Regulation of the Yin Yang 1 transcription factor binding to the murine 3' Igh enhancers hs3A and hs3B, by inducers of class switch recombination

Abstract

The 3' Igh enhancers, hs3B and/or hs4, are required for germline transcription (GT), and hence, class switch recombination (CSR) for multiple isotypes. A number of hs3-binding transcription factors have been identified by EMSA, including octamer and NF-kB family members, and Pax5. We have found that the binding of the transcription factor, Yin Yang 1 (YY1), to hs3 and to the muE1 site of the intronic enhancer, Emu, is induced in primary splenic B cells after ∼48 hrs in response to LPS and other activators of CSR. Transient transfection experiments in B cell lines indicate that YY1 is an activator of hs3. Interestingly, levels of YY1 expression are unchanged in resting and LPS-stimulated B cells. Mixing experiments followed by EMSA showed that a protein present in resting B cells prevented YY1's binding to DNA. We found that recombinant Rb protein inhibited binding of YY1 to hs3 in a dose-dependent manner; and we have identified complexes of endogenous YY1 with the retinoblastoma protein (Rb) in resting B cells, but not in LPS stimulated B cells. A difference in Rb phosphorylation state was also confirmed between resting (G0) B cells and LPS stimulated B cells. These observations suggest that the interaction of YY1 with hypo-phosphorylated Rb in resting B cells prevents YY1's interaction with DNA. After stimulation with class switching activators, such as LPS, Rb becomes hyper-phosphorylated, YY1 is released and can then bind to the hs3 enhancer and Emu. Our data provide the first evidence for the role of YY1 in regulating hs3 enhancer activity, and suggest a mechanism by which cell cycle control and the early stages of class switch recombination, mediated by the 3' Igh and intronic enhancers, can be linked at the molecular level.