YY1 Regulates the Germinal Center Reaction by Inhibiting Apoptosis.

The germinal center (GC) reaction produces high-affinity Abs for a robust adaptive immune response. When dysregulated, the same processes cause GC B cells to become susceptible to lymphomagenesis. It is important to understand how the GC reaction is regulated. In this study, we show that transcription factor YY1 is required to maintain a robust GC reaction in mice. Selective ablation of YY1 significantly decreased in the frequency and number of GC B cells during the GC reaction. This decrease of GC B cells was accompanied by increased apoptosis in these cells. Furthermore, we found that loss of YY1 disrupted the balance between dark zones and light zones, leading to a preferential decrease in dark zone cells. Collectively, these results indicate that YY1 plays an important role in regulating the balance between dark zone and light zone cells in GCs and between survival and death of GC B cells.

Knockdown of zebrafish YY1a can downregulate the phosphatidylserine (PS) receptor expression, leading to induce the abnormal brain and heart development.

BACKGROUND: Yin Yang 1 (YY1) is a ubiquitously expressed GLI-Kruppel zinc finger-containing transcriptional regulator. YY1 plays a fundamental role in normal biologic processes such as embryogenesis, differentiation, and cellular proliferation. YY1 effects on the genes involved in these processes are mediated via initiation, activation, or repression of transcription depending upon the context in which it binds. The role of the multifunctional transcription factor Yin Yang 1 (YY1) in tissue development is poorly understood. In the present, we investigated YY1a role in developing zebrafish on PSR-mediated apoptotic cell engulfment during organic morphogenesis. RESULTS: YY1a is first expressed 0.5 h post-fertilization (hpf), in the whole embryo 12 hpf, and in brain, eyes, and heart 72 hpf by in situ hybridization assay. The nucleotide sequence of zebrafish YY1a transcription factor (clone zfYY1a; HQ 166834) was found to be similar to that of zebrafish YY1a (99 % sequence identity; NM 212617). With the loss-of-function assay, YY1a knockdown by a morpholino oligonucleotide led to downregulation of the phosphatidylserine engulfing receptor zfPSR during embryonic segmentation and to the accumulation of a large number of dead apoptotic cells throughout the entire early embryo, especially in the posterior area. Up to 24 hpf, these cells interfered with embryonic cell migration and cell-cell interactions that normally occur in the brain, heart, eye, and notochord. Finally, with gain-of-function assay, defective morphants could be rescued by injecting both YY1a mRNA and PSR mRNA and trigger resumption of normal development. CONCLUSIONS: Taken together, our results suggest that YY1a regulates PS receptor expression that linked to function of PSR-phagocyte mediated apoptotic cell engulfment during development, especially the development of organs such as the brain and heart. YY1a/PSR-mediated engulfing system may involve in diseases.

mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex.

Transcriptional complexes that contain peroxisome-proliferator-activated receptor coactivator (PGC)-1alpha control mitochondrial oxidative function to maintain energy homeostasis in response to nutrient and hormonal signals. An important component in the energy and nutrient pathways is mammalian target of rapamycin (mTOR), a kinase that regulates cell growth, size and survival. However, it is unknown whether and how mTOR controls mitochondrial oxidative activities. Here we show that mTOR is necessary for the maintenance of mitochondrial oxidative function. In skeletal muscle tissues and cells, the mTOR inhibitor rapamycin decreased the gene expression of the mitochondrial transcriptional regulators PGC-1alpha, oestrogen-related receptor alpha and nuclear respiratory factors, resulting in a decrease in mitochondrial gene expression and oxygen consumption. Using computational genomics, we identified the transcription factor yin-yang 1 (YY1) as a common target of mTOR and PGC-1alpha. Knockdown of YY1 caused a significant decrease in mitochondrial gene expression and in respiration, and YY1 was required for rapamycin-dependent repression of those genes. Moreover, mTOR and raptor interacted with YY1, and inhibition of mTOR resulted in a failure of YY1 to interact with and be coactivated by PGC-1alpha. We have therefore identified a mechanism by which a nutrient sensor (mTOR) balances energy metabolism by means of the transcriptional control of mitochondrial oxidative function. These results have important implications for our understanding of how these pathways might be altered in metabolic diseases and cancer.

Yin-yang1 is required in the mammalian oocyte for follicle expansion.

The multifaceted polycomb group gene Yin-Yang1 (Yy1) has been implicated in a variety of transcriptional regulatory roles both as an activator and silencer of gene expression. Here we examine the role of Yy1 during oocyte growth by conditional deletion of the locus in the growing oocyte. Our results indicate that YY1 is required for oocyte maturation and granulosa cell expansion. In mutant oocytes, we observe severely reduced expression of both Gdf9 and Bmp15, suggesting a mechanism underlying the failure of granulosa cell expansion. Consequently, we observe infertility, failure of estrus cycling, and altered reproductive hormone levels in mutant females. Additionally, we find that YY1-deficient oocytes exhibit altered levels of several oocyte-specific factors, including Pou5f1, Figla, Lhx8, Oosp1, and Sohlh2. These results document YY1's involvement in folliculogenesis and ovarian function in the mouse and indicate that YY1 is required specifically in the oocyte for oocyte-granulosa cell communication.

In vivo YY1 knockdown effects on genomic imprinting.

The YY1 transcription factor is predicted to control several imprinted domains, including the Peg3, Gnas and Xist/Tsix regions. To test this possibility, we have used RNA interference strategies to generate transgenic mouse lines that express reduced levels of the cellular YY1 protein. As predicted, lowering YY1 levels resulted in global expression changes in these three imprinted domains. In neonatal brains, most imprinted genes of the Peg3 domain were up-regulated. In the Gnas domain, Nespas was down-regulated, whereas three other imprinted transcripts were up-regulated, including Nesp, Gnasxl and Exon1A. In the Xist/Tsix domain, no obvious change was detected in the expression levels of the two genes in female mice. However, male mice showed low-level coordinated, up- and down-regulation of Xist and Tsix, respectively, suggesting potential de-repression of Xist in a subset of male cell populations. YY1 knockdown also changed the methylation levels at the imprinting control regions (ICRs) of these domains in a target-specific manner. In addition, breeding experiments indicated that the birth weights of 20% of the transgenic females were much lower than those of normal female littermates. We surmise that this gender-specific outcome is caused by the YY1 knockdown effect on the Xist locus of females. In sum, these results demonstrate that YY1 indeed functions as a trans factor for transcriptional regulation and DNA methylation of these imprinted domains in vivo.

Yin-Yang 1 regulates effector cytokine gene expression and T(H)2 immune responses.

BACKGROUND: The transcription factor Yin-Yang 1 (YY-1) binds to the promoter regions of several T-cell cytokine genes, but the expression and contribution of this factor to cytokine gene expression and T-cell activation in vivo is not clear. OBJECTIVE: We sought to better define the role of YY-1 in T-cell gene regulation and allergic immune responses. METHODS: We studied cytokine gene expression in T lymphocytes isolated from wild-type mice and heterozygous littermates bearing 1 targeted yy-1 allele (yy-1(+/-) mice). T cells were stimulated with anti-T-cell receptor (anti-TCR) plus CD28 antibodies or with peptide antigen plus antigen-presenting cells by using newly generated yy-1(+/-) TCR transgenic mice. We also studied ovalbumin-driven allergic immune responses in a mouse model of asthma and YY-1 expression in lung tissue from human asthmatic subjects. RESULTS: CD4(+) T cells from yy-1(+/-) mice secreted significantly less IL-4 and IFN-gamma compared with wild-type littermates after TCR-dependent activation, whereas IL-2 production was not significantly affected. Both airway inflammation and recall splenocyte IL-4 production were inhibited in yy-1(+/-) mice, as was antigen-driven T-cell proliferation. YY-1 expression was higher in airway biopsy specimens from asthmatic compared with control subjects. CONCLUSION: These data indicate that YY-1 regulates T-cell cytokine gene expression and allergic immune responses in a gene dose-dependent manner.

Yin Yang 1 Orchestrates a Metabolic Program Required for Both Neural Crest Development and Melanoma Formation.

Increasing evidence suggests that cancer cells highjack developmental programs for disease initiation and progression. Melanoma arises from melanocytes that originate during development from neural crest stem cells (NCSCs). Here, we identified the transcription factor Yin Yang 1 (Yy1) as an NCSCs regulator. Conditional deletion of Yy1 in NCSCs resulted in stage-dependent hypoplasia of all major neural crest derivatives due to decreased proliferation and increased cell death. Moreover, conditional ablation of one Yy1 allele in a melanoma mouse model prevented tumorigenesis, indicating a particular susceptibility of melanoma cells to reduced Yy1 levels. Combined RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and untargeted metabolomics demonstrated that YY1 governs multiple metabolic pathways and protein synthesis in both NCSCs and melanoma. In addition to directly regulating a metabolic gene set, YY1 can act upstream of MITF/c-MYC as part of a gene regulatory network controlling metabolism. Thus, both NCSC development and melanoma formation depend on an intricate YY1-controlled metabolic program.

miR-29a regulates vascular neointimal hyperplasia by targeting YY1.

OBJECTIVES: The formation of vascular neointima is mainly related to impairment of the vascular endothelial barrier and abnormal proliferation and migration of smooth muscle cells. The objective of this study was to investigate whether miR-29a exerts any promoting effect on the vascular neointimal hyperplasia and if so, its mechanism. MATERIALS AND METHODS: RT-qPCR was performed to determine expression of miR-29a in vascular smooth muscle cells (VSMC) and vascular neointimal hyperplasia. To further understand its role, we restored its expression in VSMCs by transfection with miR-29a mimics or inhibitors. Effects of miR-29a on cell proliferation were also determined. RESULTS: In this study, we used two kinds of model to observe the role of miR-29a in neointimal hyperplasia induced by carotid ligation or balloon injury. The major findings were that: (i) miR-29a overexpression promoted neointimal hyperplasia induced by carotid ligation; (ii) miR-29a increased proliferation of VSMCs, one aspect of which was by targeting expression of Ying and yang 1 protein (YY1), a negative regulator of Cyclin D1. A further aspect, was by increasing expression of Krüppel-like factor 5, a positive regulator of Cyclin D1, thereby allowing formation a synergistic effect. (iii) Tongxinluo (TXL), a traditional Chinese medicine reduced neointimal formation in ligated vessels by inhibiting VSMC proliferation and migration. CONCLUSIONS: These findings provide a new molecular mechanism of TXL in decreasing neointima hyperplasia.

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.

Brown Adipose YY1 Deficiency Activates Expression of Secreted Proteins Linked to Energy Expenditure and Prevents Diet-Induced Obesity.

Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight.

Decreased genetic dosage of hepatic Yin Yang 1 causes diabetic-like symptoms.

Insulin sensitivity in liver is characterized by the ability of insulin to efficiently inhibit glucose production and fatty acid oxidation as well as promote de novo lipid biosynthesis. Specific dysregulation of glucose and lipid metabolism in liver is sufficient to cause insulin resistance and type 2 diabetes; this is seen by a selective inability of insulin to suppress glucose production while remaining insulin-sensitive to de novo lipid biosynthesis. We have previously shown that the transcription factor Yin Yang 1 (YY1) controls diabetic-linked glucose and lipid metabolism gene sets in skeletal muscle, but whether liver YY1-targeted metabolic genes impact a diabetic phenotype is unknown. Here we show that decreased genetic dosage of YY1 in liver causes insulin resistance, hepatic lipid accumulation, and dyslipidemia. Indeed, YY1 liver-specific heterozygous mice exhibit blunted activation of hepatic insulin signaling in response to insulin. Mechanistically, YY1, through direct recruitment to promoters, functions as a suppressor of genes encoding for metabolic enzymes of the gluconeogenic and lipogenic pathways and as an activator of genes linked to fatty acid oxidation. These counterregulatory transcriptional activities make targeting hepatic YY1 an attractive approach for treating insulin-resistant diabetes.

Yin Yang 1 deficiency in skeletal muscle protects against rapamycin-induced diabetic-like symptoms through activation of insulin/IGF signaling.

Rapamycin and its derivatives are mTOR inhibitors used in tissue transplantation and cancer therapy. A percentage of patients treated with these inhibitors develop diabetic-like symptoms, but the molecular mechanisms are unknown. We show here that chronic rapamycin treatment in mice led to insulin resistance with suppression of insulin/IGF signaling and genes associated within this pathway, such as Igf1-2, Irs1-2, and Akt1-3. Importantly, skeletal muscle-specific YY1 knockout mice were protected from rapamycin-induced diabetic-like symptoms. This protection was caused by hyperactivation of insulin/IGF signaling with increased gene expression in this cascade that, in contrast to wild-type mice, was not suppressed by rapamycin. Mechanistically, rapamycin induced YY1 dephosphorylation and recruitment to promoters of insulin/IGF genes, which promoted interaction with the polycomb protein-2 corepressor. This was associated with H3K27 trimethylation leading to decreased gene expression and insulin signaling. These results have implications for rapamycin action in human diseases and biological processes such as longevity.

Yin Yang 1 regulates the transcriptional activity of androgen receptor.

The multifunctional protein Yin Yang 1 (YY1) has an important role in epigenetic regulation of gene expression. YY1 is highly expressed in various types of cancers, including prostate cancer. Currently, the mechanism underlying the functional role of YY1 in prostate tumorigenesis remains unclear. In this report, we investigated the functional interplay between YY1 and androgen receptor (AR), and the effect of YY1 on AR-mediated transcription. We found that YY1 physically interacts with AR both in a cell-free system and in cultured cells. YY1 is required for the optimal transcriptional activity of AR in promoting the transcription of the prostate-specific antigen (PSA) promoter. However, ectopic YY1 expression in LNCaP cells did not further enhance the reporter driven by the PSA promoter, suggesting that an optimal level of YY1 is already established in prostate tumor cells. Consistently, YY1 depletion in LNCaP cells reduced endogenous PSA levels, but overexpressed YY1 did not significantly increase PSA expression. We also observed that YY1-AR interaction is essential to YY1-mediated transcription activity of AR and YY1 is a necessary component in the complex binding to the androgen response element. Thus, our study demonstrates that YY1 interacts with AR and regulates its transcriptional activity.

Loss of YY1 impacts the heterochromatic state and meiotic double-strand breaks during mouse spermatogenesis.

The progression of spermatogenesis involves global changes in chromatin structure and conformation. However, our understanding of the regulation of chromatin changes in germ cells remains limited. Here we describe both in vivo RNA interference and genetic mouse knockout studies that identify a critical role for Yin Yang 1 (YY1) in mammalian spermatogenesis. In the YY1-deficient spermatocytes, we find a significant decrease in the global level of the heterochromatin markers (H3K9me3 and HP1-gamma) and a concomitant increase in the double-strand break (DSB) signals on chromosomes (gamma-H2AX, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and Rad51) at the leptotene/zygotene stages of spermatocytes. These findings support a link between chromatin modifications and meiotic DSB formation, as has been seen in other model organisms. We propose that a depletion of YY1 may alter the structural integrity of heterochromatin, rendering it more accessible to the DSB machinery. In addition, YY1-deficient spermatocytes show univalent formation, increased aneuploidy, and pachytene cell death, which are likely due to defects in DNA repair. Taken together, this study identifies an important role for YY1 in mouse meiosis and provides new insight into mechanisms that regulate mammalian spermatogenesis.

Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis.

We know that the Yin Yang 1 protein (YY1) overexpression is positively and strongly correlated with the degree of malignancy of bone tumors. Therefore, we questioned whether we could influence cell invasiveness by deleting YY1 in human osteosarcoma cells (SaOs-2), as tested in Matrigel-coated filters and metastasis implantation of such osteosarcoma cells in vivo, by serial analysis with nuclear magnetic resonance. Moreover, we focused our work on the chemokine receptor CXCR4 and its inhibition by T22 antibody, as well as on systemic (direct in vivo assay) and computer-assisted imaging of angiogenesis-related metastasis. Results showed that cell invasiveness and metastasis implantation by wild-type SaOs-2 cells, as evaluated by histology and immunohistochemistry, are associated with up-regulation of CXCR4 expression, which in turn was significantly reduced by T22. In addition, deletion of YY1 (siRNAYY1-SaOs-2) induced a significant decrease of cell invasion and metastasis growth. This phenomenon was associated with decreased vascular endothelial growth factor (VEGF)/angiogenesis and a complex rearrangement of the gene expression profile as evaluated by microarray analysis. In conclusion, YY1 and VEGF/CXCR4 seem to intervene in the pathogenesis of the malignant phenotype of osteosarcoma by acting on cell invasiveness and metastasis growth.

Identification of a Ctcf cofactor, Yy1, for the X chromosome binary switch.

In mammals, inactivation of one X chromosome in the female equalizes gene dosages between XX females and XY males. Two noncoding loci, Tsix and Xite, together regulate X chromosome fate by controlling homologous chromosome pairing, counting, and mutually exclusive choice. Following choice, the asymmetry of Xite and Tsix expression drives divergent chromosome fates, but how this pattern becomes established is currently unknown. Although no proven trans-acting factors have been identified, a likely candidate is Ctcf, a chromatin insulator with essential function in autosomal imprinting. Here, we search for trans-factors and identify Yy1 as a required cofactor for Ctcf. Paired Ctcf-Yy1 elements are highly clustered within the counting/choice and imprinting domain of Tsix. A deficiency of Yy1 leads to aberrant Tsix and Xist expression, resulting in a deficit of male and female embryos. Yy1 and Ctcf associate through specific protein-protein interactions and together transactivate Tsix. We propose that the Ctcf-Yy1-Tsix complex functions as a key component of the X chromosome binary switch.