In FY13, we accomplished the following: 1) Continued analyses of transgenic mouse strains that contain BACs with modified IgH loci. Comparison of the epigenetic and transcriptional states of BAC transgenes with wild type (WT) and Emu-deleted (Delta E) IgH loci indicated that the Delta E BAC closely mimicked Emu-deleted endogenous IgH alleles. This permits further analysis of the consequences of recruiting specific transcription factors of chromatin remodeling enzymes to the Delta E transgenes as fusion proteins with the Gal4 DNA binding domain. Studies with pro-B cells from transgenic mice in which E was moved to different positions within the IgH locus were also initiated. Preliminary results indicate that the location of E is critical for function. These observations directly contradict that text-book dogma that enhancer function is distance- and orientation-independent. 2) Re-calibrated genome-wide DNA are I hypersensitive site methodology. Pro-B cell analysis is underway. We extended this methodology to initiate studies with primary B lymphocytes before and after activation via the B cell antigen receptor. 3) Extended the analysis of bivalent chromatin generation and resolution during hematopoiesis to incorporate two additional epigenetic features. There were the presence of H3K36me3, a mark associated with processively engaged RNA polymerase II, and the presence of H3K27me3 in the gene body (our previous analyses were restricted to the presence or absence of this mark in a 6kb window spanning the transcription initiation site). We found that resolution of bivalent chromatin can be transient. However, genes that resolved permanently to H3K4me3 only status were overwhelmingly H3K27me3 negative in the gene body during the bivalent stage. The oscillatory behavior of many genes between bivalent and monovalent states also led the realization that greater than 60% of bivalent genes present in hematopoietic stem cells (HSCS) did not resolve up to the stage of T lymphocyte lineage commitment. Additionally, novel de novo bivalent genes were generated in the thymus, with maximum generation occurring at the DN2 developmental stage. 3) We initiated a new line of investigation to assess the function of individual motifs of E. We followed two parallel approaches. In one approach, we generated competitive inhibitors of the muE1 site of the enhancer using TALEN technology. In the second approach, we generated targeting constructs to specifically mutate muE1, muA + muB, muE2+ muE5, and the IRF binding site in Emu.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000383-10
Application #
8736550
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2013
Total Cost
$171,298
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Lovely, Geoffrey A; Sen, Ranjan (2016) Evolving adaptive immunity. Genes Dev 30:873-5
Sen, Ranjan (2016) A Pioneer's Tail. Immunity 44:516-8
Montefiori, Lindsey; Wuerffel, Robert; Roqueiro, Damian et al. (2016) Extremely Long-Range Chromatin Loops Link Topological Domains to Facilitate a Diverse Antibody Repertoire. Cell Rep 14:896-906
Kumari, Gita; Sen, Ranjan (2015) Chromatin Interactions in the Control of Immunoglobulin Heavy Chain Gene Assembly. Adv Immunol 128:41-92
Feldman, Scott; Achour, Ikbel; Wuerffel, Robert et al. (2015) Constraints contributed by chromatin looping limit recombination targeting during Ig class switch recombination. J Immunol 194:2380-9
Gerasimova, Tatiana; Guo, Changying; Ghosh, Amalendu et al. (2015) A structural hierarchy mediated by multiple nuclear factors establishes IgH locus conformation. Genes Dev 29:1683-95
Selimyan, Roza; Gerstein, Rachel M; Ivanova, Irina et al. (2013) Localized DNA demethylation at recombination intermediates during immunoglobulin heavy chain gene assembly. PLoS Biol 11:e1001475
Kumar, Satyendra; Wuerffel, Robert; Achour, Ikbel et al. (2013) Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny. Genes Dev 27:2439-44
Sen, Ranjan; Fugmann, Sebastian D (2012) Transcription, splicing, and release: are we there yet? Cell 150:241-3
Subrahmanyam, Ramesh; Sen, Ranjan (2012) Epigenetic features that regulate IgH locus recombination and expression. Curr Top Microbiol Immunol 356:39-63

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