Yin Yang 1 Is a Lipopolysaccharide-Inducible Activator of the Murine 3 Igh Enhancer, hs3 1 Steven J. Gordon, Shireen Saleque, 2 and Barbara K. Birshtein 3 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, in- cluding octamer and NF-B family members, and Pax5. We have found that the binding of the transcription factor, Yin Yang 1 (YY1), to hs3 and to the E1 site of the intronic enhancer, E, is induced in primary splenic B cells after 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. The Journal of Immunology, 2003, 170: 5549 –5557. D uring B cell differentiation, the Igh locus undergoes unique DNA rearrangements and modifications, includ- ing V(D)J joining, class switch recombination, and so- matic hypermutation (1). These changes in the Igh locus are ex- quisitely regulated by a number of cis-acting elements, including the 5' V H promoters, the intronic enhancer, E, and the 3' Igh regulatory region, which is composed of four enhancers (DNase I hypersensitive site (hs) 4 3A, hs1, 2, hs3B, and hs4) (2). E is essential for early events in B cell differentiation, including V3 DJ joining and  expression (3, 4), and recent experiments have shown that the Igh 3' enhancers are essential for H chain class switching. Targeted clean deletions of hs3B and hs4 (5), but not of hs3A or hs1,2 (6), resulted in defects in class switching to many isotypes at the level of germline transcription (GT). Hs3B and hs4 together may also contribute to surface IgM expression in resting B cells (5) and are essential for maintaining high levels of 2b mRNA expression in a plasma cell line (7). The 3' Igh enhancers are also candidates for influencing the dysregulation of the expres- sion of c-myc that occurs as a result of Igh:c-myc chromosomal translocations in Burkitt’s lymphoma, and mouse and human my- eloma cells (8). In mice, hs3A and hs3B are arranged in a single 25-kb in- verted repeat centered at hs1,2 (9, 10). Although targeted deletion may have identified a distinctive role for hs3B as compared with hs3A, the sequences of hs3A (GenBank accession U65625) and hs3B (GenBank accession S74164) in mice are virtually identical, and both enhancers are equally active in transcriptional assays (9). Therefore, we have considered hs3A and hs3B to be functionally interchangeable in our studies. Hs3A, hs1,2, and hs3B appear to comprise a distinct functional unit, becoming DNase I hypersen- sitive and active in transient transfection assays only later in B cell development (9). In contrast, the most 3' enhancer, hs4, appears to be active throughout B cell differentiation (11, 12). The 3' Igh enhancers are regulated by an array of transcription factors, including but not limited to Oct-1, Oct-2, NF-B, and Pax5 (B cell-specific activating protein (BSAP)) (12–14). Pax5, in particular, functions as an early B cell-specific regulator of the hs1,2 and hs4 enhancers (14, 15). Additional motifs within the hs1,2 enhancer are bound inducibly to protein complexes after the treatment of splenic B cells with LPS (NF-B), anti-IgM, or CD40 ligand (Jun-B, Elf-1) (16, 17). Little information is currently avail- able on the factors that regulate mouse hs3. Previous studies have predicted octamer sites (13) and Maf recognition elements within hs3 (18). We wanted to identify transcription factors that might impact on a role of hs3 in CSR. B cells activated by LPS or anti-CD40 are stimulated to proliferate and to undergo class switch recombina- tion (19). Various cytokines (i.e., IL-4, IFN-, TGF-, and IL-10) direct switching to particular isotypes by enhancing expression of specific germline transcripts (19). In this study, we discovered the induction of an hs3 enhancer-binding protein, Yin Yang 1 (YY1), a zinc finger transcription factor, after 48-h treatment of primary splenic B cells with LPS, LPS + IL-4, and anti-CD40 + IL-4. YY1 binding to the E1 site of the intronic enhancer is also in- duced. We find that YY1 is a transcriptional activator of the hs3 enhancer, as has been demonstrated previously for the engagement of YY1 with the E1 site of E (20, 21). Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 Received for publication December 20, 2002. Accepted for publication March 21, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health AI13509 and by an Albert Einstein Cancer Center Grant P30 CA13330. 2 Current address: Department of Hematology and Oncology, Children’s Hospital and Harvard Medical School, Boston, MA 02115. 3 Address correspondence and reprint requests to Dr. Barbara K. Birshtein, Depart- ment of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave- nue, Bronx, NY 10461. E-mail address: birshtei@aecom.yu.edu 4 Abbreviations used in this paper: hs, DNase I hypersensitive site; BSAP, B cell- specific activating protein; GDI, guanine nucleotide dissociation inhibitor; GT, germ- line transcription; Rb, retinoblastoma protein; YY1, Yin Yang 1. 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