Here we propose to continue our studies on functionally elucidating cis-acting regulatory elements in the mouse immunoglobulin (Ig) gene locus. Namely, the elements that either inhibit or target locus accessibility to the recombinational and transcriptional machinery in B- and T-lymphocytes. During previous funding cycles of this grant we discovered the following two new regions in Ig: chromatin that contain B-cell-specific and developmentally regulated DNase I hypersensitive sites (HSs): (1) A silencer (Sis) within the intervening sequence between the joining (J?) region and the most proximal V? gene, which upon V? - J? joining is either deleted or inverted and far removed from the rearranged V? gene destined to be expressed. Importantly, we showed during the last funding cycle that this element is a recombination silencer responsible for recruiting Ikaros and targeting an Igk minilocus carried on a yeast artificial chromosome (YAC) to pericentromeric heterochromatin;(2) A powerful enhancer (Ed) in the distal downstream region, which is always preserved after normal V? gene rearrangement. Importantly, we showed during the last funding cycle that this element is required for generating maximal levels of Ig: transcripts, and maximal levels of somatic hypermutation (SHM) in germinal center (GC) B cells. In this renewal we plan to focus our studies specifically on further functional delineation of the Sis element by taking advantage of the unique reagents that we have generated over the past quarter of a century under the funding of this grant.
The specific aims are: 1. To determine the impact of deleting Sis on Ig loci nuclear organization. (Parameters investigated will include pericentromeric heterochromatin localization, Igk allele contraction and looping, and heterologous and homologous Ig allele chromosome pairing.) 2. To determine distal DNA sequences that Sis interacts with that may be involved in Igk allele contraction and looping, or in heterologous or homologous Ig allele chromosome pairing. 3. To determine the impact of deleting Sis on the Ig: repertoire, with respect to V gene usage and potential RAG protein targeting mechanisms. 4. To determine the impact of separately mutating Ikaros and CTCF binding sites within Sis on Ig loci nuclear organization, distal-Sis-interacting sequences, and repertoire.

Public Health Relevance

We have recently discovered a genetic region in the mouse kappa immunoglobulin gene that regulates antibody production and the diversity of antibody species that are generated during white blood cell ontogeny. This proposal will further delineate how this genetic element works at the molecular level.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular and Molecular Immunology - A Study Section (CMIA)
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Carter, Anthony D
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University of Texas Sw Medical Center Dallas
Schools of Medicine
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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

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