The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gordon, S. J.
Right arrow Articles by Birshtein, B. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gordon, S. J.
Right arrow Articles by Birshtein, B. K.
The Journal of Immunology, 2003, 170: 5549-5557.
Copyright © 2003 by The American Association of Immunologists

Yin Yang 1 Is a Lipopolysaccharide-Inducible Activator of the Murine 3' Igh Enhancer, hs3 1

Steven J. Gordon, Shireen Saleque2 and Barbara K. Birshtein3

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 µE1 site of the intronic enhancer, Eµ, 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 (G0) 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 Eµ.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During B cell differentiation, the Igh locus undergoes unique DNA rearrangements and modifications, including V(D)J joining, class switch recombination, and somatic hypermutation (1). These changes in the Igh locus are exquisitely regulated by a number of cis-acting elements, including the 5' VH promoters, the intronic enhancer, Eµ, 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). Eµ is essential for early events in B cell differentiation, including V->DJ joining and µ 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 {gamma}2b mRNA expression in a plasma cell line (7). The 3' Igh enhancers are also candidates for influencing the dysregulation of the expression of c-myc that occurs as a result of Igh:c-myc chromosomal translocations in Burkitt’s lymphoma, and mouse and human myeloma cells (8).

In mice, hs3A and hs3B are arranged in a single ~25-kb inverted 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 hypersensitive 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-{kappa}B, and Pax5 (B cell-specific activating protein (BSAP)) (12, 13, 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-{kappa}B), anti-IgM, or CD40 ligand (Jun-B, Elf-1) (16, 17). Little information is currently available 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 recombination (19). Various cytokines (i.e., IL-4, IFN-{gamma}, TGF-{beta}, 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 µE1 site of the intronic enhancer is also induced. We find that YY1 is a transcriptional activator of the hs3 enhancer, as has been demonstrated previously for the engagement of YY1 with the µE1 site of Eµ (20, 21).

YY1 can function as a transcriptional repressor or activator (22), most likely reflecting the differential association of YY1 with its many interaction partners. These include numerous histone deacetylases (23, 24) and histone acetyl transferases, such as p300, CREB binding protein (CBP), and p300/CBP-associated factor (PCAF) (24). YY1 has been proposed to be a mammalian polycomb protein, based on the extensive identity of its C-terminal DNA binding domain to the Drosophila pleiohomeotic protein (25). As such, YY1 interacts with the RYBP protein, which is associated with a transcriptionally repressive polycomb complex (26), and with YAF-2, which, although closely related to RYBP, is a coactivator of YY1 (27).

YY1 has been reported to be constitutively expressed (22), and we confirmed this in resting and activated splenic B cells. Because an important role of LPS is to stimulate entry of resting B cells into the cell cycle, we were interested in whether a cell cycle regulatory protein could modulate the binding of YY1 to hs3. Recent studies reported that the retinoblastoma protein (Rb) binds to YY1 in serum-starved coronary artery smooth muscle cells, but not in cycling coronary artery smooth muscle cells, suggesting that an interaction between YY1 and Rb is subject to cell cycle regulation (28). Our data show that LPS and other class-switching regimens lead to the dissociation of YY1 from Rb in primary B cells and the activation of the Igh enhancers, hs3 and Eµ. This study provides the first evidence for the role of YY1 in regulating hs3 enhancer activity, and suggests a mechanism by which cell cycle control and the early stages of class switching, mediated by the 3' Igh and intronic enhancers, can be linked at the molecular level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of cell lines and primary B cells

Cell lines included 18-81 (BALB/c, pre-B; µ), A20 (BALB/c, B cell lymphoma; {gamma},{kappa}), M12.4.1 (B cell lymphoma; membrane IgM-, {alpha}), S194 (BALB/c, myeloma; {alpha},{kappa}), and S107 (BALB/c, myeloma; {alpha},{kappa}). Splenic B cells were isolated by RBC lysis and T cell depletion. Briefly, 10 spleens from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were dissected and homogenized in 10 ml of HBSS (Life Technologies, Gaithersburg, MD). The splenocytes were pelleted (5 min at 2000 rpm) and resuspended in 10 ml of RBC lysis buffer (Puregene, Minneapolis, MN), then pelleted and resuspended in 25 ml anti-T cell Ag cocktail (TAC) and incubated on ice for 30 min (TAC contains anti-Thy-1 ascites or anti-Thy-1.2 mAb (Southern Biotechnology Associates, Birmingham, AL; 1750-01), anti-CD4 (GK1.5), and anti-CD8 (Ly-2) hybridoma or anti-CD8a mAb (BD PharMingen, San Diego, CA; 553026)). Hybridoma supernatants and ascites were all generously provided by C. Snapper (Uniformed Sciences Health Service Academy, Bethesda, MD). Cells were pelleted and resuspended in 20 ml of prewarmed complement cocktail (containing Mar 18.5 hybridoma (anti-rat {kappa}) and guinea pig complement (Life Technologies) in buffered HBSS) and incubated at 37°C for 30 min. Cells were washed with cold HBSS. More recently, B cells were further purified by negative selection using anti-CD43 magnetic beads (Miltenyi Biotec, Paris, France) for MACS, according to the manufacturer’s protocols. B cell purity was checked after purification by FACS analysis with FITC B220 and PE CD3e Abs (BD PharMingen). More than 98% of splenocytes were B220+, CD3-, CD43-.

B cells were resuspended in RPMI 1640 medium (BioWhittaker, Walkersville, MD), supplemented with 10% heat-inactivated FCS (Gemini, Woodland, CA), containing 50 µM 2-ME and 100 U/ml of penicillin-streptomycin. Cells were plated at a concentration of 2–3 x 106 cells/ml with the appropriate concentration of inducer for 48 h at 37°C in an atmosphere of 5% CO2. For induction of class switching in primary cells, LPS (Calbiochem, San Diego, CA; 437625, from Escherichia coli serotype 055:B5) was added to medium at a concentration of 15 µg/ml, anti-mouse CD40 (BD PharMingen) was used at 2.5 µg/ml, and mouse rIL-4 (R&D Systems, Minneapolis, MN) was used at 3000 U/ml. Additionally, 1 x 106 B cells were stained with 0.05 mg/ml propidium iodide in 0.1% sodium citrate, subjected to cell cycle analysis before and after induction by LPS for 48 h on FACSCalibur, and analyzed using CellQuest software (BD Biosciences, San Diego, CA).

Plasmids and site-directed mutagenesis

Murine hs3B was PCR amplified from the 13-kb C57BL/6 YAC subcloned with primers flanked by SmaI sites and inserted into the SmaI site of the vector QM293luc (9), which contains the B cell-specific V{lambda}1 promoter (14). The mutated hs3B monomer was constructed by performing two separate PCR and ligating the resultant fragments. The hs3-5' primer (5'-gggccctctagaaccacatgcgatcta-3') and mLRE antisense primer were used to amplify the 5' portion of hs3, and mLRE sense (5'-ccacactcgtgccttagaattcccatgttctgtcccaa-3') and hs3-3' primer (5'-cccgggatcattgagctccggctcta-3') were used to amplify the 3' portion of hs3. Mutated residues are in bold type. Dimers of hs3B wt and mutant were generated by ligation of individual monomers for 4 h to overnight at 16°C, followed by gel isolation and ligation with SmaI-linearized QM293luc, as described above. The Rous sarcoma virus-{beta}-galactosidase plasmid has been described (29). All plasmids were purified using Qiagen (Valencia, CA) kits, as described by the manufacturer.

EMSA and methylation interference footprinting

Nuclear extracts were prepared from cell lines, as previously described (29), and EMSA was performed, as indicated (14), with 20 µg of nuclear extracts per lane. Hs3B-derived probes used for EMSA were PCR amplified from the ~1.2-kb hs3B enhancer cloned into the QM293 vector as a template. Digestion with BsmAI yielded four enhancer fragments of 360, 323, 290, and 206 bp. The 323-bp fragment was further divided into a 5' 111-bp and a 3' 212-bp segment by digestion with StyI. The smaller 111-bp fragment was used for methylation footprinting assays described below. Subsequently, a PCR-derived 134-bp fragment (sense, 5'-gggttgtctctggct3'; antisense, 5'-cccatcacccaaggc-3') encompassing the 111-bp sequence was used as a probe for EMSA. Probes were end labeled with [{gamma}-32P]dATP using polynucleotide kinase (Roche, Indianapolis, IN), or end filled with exo (-) Klenow enzyme (Stratagene, La Jolla, CA) and [{alpha}-32P]dCTP. The sequences of double-stranded oligonucleotides were: µE1, 5'-ggatcggccatcttgactcaa-3' and its complement (30); mµE1, 5'-ggatcggccatggtgactcaa-3' and its complement (30); PU.1, 5'-gggctgcttgaggaagtataagaat-3' (Santa Cruz Biotechnology, Santa Cruz, CA; sc-2549) and its complement; Pax5, 5'-aggattgtgaagcgtgacca-3' and its complement; {kappa}B, 5'-gagaggggattccccgattagctttcggggaatcccctct-3' (self complementary); and octamer, 5'-(atttgcat)3-3' and its complement. Anti-YY1 Ab was purchased from Santa Cruz (sc-281X; C-20, polyclonal Ab against C terminus of YY1).

Methylation interference footprinting was conducted, as previously described (31), with minor modifications. Briefly, 5 x 105 cpm of hs3-111 labeled at the 5' (BsmA1) end was partially methylated for 2 min with dimethyl sulfate. A total of 2 x 104 cpm of this partially methylated probe was used in an EMSA reaction with 5 µg of nuclear extract prepared from primary splenic B cells stimulated for 48 h with LPS and IL-4. The free and bound probes were separated on a 5% acrylamide gel and then eluted onto a DEAE membrane (Schleicher & Schuell, NA45, Keene, NH). The shifted complex was purified and cleaved at methylated guanine residues with 10% piperidine (Sigma-Aldrich, St. Louis, MO). The cleaved free and bound probes were analyzed on an 8% denaturing sequencing gel, which was dried and subjected to autoradiography.

Western blotting

Nuclear and cytoplasmic extracts were prepared, as described above. A total of 50 µg of protein was loaded in each well of a 7.5 or 4–15% gradient SDS-PAGE (Bio-Rad, Hercules, CA). Proteins were transferred onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia, Buckinghamshire, U.K.). Membranes were blocked in PBST (PBS and 0.1% Tween 20) containing 5% nonfat dry milk, first incubated with primary Abs at a 1/1000 dilution for 4 h at 4°C, and then with the appropriate HRP-conjugated secondary Ab (Santa Cruz). Proteins were detected using ECL reagent and Hyperfilm ECL (Amersham Pharmacia). Abs used were against YY1 (sc-281x; C-20) and guanine nucleotide dissociation inhibitor (GDI) (kindly provided by P. Bickel, Washington University, St. Louis, MO). GDI Ab was used for normalization of total protein in each lane, as described (32). Rb mAb was from BD PharMingen (554136).

Transient transfection assays

A total of 2 x 107 cells of the appropriate M12.4.1 and S194 cell lines growing in log phase was transfected with 20 µg of reporter DNA by the DEAE-dextran method (14). Following transfection, S194 cells were treated with 100 µM chloroquine for 30 min, while M12.4.1 cells were plated directly into medium. Cells were harvested 48 h after transfection, and luciferase assays were performed, as described previously (14), with the luciferase reporter assay system (Promega, Madison, WI). A total of 1 x 107 S107 or A20 cells was electroporated at 400 V using a Bio-Rad gene pulser II (Hercules, CA). A total of 2.5 µg of Rous sarcoma virus-{beta}-galactosidase was included as an internal transfection control in all transfection experiments. Luciferase values were normalized to {beta}-galactosidase activity, which was determined using the Galacto-Light plus kit (Tropix, Bedford, MA). Each transfection experiment was done in triplicate and repeated three times.

Purification of recombinant proteins

The PGEX-2T plasmid containing GST in-frame with the large pocket sequence of Rb (aa 379–928) (33) was kindly provided by L. Zhu (Albert Einstein College of Medicine). Briefly, the plasmid was transformed into BL21 (pLysE) (Invitrogen, Carlsbad, CA), and colonies were inoculated overnight into Luria-Bertani broth, then transferred at a 1/100 dilution into Luria-Bertani/Amp liquid medium. When cells reached an OD600 of 0.6, protein expression was induced with 0.1 mM isopropyl {beta}-D-thiogalactoside and cells were grown for another 6 h at 37°C. The protein was then purified with glutathione-Sepharose beads (Amersham Pharmacia), according to the standard protocol provided in the GST gene fusion manual available at apbiotech.com, and its function was confirmed by an E1A pulldown assay from 293T cells, as described (33). Recombinant full-length Rb and its mock purification produced in SF9 cells were generously provided by K. Rehktman (Albert Einstein College of Medicine).

Immunoprecipitations

Primary B cells were lysed in nondenaturing lysis buffer (1% Triton X-100, 50 mM Tris, 300 mM NaCl, 5 mM EDTA). Sigma-Aldrich phosphatase inhibitor cocktails (P5726, P2850) and protease inhibitor cocktail (P2714) were added freshly to lysis buffer. Total cell lysates (1 mg) were incubated 4 h at 4°C with protein G-agarose (Sigma-Aldrich) and 2 µg of anti-YY1 (sc-7341; H-10, mAb). Beads were washed five times in wash buffer (0.1% Triton X-100, 50 mM Tris, 300 mM NaCl, 5 mM EDTA), and proteins were eluted with SDS gel-loading buffer (Bio-Rad; 161-0737).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the transcription factor binding sites in hs3

To identify how hs3 is regulated at the DNA level, we initiated a search for transcription factor binding sites within the hs3 enhancer sequence. We analyzed the 1.2-kb hs3B enhancer sequence with the Transfac 4.0 program (34). Four potential octamer elements were identified. Although three of the sites had previously been predicted, the most 5' octamer site (Fig. 1, probe A, and Table I) was potentially a new binding site. We also found tandem NF-{kappa}B sites, each with an 8/10-bp match to the consensus (Fig. 1A, probe D, and Table I). To assess transcription factor binding across a range of B cell lineages, we divided the hs3B enhancer into four fragments by restriction digestion (Fig. 1A) and performed EMSA on each probe with nuclear extracts from 18-81 pre-B (data not shown), A20 mature B, and S194 plasma cell lines.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 1. Predicted transcriptional regulators of murine 3' Igh enhancer, hs3. A, Schematic map of the mouse Igh locus shows V, D, J, and C region genes in addition to the 3' regulatory region. Enhancer elements are shown as gray ovals. Hs3A and hs3B are virtually identical by sequence. Our studies have used hs3B as a model for both enhancers. Hs3B was divided into four BsmAI probes for EMSA analysis. The octamer elements possessing binding in EMSA are depicted as solid ovals; those predicted octamer elements, which did not bind to octamer-binding proteins in EMSA, are indicated in dotted ovals. The {kappa}B sites are indicated by triangles. We have observed the presence of LPS-inducible complexes binding to the intronic enhancer and hs3, which we later identified as YY1 (indicated as black boxes). The relative positions of the sites within the enhancer are indicated. Pax5 sites were not localized within individual fragments of hs3. B, Identification of transcription factor binding sites within hs3 by EMSA using nuclear extracts from A20 (lanes 1–9) and S194 (lanes 10–12) cell lines. Oligonucleotide binding sites for Pax5, octamer, and {kappa}B family members were used as cold competitors to identify binding complexes, indicated by arrows. Assignment of Oct-1 and Oct-2 was based on the relative mobility of these complexes and on supershift analysis of nuclear extracts of splenic B cells with other octamer site-containing probes.

 

View this table:
[in this window]
[in a new window]
  		Table I. Binding sites for transcription factors within hs3

 
We found a great deal of binding complexity to hs3 (Fig. 1B). Using oligonucleotide competitors for our predicted transcription factor binding sites, we identified a number of specific complexes. For instance, probe A bound to Oct-1 and to two different Oct-2 isoforms, apparently through the same site (Fig. 1B, lanes 1-3), verifying a novel octamer-binding sequence at the 5' end of hs3. We also detected a weak Pax5/BSAP-binding complex (compare lanes 2 and 3) within probe A. Both Oct-1 and Oct-2 also bound to probe B (lanes 4–6), as reported (13). Additionally, an intense band in probe B in cell lines was evident. This band was later identified as YY1, and our observation that YY1 binding to hs3 is induced after LPS stimulation is the subject of this study. Probe C contains the two most conserved octamer sites (see Table I), but we could not detect octamer binding by EMSA (Fig. 1B, lanes 7–9). However, probe C contained a novel and intense Pax5 binding site. This complex was developmentally regulated, as S194, like other plasma cells, lacked this complex, as well as the weak Pax5 complex detected in probe A (data not shown). As Pax5 possesses a rather degenerate consensus sequence (15, 35), we did not attempt to define the sequence of these binding sites within hs3. Additionally, a number of bands that could be competed by an NF-{kappa}B competitor were detected in probe D (Fig. 1B, lanes 10–12). With the exception of Pax5, the other binding complexes were present in each of the B cell lines assayed.

Octamer, Pax5, and {kappa}B families of binding complexes form the constellation of transcription factors that coregulate the murine hs1,2 and hs4 enhancers (14, 15). Additionally, we identified a novel candidate regulator of hs3, YY1, which we elected to study further.

LPS and other class-switching activators induce YY1 binding to the hs3 enhancer

Given that hs3 and/or hs4 are required for GT in response to LPS and other activators, we sought to identify whether any of the complexes identified above were regulated by class-switching regimens. Consistent with previous observations that LPS induced binding of NF-{kappa}B family members p50 and c-Rel, as well as other complexes, to hs1,2 (16, 17), we also observed LPS induction of NF-{kappa}B binding to probe D (data not shown). However, upon incubation of extracts from primary B cells cultured with LPS or LPS + IL-4 for 48 h with our four EMSA probes, only a fragment derived from probe B consistently and reproducibly showed induction of a novel binding complex (Fig. 2A, compare lanes 1 and 2).



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 2. Class-switching regimens induce YY1 binding to hs3 and to Eµ. A, EMSA analysis of hs3-134, a segment of probe B (see Materials and Methods), with Ab to YY1 and oligonucleotide competitors (lanes 1–8). The major complexes formed after induction of splenocytes by LPS or CD40 + IL-4 were shown to contain YY1 by competition with the YY1 binding site (µE1) and supershift analysis with antiserum to YY1. The mutated version of the binding site (mµE1) did not compete YY1 binding. Note that both isoforms of YY1 (indicated by arrows) were supershifted by Ab. The upper band is the full-length YY1 (414 aa), while the faster migrating form most likely is a proteolytic degradation product that contains the C-terminal DNA binding domain (Zn fingers), as described in the text. B, Methylation interference footprinting of the hs3-111 probe (a truncated version of hs3-134, also derived from probe B; see Materials and Methods for complete description) following incubation with nuclear extracts from LPS + IL-4-activated splenic B cells. Probe bound by the protein complex (B). Free probe (F). The sequence of the footprinted region (with 87.5% identity to the YY1 consensus binding site) is indicated in the rectangle, with the protected guanine residues highlighted in gray. The extended YY1 binding site is bracketed on the left. C, LPS induces YY1 binding to the µE1 site of Eµ (lanes 1–3), but not to a PU.1 binding site (lanes 4 and 5), as measured by EMSA.

 
This newly acquired band in LPS-stimulated primary B cells comigrated with a band that we observed in B cell lines (Fig. 1B, lanes 4–6), which could not be competed with octamer or BSAP competitors. To identify the site protected by this binding complex, methylation interference footprinting was performed on a 111-bp probe, derived from probe B, with nuclear extracts from splenic B cells that had been cultured with LPS + IL-4. The footprint and the corresponding sequence that was protected by the protein complex (5'-GAGGCCATG-3') are depicted in Table I and Fig. 2B. The sequence that we obtained was highly homologous to the consensus binding site for YY1, which binds to a core sequence of CCAT (22). To determine whether YY1 protein itself bound to hs3, we performed EMSA analysis using a 134-bp fragment of hs3, which encompasses the 111-bp fragment used for methylation footprinting. We used oligonucleotide competitors from the µE1 high affinity YY1 binding site in the intronic enhancer Eµ to compete the protein complex. The wild-type µE1 binding site completely interfered with binding of YY1 to the hs3 probe; however, a mutated µE1 site did not compete the complex (Fig. 2A, lanes 3 and 4). Furthermore, Ab to YY1 supershifted the entire LPS-induced complex (Fig. 2A, lane 6). These data confirmed that upon stimulation of splenic B cells with LPS, YY1 acquires binding to hs3. We have consistently observed two bands that bind to both hs3 (Fig. 2A) and µE1 (Fig. 2C), each of which can be supershifted with an Ab against the C terminus of YY1 (C-20). The faster mobility YY1 isoform is, therefore, as others have observed (36, 37), most likely derived from the truncation of the amino-terminal segment, leaving behind a C-terminal segment containing the DNA binding domain.

We wanted to determine whether other class-switching regimens also resulted in induction of YY1 binding to hs3. LPS primarily causes switching to {gamma}2b and {gamma}3 (19), while LPS + IL-4 shifts the switching program to {gamma}1 and {epsilon} (19, 38). Stimulation of splenic B cells with CD40 + IL-4 also induces switching to {gamma}1 and {epsilon} (39). All three stimuli similarly induced YY1 binding to hs3 (Fig. 2, A and B). Therefore, induction of YY1 binding to hs3 does not seem to be associated with CSR to specific isotypes.

Previous studies had identified binding of YY1 to the µE1 site in the intronic enhancer in a number of cell lines (21). We observed that LPS stimulation of splenic B cells also results in inducible binding of YY1 to Eµ (Fig. 2C, lanes 1–3). As a control for transcription factor binding to DNA in uninduced vs induced splenocyte extracts, we have measured PU.1 binding in EMSA. PU.1 binding was unchanged before and after LPS treatment (Fig. 2C, lanes 4 and 5). Therefore, the induction that we observe by EMSA is specific for YY1, and not a global increase in transcription factor binding after LPS treatment.

Our data provide evidence that YY1 binding to hs3 and the intronic enhancer is increased upon treatment of primary splenic B cells with isotype-switching regimens. Previous studies had predicted that hs1,2 contained a µE1-like site (40); however, neither µE1 nor YY1 Abs were able to disrupt binding complexes in hs1,2 by EMSA (data not shown). We also examined binding of YY1 to the hs4 enhancer. Our EMSA results indicated that there is no YY1 binding to hs4 in unstimulated or LPS-induced primary B cells, although YY1 binding was detected using extracts from a myeloma cell line (data not shown). Thus, our current study indicates that induction of YY1 binding is distinctive for the hs3 3' Igh enhancer.

YY1 is a transcriptional activator of hs3 in mature B and plasma cell lines

To determine the contribution of YY1 binding to hs3 enhancer activity, we cloned wild-type and mutated hs3 enhancer dimers in a B cell-specific reporter construct and performed transient transfection assays. Previous experiments have shown hs3 to be virtually inactive in pre-B cells, and highly active in plasma cells (9, 41). In mature B cell lines (A20 and M12.4.1) and plasma cells (S107 and S194), mutation of the YY1 binding site decreased transcriptional activity by 2- to 5-fold (Fig. 3), indicating that YY1 is a transcriptional activator of hs3 in the context of these cell lines. The wild-type enhancer was approximately equal in activity in S194 and S107 plasma cell lines (data not shown). As S107 lacks constitutive {kappa}B activity (42), we surmised that NF-{kappa}B binding is not essential for hs3 enhancer activity in transient transfection. These data indicate that YY1 contributes to activation of the hs3 enhancer in mature B and plasma cells.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. YY1 is a transcriptional activator of hs3. The contribution of the YY1 binding site to hs3 enhancer activity was determined by comparing the activity of a dimerized wild-type hs3 reporter vector (hs3B)2 (shown in black) with a mutated reporter vector, m(hs3B)2 (shown in white) in transient transfection assays in several B cell lines. The normalized luciferase values of the wild-type reporter in the following cell lines are indicated in parentheses for comparison: A20 (16,323) and M12.4.1 (8835) are mature B cells, S107 (1850) and S194 (2798) are plasma cells. Representative transient transfection assays in each cell line are shown. The mean value of triplicate transfections is plotted, and all data are normalized to {beta}-galactosidase expression, as described in Materials and Methods. SD is included.

 
Acquisition of DNA binding by YY1 to hs3 is not due to an increase in YY1 protein levels

One way to account for the acquisition of DNA-binding activity by YY1 is by an increase in levels of YY1 protein. In most cases, YY1 is ubiquitously expressed (22); however, YY1 protein levels can vary under certain circumstances, e.g., fibroblast wound healing (43). We performed Western blots and determined that YY1 protein is expressed in resting B cells and LPS-stimulated cells at similar levels, even though DNA-binding activity is significantly different. YY1 protein levels were also unchanged after stimulation by LPS +/- IL-4, and CD40 + IL-4 (Fig. 4A, top panel; lanes 2–4). We confirmed that there were equal amounts of protein in each lane by normalizing with the ubiquitous protein GDI (32) (data not shown). Thus, up-regulation of YY1 protein levels does not account for the change in DNA-binding activity that we observe after treatment of primary B cells with different class-switching regimens.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4. Resting B cells contain a factor that inhibits YY1 from DNA binding. A, Protein levels and nuclear/cytoplasmic distribution of YY1 are unaffected by stimulation with class-switching regimens. Western blot of YY1 protein levels from nuclear extracts of primary B cells before and after treatment with various class-switching regimens for 48 h (top panel, lanes 1–4) and from nuclear and cytoplasmic extracts prepared from resting and LPS-stimulated B cells (bottom panel, lanes 1–4). GDI is used as a loading control. B, Nuclear extracts from resting B cells contain a factor that inhibits YY1 binding by extracts from LPS-stimulated cells to the µE1 site of Eµ (lanes 1–5) and by extracts from 18-81 pre-B cells to hs3 (lanes 6–9). Ab to YY1 is included to show that the repression of DNA binding is specific for YY1. The upper band (indicated by arrow) is preferentially competed by uninduced extracts.

 
Another possibility to account for increased DNA binding is a change in the relative cellular distribution of YY1 before and after LPS stimulation. Cytoplasmic vs nuclear distributions have been classically observed to differ for NF-{kappa}B after lymphocyte stimulation by LPS (44, 45). YY1 is predominantly a nuclear protein (46), although, in Xenopus oocytes, YY1 can be cytoplasmic (47). By Western blot analysis, we observed that YY1 was absent from the cytoplasm, and entirely nuclear in primary B cells. Protein loading was constant throughout all of the extracts, as measured by GDI (Fig. 4A, bottom panel; lanes 1–4). Therefore, our results indicate that neither an increase in protein levels nor a change in cellular distribution of YY1 can account for its acquisition of DNA binding to hs3 after LPS stimulation. Likewise, we did not observe a change in electrophoretic mobility after LPS treatment, suggesting that YY1 may not be posttranslationally modified in stimulated cells. However, this does not rule out the possibility because YY1 migrates anomalously on SDS-PAGE (22).

A protein in nuclear extracts from uninduced B cells inhibits YY1 binding to the hs3 enhancer

One possibility to account for limitations in YY1 binding to hs3 in resting cells is that YY1 may reversibly interact with proteins that sequester it from binding to DNA. To test this hypothesis, we used EMSA to titrate the effect of mixing nuclear extracts from unstimulated B cells with LPS-stimulated nuclear extracts. We observed a dose-dependent decrease in YY1 binding to hs3 and the intronic enhancer when unstimulated extracts were mixed with LPS-treated extracts (Fig. 4B, lanes 1–5). Inhibition of YY1 binding became visible at a 1:4 mass ratio of resting B cell extract to LPS-stimulated extract (5:20 µg), and at a 1:1 ratio, DNA binding of the upper YY1 complex was reduced to basal levels present in unstimulated cells. Of the EMSA bands containing YY1, the upper band, representing intact YY1 (double arrow), is the major form competed by resting B cell extracts. This suggests that the protein responsible for inhibition of YY1 binding in resting B cell extracts preferentially interacts with the N-terminal region of YY1. As indicated earlier, cell lines have constitutive YY1-binding activity to the hs3 enhancer. We observed that extracts from resting B cells could also inhibit binding of YY1 in 18-81 cells to hs3 in a dose-dependent manner (Fig. 4B, lanes 6–9). Collectively, these data indicated that a nuclear protein was responsible for dose-dependent inhibition of YY1 binding.

Rb inhibits binding of YY1 to hs3

The mixing experiments implied that a YY1-interacting protein present in uninduced B cell extracts inhibited DNA binding by YY1. This protein might be cell cycle regulated as we observed a shift from less than 1 to ~40% of B cells in S phase after LPS stimulation (Fig. 5A). The Rb tumor suppressor protein was a good candidate, as Rb is known to bind to and sequester transcription factors, such as E2F, in a cell cycle-regulated manner (48), and Rb has been shown to associate with YY1 (28). Rb becomes progressively phosphorylated upon entry into the cell cycle, releasing it from E2F. The phosphorylation state of Rb was measured before and after LPS-mediated entry into the cell cycle. Nuclear extracts from unstimulated and LPS-treated B cells were separated by SDS-PAGE, and Rb isoforms were detected by a Rb mAb. Resting primary B cells have a faster migrating, predominantly hypophosphorylated form of Rb that becomes hyperphosphorylated after 48 h of LPS treatment (Fig. 5B, top; lanes 1 and 2).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 5. Rb associates with YY1 in primary resting B cells and inhibits DNA binding. A, Cell cycle analysis on resting (top) and LPS-stimulated B cells (bottom). B, Western blot analysis of Rb protein in resting and LPS-stimulated primary B cells shows hyperphosphorylation of Rb after 48 h of LPS stimulation (top panel, lanes 1 and 2). Hypophosphorylated Rb associates with YY1 in resting B cells, as assessed by coimmunoprecipitation with YY1 Ab, and Western blotting with an Rb Ab that can recognize all Rb isoforms (pan-Rb) (lower panel, lanes 1 and 2). C, Recombinant Rb protein inhibits YY1 binding to hs3 in EMSA when added to LPS-stimulated extracts (lanes 1–5). Recombinant Rb and GST-Rb large pocket, but not a mock purification of Rb or GST alone, inhibited YY1 binding to hs3 (lanes 6–11).

 
To test whether Rb physically associated with YY1 in primary B cells, we performed endogenous coimmunoprecipitations with B cell lysates. Using Ab to YY1, we could coprecipitate Rb protein (Fig. 5B, lower panel; lane 1). We found that Rb associated with YY1 only in resting, but not in LPS-treated B cells (compare lanes 1 and 2). Accordingly, only the lower mobility, hypophosphorylated form of Rb was able to associate with YY1. As a control, equal amounts of YY1 were immunoprecipitated in each sample (Fig. 5B, lower panel; compare YY1 levels in lanes 1 and 2); therefore, the differential binding of Rb is specific and not due to unequal immunoprecipitation efficiencies.

Next, we wanted to determine whether Rb could directly inhibit the YY1 binding to hs3. To do this, we used EMSA to measure the effect of titrating LPS-stimulated extracts with full-length recombinant Rb protein. In Fig. 5C, lanes 1–5, we observed that recombinant Rb protein inhibited binding of YY1 to hs3 in a dose-dependent manner. As an important control, we tested a mock purification, containing the same components of the Rb purification, except for the Rb protein itself. The mock purification cocktail did not inhibit YY1 binding (Fig. 5C, lane 7). A GST-fusion protein with the carboxyl-terminal large pocket (E2F binding domain) of Rb also inhibited YY1 binding, while GST alone had no effect (compare lanes 9 and 10). Therefore, Rb most likely uses its C-terminal domain to bind to YY1 in B cells, as had been suggested in previous studies (28), and thereby inhibits the binding of YY1 to the hs3 enhancer.

Previous experiments have shown that aa 297–414 of YY1 can bind to Rb (28). Our data show that DNA binding by full-length YY1 is more easily inhibited by both recombinant Rb and nuclear extracts from resting cells than that of the truncated YY1, and that recombinant Rb appears to be a stronger inhibitor of YY1 binding to DNA than nuclear extracts from unstimulated splenic B cells (compare Figs. 4B and 5C). These data suggest the possibility that the truncated YY1 form we observe binds more strongly to DNA than does full-length YY1, and/or the interaction of Rb with proteins in nuclear extracts biases the interaction of Rb toward full-length YY1. We conclude from these data that a nuclear protein responsible for inhibition of YY1 binding in our mixing experiments (Fig. 4B) is most likely to be the hypophosphorylated form of Rb. We predict that repression of YY1 binding to DNA is relieved by dissociation of Rb from YY1 that results from LPS-induced cell cycle-mediated hyperphosphorylation of Rb.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our experiments have shown that in response to LPS and other CSR activators, splenic B cells show inducible binding of YY1 to hs3, uniquely among the 3' Igh enhancers. YY1 also acquires binding to the µE1 site of Eµ in response to these stimuli. Furthermore, transient transfection experiments have shown that YY1 is a transcriptional activator of both enhancers ((20, 49); this study). In resting primary B cells, YY1 is inhibited from binding to hs3 and Eµ through its association with hypophosphorylated Rb. LPS stimulates hyperphosphorylation of Rb, causing Rb to dissociate from YY1, and permitting YY1 to bind to these enhancers. Therefore, the regulation of GT during the early stages of class switching by 3' enhancers, such as hs3 (5), is apparently synchronized with changes in Rb phosphorylation state that are associated with B lymphocyte proliferation.

There have been previous reports suggesting that Rb may interfere with DNA binding of other transcription factors that affect Igh gene expression. For example, an association of hypophosphorylated Rb with Pax5 has been reported (50), which appears to affect binding to low affinity Pax5 binding sites within the {kappa} gene cluster in early B cell lines (51). Furthermore, Oct-1 binding to an MHC class I promoter was also displaced by Rb, perhaps through an indirect mechanism (52).

Enhancers of the 3' regulatory region are required to promote GT of several specific isotypes (5). There is no apparent isotype specificity in the induction of YY1 binding, as we have observed induction both with anti-CD40 + IL-4 and with LPS, regimens that promote {gamma}1, {gamma}2b, {gamma}3, and {epsilon} expression. This convergence of signaling pathways is not completely unexpected. Both CD40 and LPS regulate a common pool of genes (53). Among the genes that are induced by CD40 signaling are cyclin E and CDK4/6, which are the endogenous kinases for Rb. The Rb-related pocket protein, p130, is down-regulated by CD40 signaling, and this might be a general mechanism for releasing B cells from quiescence (53). We observed that p130 was markedly down-regulated by LPS stimulation of primary B cells (data not shown), and these results were consistent with studies in a myeloid cell line (54), which showed that p130 was associated with differentiated, but not proliferating cells. Rb-deficient mice have no obvious immunological defects; thus, there may be some functional redundancy between Rb and its family members, such as p130, in controlling lymphocyte proliferation (55).

There is a precedent for cell cycle regulation of class switching. First, isotype switching has been shown to be dependent on cell division number (56). Class switching also occurs in the S phase of cells stimulated with CD40 ligand and IL-4 (57). We observed an analogous time course for the induction of YY1 binding to hs3 and Eµ in response to stimulation by LPS and anti-CD40 + IL-4. Binding is minimal at 24 h, but at 48 h, YY1 binds substantially to hs3 and µE1 and continues to be induced for as long as 96 h of LPS stimulation (data not shown). This time period might reflect the interval required for Rb to become hyperphosphorylated and to dissociate from YY1, and/or the time required for cells to undergo sufficient numbers of cell divisions.

The levels of YY1 protein are unchanged after LPS induction of B cells. Similarly, YY1 levels remained uniformly unchanged during LPS-induced 70Z/3 B cell differentiation (58), although up-regulated in other experimental systems (43). In addition, we have not detected a change in the electrophoretic mobility of YY1 in SDS-PAGE. However, we cannot rule out direct modification of YY1, or of YY1-interacting proteins, other than Rb, as a means to control DNA binding. For example, studies indicate that YY1 is a phosphoprotein in 3T3 mouse fibroblasts (58) and that phosphorylation of YY1 inhibits DNA binding (59, 60). We have observed that {lambda} protein phosphatase treatment of nuclear extracts from resting B cells dramatically increases YY1 binding to µE1 (S. Gordon, unpublished data). Phosphatases may act directly on YY1 or on other proteins in nuclear extracts to facilitate binding. To complicate matters, LPS both activates protein kinase C (61) and, conversely, induces expression of nuclear alkaline phosphatases in B cells (62). Therefore, some proteins are likely to lose phosphate moieties upon LPS stimulation, whereas other proteins, such as Rb, become phosphorylated. Our preliminary studies have revealed no evidence for tyrosine phosphorylation of YY1 after LPS stimulation, suggesting that phosphorylation may be focused on serine/threonine residues. Other studies have shown that acetylation of YY1 can also affect DNA binding (24).

The function of YY1 in B cell development is currently unknown, as targeted deletion of YY1 leads to early embryonic lethality (63). YY1 is ubiquitously expressed and has an array of binding sites throughout the genome, including several that are B-lineage specific, such as the Ig{kappa} 3' enhancer (64), the Igh intronic enhancer Eµ (20, 21), and now the hs3 enhancer of the 3' Igh regulatory region.

The cascade of events initiated by LPS that results in activation of GT by 3' enhancers may open the Igh locus at the chromatin level. LPS has been reported to down-regulate the Bach2 repressor of hs3 and potentially other 3' Igh enhancers (18). In our studies, we show that LPS induces YY1 binding to both hs3 and the intronic enhancer, both of which are candidates for involvement in CSR. Addition of Eµ to a combination of all four 3' Igh enhancers results in strong transcriptional synergy at all stages of B cell differentiation (41, 65). YY1 may also bind to some switch promoters via interaction with the late SV40 factor (LSF) (66), bringing these regions into proximity with the Igh enhancers. Various models have been proposed to explain how a distant enhancer locus, such as the 3' Igh regulatory region, is able to mediate long distance regulation. Perhaps YY1 serves as a nucleating protein for an Igh holocomplex (67), in which LPS stimulation results in a looping out of the intervening sequence and places the 3' regulatory region in close proximity with the switch promoters, intronic enhancer, and/or VH promoters. YY1 interacts with components of the basal transcription machinery, such as TFIIB, TBP, and TAFII55 (58, 68), that may also be associated with such a complex.


   Acknowledgments
 
We thank Dr. Liang Zhu and Katya Rehktman for the GST-Rb construct, recombinant Rb, and mock purifications; Dr. Perry Bickel for the GDI Ab; and Dr. Clifford Snapper for providing anti-T cell supernatants and T cell depletion protocols. We thank Drs. Liang Zhu, Michael Pappetti, and Phillip Bardwell, and Alejandro Sepulveda for reviewing the manuscript and for helpful advice, and Nasrin Ashouian for expert technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health AI13509 and by an Albert Einstein Cancer Center Grant P30 CA13330. Back

2 Current address: Department of Hematology and Oncology, Children’s Hospital and Harvard Medical School, Boston, MA 02115. Back

3 Address correspondence and reprint requests to Dr. Barbara K. Birshtein, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: birshtei{at}aecom.yu.edu Back

4 Abbreviations used in this paper: hs, DNase I hypersensitive site; BSAP, B cell-specific activating protein; GDI, guanine nucleotide dissociation inhibitor; GT, germline transcription; Rb, retinoblastoma protein; YY1, Yin Yang 1. Back

Received for publication December 20, 2002. Accepted for publication March 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Max, E. E.. 1999. Immunoglobulins: molecular genetics. W. E. Paul, ed. Fundamental Immunology 4th Ed.183. Lippincott-Raven Publishers, Philadelphia.
  2. Khamlichi, A. A., E. Pinaud, C. Decourt, C. Chauveau, M. Cogne. 2000. The 3' IgH regulatory region: a complex structure in a search for a function. Adv. Immunol. 75:317.[Medline]
  3. Serwe, M., F. Sablitzky. 1993. V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer. EMBO J. 12:2321.[Medline]
  4. Chen, J., F. Young, A. Bottaro, V. Stewart, R. K. Smith, F. W. Alt. 1993. Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus. EMBO J. 12:4635.[Medline]
  5. Pinaud, E., A. A. Khamlichi, C. Le Morvan, M. Drouet, V. Nalesso, M. Le Bert, M. Cogne. 2001. Localization of the 3' IgH locus elements that effect long-distance regulation of class switch recombination. Immunity 15:187.[Medline]
  6. Manis, J. P., N. van der Stoep, M. Tian, R. Ferrini, L. Davidson, A. Bottaro, F. W. Alt. 1998. Class switching in B cells lacking 3' immunoglobulin heavy chain enhancers. J. Exp. Med. 188:1421.[Abstract/Free Full Text]
  7. Shi, X., L. A. Eckhardt. 2001. Deletional analyses reveal an essential role for the hs3b/hs4 IgH 3' enhancer pair in an Ig-secreting but not an earlier-stage B cell line. Int. Immunol. 13:1003.[Abstract/Free Full Text]
  8. Birshtein, B. K. C. C., S. Saleque, J. S. Michaelson, M. Singh, R. D. Little. 1997. Murine and human 3' IgH regulatory sequences. Curr. Top. Microbiol. Immunol. 224:73.[Medline]
  9. Saleque, S., M. Singh, R. D. Little, S. L. Giannini, J. S. Michaelson, B. K. Birshtein. 1997. Dyad symmetry within the mouse 3' IgH regulatory region includes two virtually identical enhancers (C{alpha} 3'E and hs3). J. Immunol. 158:4780.[Abstract]
  10. Chauveau, C., M. Cogne. 1996. Palindromic structure of the IgH 3' locus control region. Nat. Genet. 14:15.[Medline]
  11. Michaelson, J. S., S. L. Giannini, B. K. Birshtein. 1995. Identification of 3' {alpha}-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation. Nucleic Acids Res. 23:975.[Abstract/Free Full Text]
  12. Madisen, L., M. Groudine. 1994. Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt’s lymphoma cells. Genes Dev. 8:2212.[Abstract/Free Full Text]
  13. Matthias, P., D. Baltimore. 1993. The immunoglobulin heavy chain locus contains another B-cell-specific 3' enhancer close to the {alpha} constant region. Mol. Cell. Biol. 13:1547.[Abstract/Free Full Text]
  14. Singh, M., B. K. Birshtein. 1993. NF-HB (BSAP) is a repressor of the murine immunoglobulin heavy-chain 3' {alpha} enhancer at early stages of B-cell differentiation. Mol. Cell. Biol. 13:3611.[Abstract/Free Full Text]
  15. Michaelson, J. S., M. Singh, B. K. Birshtein. 1996. B cell lineage-specific activator protein (BSAP): a player at multiple stages of B cell development. J. Immunol. 156:2349.[Abstract]
  16. Grant, P. A., T. Andersson, M. F. Neurath, V. Arulampalam, A. Bauch, R. Muller, M. Reth, S. Pettersson. 1996. A T cell controlled molecular pathway regulating the IgH locus: CD40-mediated activation of the IgH 3' enhancer. EMBO J. 15:6691.[Medline]
  17. Linderson, Y., D. Cross, M. F. Neurath, S. Pettersson. 1997. NFE, a new transcriptional activator that facilitates p50 and c-Rel-dependent IgH 3' enhancer activity. Eur. J. Immunol. 27:468.[Medline]
  18. Muto, A., H. Hoshino, L. Madisen, N. Yanai, M. Obinata, H. Karasuyama, N. Hayashi, H. Nakauchi, M. Yamamoto, M. Groudine, K. Igarashi. 1998. Identification of Bach2 as a B-cell-specific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3' enhancer. EMBO J. 17:5734.[Medline]
  19. Snapper, C. M., F. D. Finkelman. 1999. Immunoglobulin class switching. W. E. Paul, ed. Fundamental Immunology 4th Ed.831. Lippincott-Raven Publishers, Philadelphia.
  20. Lenardo, M., J. W. Pierce, D. Baltimore. 1987. Protein-binding sites in Ig gene enhancers determine transcriptional activity and inducibility. Science 236:1573.[Abstract/Free Full Text]
  21. Weinberger, J., D. Baltimore, P. A. Sharp. 1986. Distinct factors bind to apparently homologous sequences in the immunoglobulin heavy-chain enhancer. Nature 322:846.[Medline]
  22. Shi, Y., J. S. Lee, K. M. Galvin. 1997. Everything you have ever wanted to know about Yin Yang 1. Biochim. Biophys. Acta 1332:F49.[Medline]
  23. Yang, W. M., Y. L. Yao, J. M. Sun, J. R. Davie, E. Seto. 1997. Isolation and characterization of cDNAs corresponding to an additional member of the human histone deacetylase gene family. J. Biol. Chem. 272:28001.[Abstract/Free Full Text]
  24. Yao, Y. L., W. M. Yang, E. Seto. 2001. Regulation of transcription factor YY1 by acetylation and deacetylation. Mol. Cell. Biol. 21:5979.[Abstract/Free Full Text]
  25. Brown, J. L., D. Mucci, M. Whiteley, M. L. Dirksen, J. A. Kassis. 1998. The Drosophila Polycomb group gene pleiohomeotic encodes a DNA binding protein with homology to the transcription factor YY1. Mol. Cell 1:1057.[Medline]
  26. Garcia, E., C. Marcos-Gutierrez, M. del Mar Lorente, J. C. Moreno, M. Vidal. 1999. RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1. EMBO J. 18:3404.[Medline]
  27. Sawa, C., T. Yoshikawa, F. Matsuda-Suzuki, S. Delehouzee, M. Goto, H. Watanabe, J. Sawada, K. Kataoka, H. Handa. 2002. YEAF1/RYBP and YAF-2 are functionally distinct members of a cofactor family for the YY1 and E4TF1/hGABP transcription factors. J. Biol. Chem. 277:22484.[Abstract/Free Full Text]
  28. Petkova, V., M. J. Romanowski, I. Sulijoadikusumo, D. Rohne, P. Kang, T. Shenk, A. Usheva. 2001. Interaction between YY1 and the retinoblastoma protein: regulation of cell cycle progression in differentiated cells. J. Biol. Chem. 276:7932.[Abstract/Free Full Text]
  29. Michaelson, J. S., M. Singh, C. M. Snapper, W. C. Sha, D. Baltimore, B. K. Birshtein. 1996. Regulation of 3' IgH enhancers by a common set of factors, including {kappa}B-binding proteins. J. Immunol. 156:2828.[Abstract]
  30. Shrivastava, A., K. Calame. 1994. An analysis of genes regulated by the multi-functional transcriptional regulator Yin Yang-1. Nucleic Acids Res. 22:5151.[Free Full Text]
  31. Garcia-Blanco, M. A., S. F. Jamison, P. A. Sharp. 1989. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev. 3:1874.[Abstract/Free Full Text]
  32. Scherer, P. E., R. Y. Lewis, D. Volonte, J. A. Engelman, F. Galbiati, J. Couet, D. S. Kohtz, E. van Donselaar, P. Peters, M. P. Lisanti. 1997. Cell-type and tissue-specific expression of caveolin-2: caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J. Biol. Chem. 272:29337.[Abstract/Free Full Text]
  33. Kaelin, W. G., Jr, D. C. Pallas, J. A. DeCaprio, F. J. Kaye, D. M. Livingston. 1991. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 64:521.[Medline]
  34. Heinemeyer, T., E. Wingender, I. Reuter, H. Hermjakob, A. E. Kel, O. V. Kel, E. V. Ignatieva, E. A. Ananko, O. A. Podkolodnaya, F. A. Kolpakov, et al 1998. Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res. 26:362.[Abstract/Free Full Text]
  35. Hagman, J., W. Wheat, D. Fitzsimmons, W. Hodsdon, J. Negri, F. Dizon. 2000. Pax-5/BSAP: regulator of specific gene expression and differentiation in B lymphocytes. Curr. Top. Microbiol. Immunol. 245:169.[Medline]
  36. Lee, T.-C., R. J. Schwartz. 1992. Using proteases to avoid false identification of DNA-protein complexes in gel shift assays. BioTechniques 12:486.[Medline]
  37. Lee, T. C., Y. Shi, R. J. Schwartz. 1992. Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal {alpha}-actin transcription in embryonic myoblasts. Proc. Natl. Acad. Sci. USA 89:9814.[Abstract/Free Full Text]
  38. Rothman, P., S. C. Li, B. Gorham, L. Glimcher, F. Alt, M. Boothby. 1991. Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive element located at the promoter of germ line {epsilon} transcripts. Mol. Cell. Biol. 11:5551.[Abstract/Free Full Text]
  39. Iciek, L. A., S. A. Delphin, J. Stavnezer. 1997. CD40 cross-linking induces Ig {epsilon} germline transcripts in B cells via activation of NF-{kappa}B: synergy with IL-4 induction. J. Immunol. 158:4769.[Abstract]
  40. Grant, P. A., C. B. Thompson, S. Pettersson. 1995. IgM receptor-mediated transactivation of the IgH 3' enhancer couples a novel Elf-1-AP-1 protein complex to the developmental control of enhancer function. EMBO J. 14:4501.[Medline]
  41. Ong, J., S. Stevens, R. G. Roeder, L. A. Eckhardt. 1998. 3' IgH enhancer elements shift synergistic interactions during B cell development. J. Immunol. 160:4896.[Abstract/Free Full Text]
  42. Baumann, B., B. Kistler, A. Kirillov, Y. Bergman, T. Wirth. 1998. The mutant plasmacytoma cell line S107 allows the identification of distinct pathways leading to NF-{kappa}B activation. J. Biol. Chem. 273:11448.[Abstract/Free Full Text]
  43. Santiago, F. S., H. C. Lowe, Y. V. Bobryshev, L. M. Khachigian. 2001. Induction of the transcriptional repressor Yin Yang-1 by vascular cell injury: autocrine/paracrine role of endogenous fibroblast growth factor-2. J. Biol. Chem. 276:41143.[Abstract/Free Full Text]
  44. Bauerle, P. A., D. Baltimore. 1988. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-{kappa}B transcription factor. Cell 53:211.[Medline]
  45. Bauerle, P. A., D. Baltimore. 1988. I{kappa}B: a specific inhibitor of the NF-{kappa}B transcription factor. Science 242:540.[Abstract/Free Full Text]
  46. Guo, B., P. R. Odgren, A. J. van Wijnen, T. J. Last, J. Nickerson, S. Penman, J. B. Lian, J. L. Stein, G. S. Stein. 1995. The nuclear matrix protein NMP-1 is the transcription factor YY1. Proc. Natl. Acad. Sci. USA 92:10526.[Abstract/Free Full Text]
  47. Ficzycz, A., C. Eskiw, D. Meyer, K. Eliassen-Marley, M. Hurt, N. Ovsenek. 2001. Expression, activity and subcellular localization of the Yin Yang 1 transcription factor in Xenopus oocytes and embryos. J. Biol. Chem. 6:6.
  48. Dagnino, L., L. Zhu, K. L. Skorecki, H. L. Moses. 1995. E2F-independent transcriptional repression by p107, a member of the retinoblastoma family of proteins. Cell Growth Differ. 6:191.[Abstract]
  49. Tsao, B. P., X. F. Wang, C. L. Peterson, K. Calame. 1988. In vivo functional analysis of in vitro protein binding sites in the immunoglobulin heavy chain enhancer. Nucleic Acids Res. 16:3239.[Abstract/Free Full Text]
  50. Eberhard, D., M. Busslinger. 1999. The partial homeodomain of the transcription factor Pax-5 (BSAP) is an interaction motif for the retinoblastoma and TATA-binding proteins. Cancer Res. 59:1716.s.
  51. Sato, H., D. Wang, A. Kudo. 2001. Dissociation of Pax-5 from KI and KII sites during {kappa}-chain gene rearrangement correlates with its association with the underphosphorylated form of retinoblastoma. J. Immunol. 166:6704.[Abstract/Free Full Text]
  52. Zhang, H., A. T. Shepherd, D. D. Eason, S. Wei, J. I. Diaz, J. Y. Djeu, G. D. Wu, G. Blanck. 1999. Retinoblastoma protein expression leads to reduced Oct-1 DNA binding activity and enhances interleukin-8 expression. Cell Growth Differ. 10:457.[Abstract/Free Full Text]
  53. Dadgostar, H., B. Zarnegar, A. Hoffmann, X. F. Qin, U. Truong, G. Rao, D. Baltimore, G. Cheng. 2002. Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes. Proc. Natl. Acad. Sci. USA 99:1497.[Abstract/Free Full Text]
  54. Erkeland, S. J., M. Valkhof, C. Heijmans-Antonissen, R. Delwel, P. J. Valk, M. H. Hermans, I. P. Touw. 2002. The gene encoding the transcriptional regulator Yin Yang 1(YY1) is a myeloid transforming gene interfering with neutrophilic differentiation. Blood 12:12.
  55. Classon, M., E. Harlow. 2002. The retinoblastoma tumor suppressor in development and cancer. Nat. Rev. Cancer 2:910.[Medline]
  56. Hodgkin, P. D., J. H. Lee, A. B. Lyons. 1996. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 184:277.[Abstract/Free Full Text]
  57. Tangye, S. G., A. Ferguson, D. T. Avery, C. S. Ma, P. D. Hodgkin. 2002. Isotype switching by human B cells is division-associated and regulated by cytokines. J. Immunol. 169:4298.[Abstract/Free Full Text]
  58. Austen, M., B. Luscher, J. M. Luscher-Firzlaff. 1997. Characterization of the transcriptional regulator YY1: the bipartite transactivation domain is independent of interaction with the TATA box-binding protein, transcription factor IIB, TAFII55, or cAMP-responsive element-binding protein (CPB)-binding protein. J. Biol. Chem. 272:1709.[Abstract/Free Full Text]
  59. Kaludov, N. K., T. L. Bowman, E. M. Sikorski, M. M. Hurt. 1996. Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene. Proc. Natl. Acad. Sci. USA 93:4465.[Abstract/Free Full Text]
  60. Eliassen, K. A., A. Baldwin, E. M. Sikorski, M. M. Hurt. 1998. Role for a YY1-binding element in replication-dependent mouse histone gene expression. Mol. Cell. Biol. 18:7106.[Abstract/Free Full Text]
  61. Tang, H., P. A. Sharp. 1999. Transcriptional regulation of the murine 3' IgH enhancer by OCT-2. Immunity 11:517.[Medline]
  62. Kenter, A. L., L. Morgan. 1995. Mitogen activation of a nuclear alkaline phosphatase in normal activated lymphocytes. J. Immunol. 154:2041.[Abstract]
  63. Donohoe, M. E., X. Zhang, L. McGinnis, J. Biggers, E. Li, Y. Shi. 1999. Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality. Mol. Cell. Biol. 19:7237.[Abstract/Free Full Text]
  64. Park, K., M. L. Atchison. 1991. Isolation of a candidate repressor/activator, NF-E1 (YY-1, {delta}), that binds to the immunoglobulin {kappa} 3' enhancer and the immunoglobulin heavy-chain µ E1 site. Proc. Natl. Acad. Sci. USA 88:9804.[Abstract/Free Full Text]
  65. Chauveau, C., E. A. Jansson, S. Muller, M. Cogne, S. Pettersson. 1999. Cutting edge: Ig heavy chain 3' HS1–4 directs correct spatial position-independent expression of a linked transgene to B lineage cells. J. Immunol. 163:4637.[Abstract/Free Full Text]
  66. Drouin, E. E., C. E. Schrader, J. Stavnezer, U. Hansen. 2002. The ubiquitously expressed DNA-binding protein late SV40 factor binds Ig switch regions and represses class switching to IgA. J. Immunol. 168:2847.[Abstract/Free Full Text]
  67. Arulampalam, V., L. Eckhardt, S. Pettersson. 1997. The enhancer shift: a model to explain the developmental control of IgH gene expression in B-lineage cells. Immunol. Today 18:549.[Medline]
  68. Usheva, A., T. Shenk. 1996. YY1 transcriptional initiator: protein interactions and association with a DNA site containing unpaired strands. Proc. Natl. Acad. Sci. USA 93:13571.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Genes Dev.Home page
K. Calame and M. Atchison
YY1 helps to bring loose ends together
Genes & Dev., May 15, 2007; 21(10): 1145 - 1152.
[Full Text] [PDF]

Home page
 Genes Dev.Home page
H. Liu, M. Schmidt-Supprian, Y. Shi, E. Hobeika, N. Barteneva, H. Jumaa, R. Pelanda, M. Reth, J. Skok, K. Rajewsky, et al.
Yin Yang 1 is a critical regulator of B-cell development
Genes & Dev., May 15, 2007; 21(10): 1179 - 1189.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
F. E. Garrett, A. V. Emelyanov, M. A. Sepulveda, P. Flanagan, S. Volpi, F. Li, D. Loukinov, L. A. Eckhardt, V. V. Lobanenkov, and B. K. Birshtein
Chromatin Architecture near a Potential 3' End of the Igh Locus Involves Modular Regulation of Histone Modifications during B-Cell Development and In Vivo Occupancy at CTCF Sites
Mol. Cell. Biol., February 15, 2005; 25(4): 1511 - 1525.
[Abstract]