YY1 control of mitochondrial-related genes does not account for regulation of immunoglobulin class switch recombination in mice.

Immunoglobulin class switch recombination (CSR) occurs in activated B cells with increased mitochondrial mass and membrane potential. Transcription factor Yin Yang 1 (YY1) is critical for CSR and for formation of the DNA loops involved in this process. We therefore sought to determine if YY1 knockout impacts mitochondrial gene expression and mitochondrial function in murine splenic B cells, providing a potential mechanism for regulating CSR. We identified numerous genes in splenic B cells differentially regulated when cells are induced to undergo CSR. YY1 conditional knockout caused differential expression of 1129 genes, with 59 being mitochondrial-related genes. ChIP-seq analyses showed YY1 was directly bound to nearly half of these mitochondrial-related genes. Surprisingly, at the time when YY1 knockout dramatically reduces DNA loop formation and CSR, mitochondrial mass and membrane potential were not significantly impacted, nor was there a significant change in mitochondrial oxygen consumption, extracellular acidification rate, or mitochondrial complex I or IV activities. Our results indicate that YY1 regulates numerous mitochondrial-related genes in splenic B cells, but this does not account for the impact of YY1 on CSR or long-distance DNA loop formation.

Transcription factor YY1 can control AID-mediated mutagenesis in mice.

Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1f/f ) or that express normal AID levels (yy1f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sµ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination.

YY1 Is Required for Germinal Center B Cell Development.

YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction.

Function of YY1 in Long-Distance DNA Interactions.

During B cell development, long-distance DNA interactions are needed for V(D)J somatic rearrangement of the immunoglobulin (Ig) loci to produce functional Ig genes, and for class switch recombination (CSR) needed for antibody maturation. The tissue-specificity and developmental timing of these mechanisms is a subject of active investigation. A small number of factors are implicated in controlling Ig locus long-distance interactions including Pax5, Yin Yang 1 (YY1), EZH2, IKAROS, CTCF, cohesin, and condensin proteins. Here we will focus on the role of YY1 in controlling these mechanisms. YY1 is a multifunctional transcription factor involved in transcriptional activation and repression, X chromosome inactivation, Polycomb Group (PcG) protein DNA recruitment, and recruitment of proteins required for epigenetic modifications (acetylation, deacetylation, methylation, ubiquitination, sumoylation, etc.). YY1 conditional knock-out indicated that YY1 is required for B cell development, at least in part, by controlling long-distance DNA interactions at the immunoglobulin heavy chain and Igκ loci. Our recent data show that YY1 is also required for CSR. The mechanisms implicated in YY1 control of long-distance DNA interactions include controlling non-coding antisense RNA transcripts, recruitment of PcG proteins to DNA, and interaction with complexes involved in long-distance DNA interactions including the cohesin and condensin complexes. Though common rearrangement mechanisms operate at all Ig loci, their distinct temporal activation along with the ubiquitous nature of YY1 poses challenges for determining the specific mechanisms of YY1 function in these processes, and their regulation at the tissue-specific and B cell stage-specific level. The large numbers of post-translational modifications that control YY1 functions are possible candidates for regulation.

YY1 DNA binding and interaction with YAF2 is essential for Polycomb recruitment.

Polycomb Group (PcG) proteins are crucial for epigenetic inheritance of cell identity and are functionally conserved from Drosophila to humans. PcG proteins regulate expression of homeotic genes and are essential for axial body patterning during development. Earlier we showed that transcription factor YY1 functions as a PcG protein. YY1 also physically interacts with YAF2, a homolog of RYBP. Here we characterize the mechanism and physiologic relevance of this interaction. We found phenotypic and biochemical correction of dRYBP mutant flies by mouse YAF2 demonstrating functional conservation across species. Further biochemical analysis revealed that YAF2 bridges interaction between YY1 and the PRC1 complex. ChIP assays in HeLa cells showed that YAF2 is responsible for PcG recruitment to DNA, which is mediated by YY1 DNA binding. Knock-down of YY1 abrogated PcG recruitment, which was not compensated by exogenous YAF2 demonstrating that YY1 DNA binding is a priori necessary for Polycomb assembly on chromatin. Finally, we found that although YAF2 and RYBP regulate a similar number of Polycomb target genes, there are very few genes that are regulated by both implying functional distinction between the two proteins. We present a model of YAF2-dependent and independent PcG DNA recruitment by YY1.

YY1 controls Igκ repertoire and B-cell development, and localizes with condensin on the Igκ locus.

Conditional knock-out (KO) of Polycomb Group (PcG) protein YY1 results in pro-B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino-acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild-type YY1 rescued B-cell development, YY1ΔREPO failed to rescue the B-cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co-localize at numerous sites across the Ig kappa locus. Knock-down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1-condensin complexes in Igκ locus structure and rearrangement.

YY1 controls immunoglobulin class switch recombination and nuclear activation-induced deaminase levels.

Activation-induced deaminase (AID) is an enzyme required for class switch recombination (CSR) and somatic hypermutation (SHM), processes that ensure antibody maturation and expression of different immunoglobulin isotypes. AID function is tightly regulated by tissue- and stage-specific expression, nuclear localization, and protein stability. Transcription factor YY1 is crucial for early B cell development, but its function at late B cell stages is unknown. Here, we show that YY1 conditional knockout in activated splenic B cells interferes with CSR. Knockout of YY1 did not affect B cell proliferation, transcription of the AID and IgM genes, or levels of various switch region germ line transcripts. However, we show that YY1 physically interacts with AID and controls the accumulation of nuclear AID, at least in part, by increasing nuclear AID stability. We show for the first time that YY1 plays a novel role in CSR and controls nuclear AID protein levels.

Increased expression of PcG protein YY1 negatively regulates B cell development while allowing accumulation of myeloid cells and LT-HSC cells.

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs.

CtBP levels control intergenic transcripts, PHO/YY1 DNA binding, and PcG recruitment to DNA.

Carboxy-terminal binding protein (CtBP) is a well-known corepressor of several DNA binding transcription factors in Drosophila as well as in mammals. CtBP is implicated in Polycomb Group (PcG) complex-mediated transcriptional repression because it can bind to some PcG proteins, and mutation of the ctbp gene in flies results in lost PcG protein recruitment to Polycomb Response Elements (PREs) and lost PcG repression. However, the mechanism of reduced PcG DNA binding in CtBP mutant backgrounds is unknown. We show here that in a Drosophila CtBP mutant background, intergenic transcripts are induced across several PRE sequences and this corresponds to reduced DNA binding by PcG proteins Pleiohomeotic (PHO) and Polycomb (Pc), and reduced trimethylation of histone H3 on lysine 27, a hallmark of PcG repression. Restoration of CtBP levels by expression of a CtBP transgene results in repression of intergenic transcripts, restored PcG binding, and elevated trimethylation of H3 on lysine 27. Our results support a model in which CtBP regulates expression of intergenic transcripts that controls DNA binding by PcG proteins and subsequent histone modifications and transcriptional activity.

PcG recruitment by the YY1 REPO domain can be mediated by Yaf2.

The Polycomb Group (PcG) complex of transcriptional repressors is critical for the maintenance of stage-specific developmental gene expression, stem cell maintenance and for large-scale chromosomal dynamics. Functional deficiency of a single PcG gene can severely compromise PcG function, leading to developmental defects, embryonic lethality, or a number of malignancies. Despite the critical nature of PcG proteins, the mechanisms by which these complexes mediate their effects are relatively uncharacterized. Nearly all vertebrate PcG proteins lack inherent DNA binding capacity, making it unclear how they are targeted to Polycomb response element (PRE) sequences. Transcription factor YY1 is a functional ortholog of a Drosophila PcG protein, Pleiohomeotic (PHO), one of the few PcG proteins with specific DNA binding capability, and YY1 can recruit PcG proteins to specific DNA sequences. A small 25 amino acid YY1 domain (the REPO domain) is necessary and sufficient for recruitment of PcG proteins to DNA and for transcriptional repression. We show here that the YY1 REPO domain interacts with PcG protein Yaf2 and recruits Yaf2 to DNA. Interaction is lost when the YY1 REPO domain is deleted. In addition we show that Yaf2, when linked to a heterologous DNA binding domain, can recruit PcG proteins to DNA leading to transcriptional repression. When the Drosophila homolog of Yaf2 (dRYBP) is mutated, PcG recruitment to DNA is reduced. Taken together, our results suggest that Yaf2 serves as a molecular bridge between YY1 and other PcG complex proteins.

Polycomb recruitment to DNA in vivo by the YY1 REPO domain.

Polycomb group (PcG) proteins are responsible for maintaining transcriptional repression of developmentally important genes. However, the mechanism of PcG recruitment to specific DNA sequences is poorly understood. Transcription factor YY1 is one of the few PcG proteins with sequence-specific DNA binding activity. We previously showed that YY1 can recruit other PcG proteins to DNA, leading to histone posttranslational modifications and stable transcriptional repression. Using Drosophila transgenic approaches, we identified YY1 sequences 201-226 as necessary and sufficient for PcG transcriptional repression in vivo. When fused to a heterologous DNA-binding domain, this short 26-aa motif was sufficient for transcriptional repression, recruitment of PcG proteins to DNA, and methylation of histone H3 lysine 27. Deletion of this short YY1 motif did not affect transient transcriptional repression but ablated PcG repression, PcG protein recruitment to DNA, and methylation of H3 lysine 27. We propose that this motif be named the REPO domain for its function in recruitment of Polycomb. The REPO domain is well conserved in YY1 orthologs and in related proteins.

Transient requirements of YY1 expression for PcG transcriptional repression and phenotypic rescue.

A hallmark of PcG transcriptional repression is stability of the repressed state, although the mechanism of this stability is unclear. The mammalian transcription factor YY1 can function as a PcG protein in Drosophila resulting in transcriptional repression and correction of phenotypic defects caused by mutation of its homologue, Pleiohomeotic (PHO). To discern the temporal requirements of YY1 expression for these functions, and to study its mechanism of stable repression in vivo, we used a Drosophila larval wing imaginal disc transcriptional repression system. We found that YY1 was needed transiently at day 3 or later of embryonic development to stably repress a reporter transgene at day 8 in wing imaginal discs. Stable transcriptional repression did not correlate with maintenance of YY1 or Polycomb DNA binding, but did correlate with persistence of histone H3 methylation on lysine 27. We also found that YY1 expressed during the first 7 days of development was sufficient for rescue of fly development (a 14 day process) in pho mutant flies. Therefore, YY1 was transiently required for correction of fly defects and was dispensable past the pharate adult stage. Possible mechanisms of repression by YY1 are discussed.

YY1 DNA binding and PcG recruitment requires CtBP.

We found that mammalian Polycomb group (PcG) protein YY1 can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA.

Transcription factor YY1 functions as a PcG protein in vivo.

Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, pleiohomeotic (PHO). YY1 might therefore be a vertebrate PcG protein. We used Drosophila embryo and larval/imaginal disc transcriptional repression systems to determine whether YY1 repressed transcription in a manner consistent with PcG function in vivo. YY1 repressed transcription in Drosophila, and this repression was stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablated YY1 repression, and YY1 could substitute for PHO in repressing transcription in wing imaginal discs. YY1 functionally compensated for loss of PHO in pho mutant flies and partially corrected mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, we found that YY1, as well as Polycomb, required the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1.