Victor G (2014) Lost in transition: dynamic enhancer organization across naive and primed stem cell states. Cell Stem Cell 14:693-4
Gómez-Díaz, Elena; Corces, Victor G (2014) Architectural proteins: regulators of 3D genome organization in cell fate. Trends Cell Biol 24:703-11
Van Bortle, Kevin; Nichols, Michael H; Li, Li et al. (2014) Insulator function and topological domain border strength scale with architectural protein occupancy. Genome Biol 15:R82
Gurudatta, B V; Yang, Jingping; Van Bortle, Kevin et al. (2013) Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle. Cell Cycle 12:1605-15
Ong, Chin-Tong; Van Bortle, Kevin; Ramos, Edward et al. (2013) Poly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in Drosophila. Cell 155:148-59
Timmerman, Christina; Suppiah, Somu; Gurudatta, Baraka V et al. (2013) The Drosophila transcription factor Adf-1 (nalyot) regulates dendrite growth by controlling FasII and Staufen expression downstream of CaMKII and neural activity. J Neurosci 33:11916-31
Phillips-Cremins, Jennifer E; Corces, Victor G (2013) Chromatin insulators: linking genome organization to cellular function. Mol Cell 50:461-74
Yang, Jingping; Sung, Elizabeth; Donlin-Asp, Paul G et al. (2013) A subset of Drosophila Myc sites remain associated with mitotic chromosomes colocalized with insulator proteins. Nat Commun 4:1464
Van Bortle, Kevin; Corces, Victor G (2013) The role of chromatin insulators in nuclear architecture and genome function. Curr Opin Genet Dev 23:212-8

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