Employing chromosome conformation capture (3C) technology, we will determine how the long-range spatial organization of the mouse Igk gene locus changes as a function of B cell development and gene activation. We will explore the modulation of interactions between known cis-acting elements in the locus before and after gene rearrangement and transcriptional activation. We will also determine which of several transcription factors that are known to interact with the enhancers are present in looped complexes. We will further determine which of several selected histone post-translational modifications are present in such looped complexes. For these studies we will use a panel of cell lines arrested at various stages of B cell differentiation, as well as primary pro-, pre- and mature-B cells from animals. We will also determine whether NF-kB or E2A (or both) are required for loop formation in pre-B cells. Finally, we will functionally elucidate a novel B-cell specific hypersensitive site at the 3'boundary of the locus that we first discovered by the 3C technology. Completion of these studies will provide new mechanistic insights on Ig gene regulation at the level of higher-order chromatin structure, transcription, recombination, and communication between cis-acting elements and trans-acting factors. The following four specific aims are proposed: 1. To elucidate separately the higher-order chromatin organization of germline and rearranged Igk alleles as a function of B cell development and transcription. 2. To determine if certain transcription factors or specific post-translational modifications of histone H3 are present in looped complexes between Igk gene enhancers and rearranged V gene or germline promoters. 3. Define the importance of NF-kB or E2A (or both) in the formation of looped complexes between cis-acting sequences in pre-B cells. 4. To functionally elucidate a novel B-cell specific hypersensitive site at the 3'boundary of the Igk gene locus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI067906-04
Application #
7803725
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Nasseri, M Faraz
Project Start
2007-05-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
4
Fiscal Year
2010
Total Cost
$381,193
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Xiang, Yougui; Park, Sung-Kyun; Garrard, William T (2014) A major deletion in the V?-J? intervening region results in hyperelevated transcription of proximal V? genes and a severely restricted repertoire. J Immunol 193:3746-54
Park, Sung-Kyun; Xiang, Yougui; Feng, Xin et al. (2014) Pronounced cohabitation of active immunoglobulin genes from three different chromosomes in transcription factories during maximal antibody synthesis. Genes Dev 28:1159-64
Zhou, Xiaorong; Xiang, Yougui; Ding, Xiaoling et al. (2013) Loss of an Igýý gene enhancer in mature B cells results in rapid gene silencing and partial reversible dedifferentiation. Mol Cell Biol 33:2091-101
Xiang, Yougui; Park, Sung-Kyun; Garrard, William T (2013) Výý gene repertoire and locus contraction are specified by critical DNase I hypersensitive sites within the Výý-Jýý intervening region. J Immunol 190:1819-26
Zhou, Xiaorong; Xiang, Yougui; Ding, Xiaoling et al. (2012) A new hypersensitive site, HS10, and the enhancers, E3' and Ed, differentially regulate Ig? gene expression. J Immunol 188:2722-32
Xiang, Yougui; Zhou, Xiaorong; Hewitt, Susannah L et al. (2011) A multifunctional element in the mouse Ig* locus that specifies repertoire and Ig loci subnuclear location. J Immunol 186:5356-66
Zhou, Xiaorong; Xiang, Yougui; Garrard, William T (2010) The Ig? gene enhancers, E3' and Ed, are essential for triggering transcription. J Immunol 185:7544-52
Liu, Zhe; Ma, Zhenyi; Terada, Lance S et al. (2009) Divergent roles of RelA and c-Rel in establishing chromosomal loops upon activation of the Igkappa gene. J Immunol 183:3819-30
Xiang, Yougui; Garrard, William T (2008) The Downstream Transcriptional Enhancer, Ed, positively regulates mouse Ig kappa gene expression and somatic hypermutation. J Immunol 180:6725-32
Xiao, Fei; Widlak, Piotr; Garrard, William T (2007) Engineered apoptotic nucleases for chromatin research. Nucleic Acids Res 35:e93