Lymphoid neoplasms (leukemias and lymphomas) are among the most common malignancies in humans, particularly children. Although they arise from diverse etiologies, the majority are linked to alterations in expression of oncogenes, tumor suppressors and / or transcription factors that are important for lineage specification. In order to identify te underlying cause of the various forms of these cancers it is important to understand the mechanisms underlying the different cellular processes that regulate gene expression in normal cells. We have focused our efforts on one particular aspect of gene regulation that remains poorly understood: enhancer mediated gene regulatory networks. Identifying the enhancer-mediated gene regulatory networks that underpin differentiation remains a challenge because it has been difficult to define the targets of enhancers. Indeed, individual enhancers can in some instances control multiple genes that are not necessarily located in close physical proximity in cis. Some studies have tried to address this using chromosome conformation capture to identify interactions between enhancers and promoters. However, it is not clear whether interactions are predictive of functional regulation so these approaches do not definitively determine the regulatory targets of individual enhancers and the extent to which they contribute to gene expression. To examine the effect of individual enhancers it is necessary to mutate these elements and characterize changes in interactions coupled with gene activity and binding of transcription factors. In this application we aim to perform a series of genome wide analyses to examine these aspects of control using mice that harbor deletions of enhancers associated with immunoglobulin and T cell receptor loci. The data from experiments we have performed to date support a model in which each enhancer participates in transcription by physically relocating each locus into specific sites, perturbation of which (e.g. by deleting a specific enhancer) can have extensive consequences on many loci (both in cis and in trans) that are expressed or repressed at the same site. Based on these findings and data from our lab and others showing that co-regulated genes come together in the nucleus, we hypothesize that the binding of transcription factors or regulators induces a distinct pattern of cell type, stage specific interactions, which form a network that contributes to the control of gene expression. In this application we aim to explore this further and test the limitations of our model by addressing the following questions: (1) What are the mechanisms underlying enhancer-mediated gene regulatory networks? (2) What is the contribution of RAG in controlling antigen receptor enhancer mediated networks? Can we genetically manipulate E? and observe changes in the enhancer mediated regulatory network? Overall the work we propose here will break new ground in our understanding of cell type-specific gene networks and the implications of coordinate locus expression during lymphoid development in cells undergoing normal and aberrant DNA recombination events.

Public Health Relevance

Although lymphoid neoplasms arise from diverse etiologies, the majority are linked to alterations in expression of oncogenes, tumor suppressors and / or transcription factors that are important for lineage specification. In this application we will focs our efforts on one particular aspect of gene regulation that remains poorly understood: enhancer mediated gene regulatory networks. Overall the work we propose here will break new ground in our understanding of cell type-specific gene networks and the implications of coordinate locus expression during lymphoid development in cells undergoing normal and aberrant DNA recombination events.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM112192-01
Application #
8748701
Study Section
Special Emphasis Panel (ZRG1-IMM-N (03))
Program Officer
Marino, Pamela
Project Start
2014-09-05
Project End
2018-08-31
Budget Start
2014-09-05
Budget End
2015-08-31
Support Year
1
Fiscal Year
2014
Total Cost
$525,166
Indirect Cost
$212,516
Name
New York University
Department
Pathology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Proudhon, Charlotte; Snetkova, Valentina; Raviram, Ramya et al. (2016) Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation. Cell Rep 15:2159-69
Rocha, Pedro P; Raviram, Ramya; Fu, Yi et al. (2016) A Damage-Independent Role for 53BP1 that Impacts Break Order and Igh Architecture during Class Switch Recombination. Cell Rep 16:48-55
Fu, Yi; Rocha, Pedro P; Luo, Vincent M et al. (2016) CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. Nat Commun 7:11707
Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P et al. (2016) Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination. J Exp Med 213:303-12
Raviram, Ramya; Rocha, Pedro P; Müller, Christian L et al. (2016) 4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments. PLoS Comput Biol 12:e1004780
Narendra, Varun; Rocha, Pedro P; An, Disi et al. (2015) CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation. Science 347:1017-21
Blumenberg, Lili; Skok, Jane A (2015) RAG Off-Target Activity Is in the Loop. Trends Mol Med 21:733-5
Proudhon, Charlotte; Hao, Bingtao; Raviram, Ramya et al. (2015) Long-Range Regulation of V(D)J Recombination. Adv Immunol 128:123-82
Mullenders, Jasper; Aranda-Orgilles, Beatriz; Lhoumaud, Priscillia et al. (2015) Cohesin loss alters adult hematopoietic stem cell homeostasis, leading to myeloproliferative neoplasms. J Exp Med 212:1833-50
Rocha, Pedro P; Raviram, Ramya; Bonneau, Richard et al. (2015) Breaking TADs: insights into hierarchical genome organization. Epigenomics 7:523-6

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