1: J Immunol. 2004 Jan 15;172(2):1054-64. 

NF-kappa B and Oct-2 synergize to activate the human 3' Igh hs4 enhancer in B
cells.

Sepulveda MA, Emelyanov AV, Birshtein BK.

Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris
Park Avenue, Bronx, NY 10461, USA.

In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs)
sites with enhancer activity. These include the 3' Igh enhancers, which are
located downstream of the Calpha gene(s) in both mouse and human. In vivo
experiments have implicated murine 3' enhancers, hs3B and/or hs4, in class
switching and somatic hypermutation. We previously reported that murine hs4 was
regulated by NF-kappaB, octamer binding proteins, and Pax5 (B cell-specific
activator protein). In this study we report that human hs4 is regulated
differently. EMSAs and Western analysis of normal B cells before and after
stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern
formed by NF-kappaB, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern
was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in
diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1)
binding was also observed. We have confirmed the in vivo association of these
transcription factors with hs4 in B cells by chromatin immunoprecipitation
assays. The diffuse large B cell lymphoma cell lines had a distinctive
slow-migrating complex containing YY1 associated with Rel-B. We have confirmed
by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not
with other NF-kappaB family members. Transient transfection assays showed robust
hs4 enhancer activity in the mature B cell lines, which was dependent on
synergistic interactions between NF-kappaB and octamer binding proteins. In
addition, human hs4 enhancer activity required Oct-2 and correlated with
expression of Oct coactivator from B cells (OCA-B).

PMID: 14707079 [PubMed - indexed for MEDLINE]


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2: J Immunol. 2003 Jun 1;170(11):5549-57. 

Yin Yang 1 is a lipopolysaccharide-inducible activator of the murine 3' Igh
enhancer, hs3.

Gordon SJ, Saleque S, Birshtein BK.

Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY
10461, USA.

The 3' Igh enhancers, DNase I hypersensitive site (hs) 3B and/or hs4, are
required for germline transcription, and hence, class switch recombination for
multiple isotypes. A number of hs3-binding transcription factors have been
identified by EMSA, including octamer and NF-kappa B family members, and Pax5.
We have found that the binding of the transcription factor, Yin Yang 1 (YY1), to
hs3 and to the mu E1 site of the intronic enhancer, E mu, is induced in primary
splenic B cells after approximately 48 h in response to LPS and other activators
of class switch recombination. 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 binding of YY1 to DNA. We found that recombinant retinoblastoma
protein (Rb) inhibited binding of YY1 to hs3 in a dose-dependent manner, and we
have identified complexes of endogenous YY1 with the Rb in resting B cells, but
not in LPS-stimulated B cells. A difference in Rb phosphorylation state was also
confirmed between resting (G(0)) B cells and LPS-stimulated B cells. These
observations suggest that the interaction of YY1 with hypophosphorylated Rb in
resting B cells prevents interaction of YY1 with DNA. After stimulation with
class-switching activators, such as LPS, Rb becomes hyperphosphorylated and YY1
is released and can then bind to the hs3 enhancer and E mu.

PMID: 12759432 [PubMed - indexed for MEDLINE]


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