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.

Activation-induced deaminase and its splice variants associate with trisomy 12 in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X.

Females have a greater immunological advantage than men, yet they are more prone to autoimmune disorders. The basis for this sex bias lies in the X chromosome, which contains many immunity-related genes. Female mammals use X chromosome inactivation (XCI) to generate a transcriptionally silent inactive X chromosome (Xi) enriched with heterochromatic modifications and XIST/Xist RNA, which equalizes gene expression between the sexes. Here, we examine the maintenance of XCI in lymphocytes from females in mice and humans. Strikingly, we find that mature naïve T and B cells have dispersed patterns of XIST/Xist RNA, and they lack the typical heterochromatic modifications of the Xi. In vitro activation of lymphocytes triggers the return of XIST/Xist RNA transcripts and some chromatin marks (H3K27me3, ubiquitin-H2A) to the Xi. Single-cell RNA FISH analysis of female T cells revealed that the X-linked immunity genes CD40LG and CXCR3 are biallelically expressed in some cells. Using knockout and knockdown approaches, we find that Xist RNA-binding proteins, YY1 and hnRNPU, are critical for recruitment of XIST/Xist RNA back to the Xi. Furthermore, we examined B cells from patients with systemic lupus erythematosus, an autoimmune disorder with a strong female bias, and observed different XIST RNA localization patterns, evidence of biallelic expression of immunity-related genes, and increased transcription of these genes. We propose that the Xi in female lymphocytes is predisposed to become partially reactivated and to overexpress immunity-related genes, providing the first mechanistic evidence to our knowledge for the enhanced immunity of females and their increased susceptibility for autoimmunity.

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 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.

A developmentally controlled competitive STAT5-PU.1 DNA binding mechanism regulates activity of the Ig κ E3' enhancer.

Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal V(H) genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3' enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show in this study that STAT5 can repress κE3' enhancer activity. We find that STAT5 binds to a site that overlaps the κE3' PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3' enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3' enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates κE3' enhancer function.

Unusual lineage plasticity revealed by YY1 knockout in pro-B cells.

During B cell development, cells progress through multiple developmental stages with the pro-B cell stage defining commitment to the B cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We find here that knockout of YY1 at the pro-B cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9- DL4 feeder system, as well as in vivo after injection into sub-lethally irradiated Rag1 -/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T cell lineage profile. Single cell-RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages indicating unusual lineage plasticity. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 likely regulates commitment in multiple cell lineages.

Multiple lineage-specific epigenetic landscapes at the antigen receptor loci.

Antigen receptors (AgRs) expressed on B and T cells provide the adaptive immune system with ability to detect numerous foreign antigens. Epigenetic features of B cell receptor (BCR) and T cell receptor (TCR) genes were previously studied in lymphocytes, but little is known about their epigenetic features in other cells. Here, we explored histone modifications and transcription markers at the BCR and TCR loci in lymphocytes (pro-B, DP T cells, and mature CD4+ T cells), compared to embryonic stem (ES) cells and neurons. In B cells, the BCR loci exhibited active histone modifications and transcriptional markers indicative of active loci. Similar results were observed at the TCR loci in T cells. All loci were largely inactive in neurons. Surprisingly, in ES cells all AgR loci displayed a high degree of active histone modifications and markers of active transcription. Locations of these active histone modifications in ES cells were largely distinct from those in pro-B cells, and co-localized at numerous binding locations for transcription factors Oct4, Sox2, and Nanog. ES and pro-B cells also showed distinct binding patterns for the ubiquitous transcription factor YY1 and chromatin remodeler Brg1. On the contrary, there were many overlapping CCCTC-binding factor (CTCF) binding patterns when comparing ES cells, pro-B cells, and neurons. Our study identifies epigenetic features in ES cells and lymphocytes that may be related to ES cell pluripotency and lymphocyte tissue-specific activation at the AgR loci.

Loss of AID exacerbates the malignant progression of CLL.

Activation-induced cytidine deaminase (AID) has been implicated as both a positive and a negative factor in the progression of B cell chronic lymphocytic leukemia (CLL), but the role that it plays in the development and progression of this disease is still unclear. We generated an AID knockout CLL mouse model, AID-/-/Eµ-TCL1, and found that these mice die significantly earlier than their AID-proficient counterparts. AID-deficient CLL cells exhibit a higher ER stress response compared to Eµ-TCL1 controls, particularly through activation of the IRE1/XBP1s pathway. The increased production of secretory IgM in AID-deficient CLL cells contributes to their elevated expression levels of XBP1s, while secretory IgM-deficient CLL cells express less XBP1s. This increase in XBP1s in turn leads AID-deficient CLL cells to exhibit higher levels of B cell receptor signaling, supporting leukemic growth and survival. Further, AID-/-/Eµ-TCL1 CLL cells downregulate the tumor suppressive SMAD1/S1PR2 pathway and have altered homing to non-lymphoid organs. Notably, CLL cells from patients with IgHV-unmutated disease express higher levels of XBP1s mRNA compared to those from patients with IgHV-mutated CLL. Our studies thus reveal novel mechanisms by which the loss of AID leads to worsened CLL and may explain why unmutated CLL is more aggressive than mutated CLL.

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.

B cell activator PAX5 promotes lymphomagenesis through stimulation of B cell receptor signaling.

The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5-tamoxifen receptor fusion protein (Pax5ER(TAM)) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.

Altered 3D chromatin structure permits inversional recombination at the IgH locus.

Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombination activating gene products (RAG) 1 and 2. Diversity (DH) gene segments rearrange first, followed by variable (VH) gene rearrangements. Here, we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, which recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on wild-type IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by distinct mechanisms from those that apply to DH gene segments. These distinctions may underlie differential allelic choice associated with each step of IgH gene assembly.

Factors that attract veterinarians to or discourage them from research careers: a program director's perspective.

There is a nationwide shortage of veterinarian-scientists in the United States. Barriers to recruiting veterinary students into research careers need to be identified, and mechanisms devised to reduce these barriers. Barriers to attracting veterinary students into research careers include ignorance of available research careers and of the training opportunities. Once admitted, students in research training programs often feel isolated, fitting into neither the veterinary environment nor the research environment. To address the above issues, it is necessary to advertise and educate the public about opportunities for veterinarian-scientists. Schools need to develop high-quality training programs that are well structured but retain appropriate flexibility. Sufficient resources are needed to operate these programs so that students do not graduate with significant debt. A community of veterinarian-scientists needs to be developed so that students do not feel isolated but, rather, are part of a large community of like-minded individuals. Because of the complexities of programs that train veterinarian-scientists, it is necessary to provide extensive advising and for faculty to develop a proactive, servant-leadership attitude. Finally, students must be made aware of career options after graduation.

Veterinarians in biomedical research: building national capacity.

This Executive Summary provides the conclusions from the presentations and discussions at the conference Veterinarians in Biomedical Research-Building National Capacity, a meeting coordinated by the AAVMC and held at the National Institutes of Health (NIH), Bethesda, MD, August 1-4, 2007.

Mechanism of e47-Pip interaction on DNA resulting in transcriptional synergy and activation of immunoglobulin germ line sterile transcripts.

E47 and Pip are proteins crucial for proper B-cell development. E47 and Pip cooperatively bind to adjacent sites in the immunoglobulin kappa chain 3' enhancer and generate a potent transcriptional synergy. We generated protein-DNA computer models to visualize E47 and Pip bound to DNA. These models predict precise interactions between the two proteins. We tested predictions deduced from these models by mutagenesis studies and found evidence for novel direct interactions between the E47 helix-loop-helix domain (Arg 357 or Asp 358) and the Pip N terminus (Leu 24). We also found that precise spatial alignment of the binding sites was necessary for transcriptional synergy and cooperative DNA binding. A Pip dominant negative mutant that cannot synergize with E47 inhibited enhancer activity in plasmacytoma cells and could not activate transcription in pre-B cells. Using electrophoretic mobility shift assays, we found that Pip can bind to the heavy-chain intron enhancer region. In addition, we found that in fibroblasts Pip greatly increased E47 induction of germ line I micro transcripts associated with somatic rearrangement and isotype class switching. However, a Pip dominant negative mutant inhibited germ line I micro transcripts. The importance of these results for late B-cell functions is discussed.

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 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.