Dynamic Protein Interactions with the 3' Regulatory Region of the IgH locus during B cell Development and Class Switching
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The immunoglobulin heavy (IgH) chain locus is subject to several tightly regulated processes, i.e. VDJ joining, somatic hyper mutation and class switching (CSR). One of two key cis regulatory elements in this locus, the 3' regulatory region (3' RR) contains multiple hypersensitive sites and is required for CSR and IgH expression. Chromatin conformation capture assays revealed that all the 3' RR hypersensitive sites interact with each other and with distal VH and I region sequences while driving non-coding transcripts required for CSR and maintaining IgH expression. In this thesis, I have identified mechanisms that enable the 3' RR to act at long distances on target IgH sequences.;I used chromatin immuno-precipitation (ChIP) assays to analyze binding of key factors to the 3' RR during B cell development and class switching. These include YY1, Pax5, CTCF, cohesin, CBP and RNA polymerase II (PolII).;We found that Pax5 and CTCF binding to 3' RR were developmentally regulated. Pax5 binds to hs1.2 in pro-B cells while in B cell lines Pax5 mainly occupies hs4. CTCF binds to the hs5/6/7 region at early stages of B cell development; however, in a plasma cell line, it additionally binds to the palindromic enhancer hs 1.2.;ChIP experiments were done in resting splenic B cells, and in cells stimulated for 48 hr when germ line transcripts are expressed and 96 hr when CSR has occurred. These experiments revealed temporal changes in binding of Pax5, YY1, PolII and cohesin to the 3' RR during LPS stimulation. The pattern for Pax5 and YY1 binding was different in cells stimulated by anti-IgM+IL4, which express normal levels of germ line transcripts (GT) without CSR. These data imply a role for Pax5 and YY1 in the CSR process. The binding profile of other proteins was similar in LPS and anti-IgM stimulated cells, suggesting their contribution to GT but an indirect role in CSR.;Our data showed that dynamic changes in protein binding to 3' RR occur during B cell development and stimulation for CSR. These proteins, together, form a scaffold to facilitate B cell specific processes, such as GT and CSR.
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