Yin Yang 1 Protein Research Articles

The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

IntroductionOverexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2α, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line.MethodsERBB2, AP-2α, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a χ2 test at a p value of less than 0.05. The functional role of AP-2α and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting.ResultsWe observed a statistically significant correlation between ERBB2 and AP-2α levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2α and YY1 (p < 0.02) as well as between the expression of AP-2α and YY1 (p < 0.001). Furthermore, the levels of both AP-2α and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2α and AP-2γ mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene.ConclusionThis study highlights the role of both AP-2α and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels.

Photoreceptor‐specific expression, light‐dependent localization, and transcriptional targets of the zinc‐finger protein Yin Yang 1 in the chicken retina

The zinc-finger transcription factor Yin Yang 1 (YY1) is a multifunctional protein that plays a critical role in embryonic development. Although it has been shown to play a role in eye development, its expression in the retina was not previously described. Here, we investigated YY1 expression in chicken tissues and we identified the neural retina as one of the tissues with highest YY1 protein levels. Immunohistochemical detection of YY1 in the retina revealed a clear-cut photoreceptor specificity and day/night differences in the cytoplasmic localization of the protein. YY1 was also present at high concentration in the nuclei of some photoreceptors. Gel-shift assays indicated YY1 bound to regulatory regions of several genes specifically expressed in photoreceptors. One of these genes, hydroxyindole-O-methyltransferase (EC 2.1.1.4), encodes the last enzyme of the melatonin synthesis pathway. Although over-expression of chicken YY1 was not sufficient to activate the chicken hydroxyindole-O-methyltransferase promoter in HEK293 cells, the YY1-binding site contained in this promoter was clearly required for full transcriptional activity in chicken embryonic retinal cells. These results suggest a role of YY1 in regulating the melatoninergic function of retinal photoreceptors.

Prostaglandin F2α Suppresses Rat Steroidogenic Acute Regulatory Protein Expression via Induction of Yin Yang 1 Protein and Recruitment of Histone Deacetylase 1 Protein

Prostaglandin F2alpha (PGF2alpha) plays a pivotal role in ovarian luteolysis by inhibiting the expression of steroidogenic acute regulatory (StAR) protein, leading to a decrease in intracellular cholesterol transport and luteal steroid production. Previously we have demonstrated that the transcription factor Yin Yang 1 (YY1) bound to three regions in the StAR promoter in vitro and repressed promoter activity. This study further defined the YY1-mediated PGF2alpha effect on the inhibition of StAR protein expression through YY1 interaction with a single region in the StAR promoter in vivo. PGF2alpha consistently suppressed StAR mRNA and protein expression in cultured luteal cells in a dose-dependent manner. PGF2alpha also enhanced YY1 protein expression and binding to its cis-element in a time-dependent pattern that preceded the decline in StAR protein levels. The StAR promoter region bound by YY1 was also associated with histone deacetylase 1 (HDAC1). PGF2alpha treatment promoted HDAC1 binding to and suppressed the histone H3 acetylation in this region. On the contrary, YY1 knockdown decreased HDAC1 binding, increased histone H3 acetylation, enhanced StAR protein expression, and negated PGF2alpha effect on StAR protein expression. Luciferase assays showed that YY1 overexpression inhibited StAR promoter activity and the addition of a HDAC inhibitor, trichostatin A, abrogated the effect of YY1. Trichostatin A-treated luteal cells displayed increased StAR protein expression. These data indicate that PGF2alpha enhances a direct YY1/StAR promoter interaction and the recruitment of HDAC1 to the promoter, thereby preventing transcriptional activation of the StAR gene.

PIASy-Mediated Sumoylation of Yin Yang 1 Depends on Their Interaction but Not the RING Finger

As a multifunctional protein, Yin Yang 1 (YY1) has been demonstrated to regulate both gene expression and protein posttranslational modifications. However, gaps still exist in our knowledge of how YY1 can be modified and what the consequences of its modifications are. Here we report that YY1 protein can be sumoylated both in vivo and in vitro. We have identified lysine 288 as the major sumoylation site of YY1. We also discovered that PIASy, a SUMO E3 ligase, is a novel YY1-interacting protein and can stimulate the sumoylation of YY1 both in vitro and in vivo. Importantly, the effects of PIASy mutants on in vivo YY1 sumoylation correlate with the YY1-PIASy interaction but do not depend on the RING finger domain of PIASy. This regulation is unique to YY1 sumoylation because PIASy-mediated p53 sumoylation still relies on the integrity of PIASy, which is also true of all of the previously identified substrates of PIASy. In addition, PIASy colocalizes with YY1 in the nucleus, stabilizes YY1 in vivo, and differentially regulates YY1 transcriptional activity on different target promoters. This study demonstrates that YY1 is a target of SUMOs and reveals a novel feature of a SUMO E3 ligase in the PIAS family that selectively stimulates protein sumoylation independent of the RING finger domain.

Over Expression of YY1 and RKIP in Multiple Myeloma (Cell Lines and MM Tissues).

There is a need to identify underlying molecular mechanisms and gene products (targets for intervention and biomarkers) involved in MM chemo- or immuno-resistance. Recent findings from our laboratory have reported high expression of the transcription factor Yin Yang 1 (YY1) in several tumor types and have demonstrated its fundamental role in tumor cell chemo/immuno-resistance (Gordon, S., et al., Oncogene 25:1125, 2006). In contrast, low levels of the Raf kinase inhibitor protein (RKIP) has been shown to play a pivotal role in the negative regulation of cell survival signaling pathways, and has been considered a metastasis tumor suppressor. The overall objective of our studies is to examine whether MM cell lines and tissues derived from MM patients present deregulated expression patterns of RKIP and YY1, and to demonstrate the roles of YY1 and RKIP in MM pathogenesis and response to various therapies. In the present study, the myeloma cell lines RPMI-8226 and MM-1S were screened for RKIP and YY1 expression at the protein and m-RNA levels. In addition, fresh tissues from MM patients were also examined for RKIP and YY1 expression by IHC and their patterns compared with those observed in normal bone marrow cells. The findings demonstrate that both MM cell lines express remarkably higher levels of RKIP protein and transcripts compared to other tumor cell lines examined (lymphomas and prostate cancer), as assessed by western, RT-PCR and IHC. There was significantly higher expression of RKIP in MM patient's samples compared to normal bone marrow cells. YY1 protein and m-RNA levels were detected in the MM cell lines; however, they were lower compared to Ramos and PC-3 cells. YY1 expression in MM tissues was significantly elevated compared to normal bone marrow cells. RKIP was basically present in the cytoplasm while YY1 was mainly accumulated in the nucleus. In contrast, normal bone marrow cells presented primarily cytoplasmic YY1 and RKIP expression. The YY1 expressing population was the CD38+/CD138− cell subset, which has been reported to be more susceptible to apoptosis (Mitsiadis, S.C., et al., Blood 98:795, 2001). Overall, the present findings support the potential involvement of two new gene products, such as YY1 and RKIP, in MM pathogenesis and their implication in regulating MM resistance to conventional therapeutics. Moreover, these gene products suggest their potential use as new therapeutic targets and/or biomarkers in multiple myeloma. Future studies with large cohorts of tissues will elucidate the importance of the above findings and will give new insights in the unexpected RKIP over expression and activity in multiple myeloma.

Yin Yang 1 Physically Interacts with Hoxa11 and Represses Hoxa11-dependent Transcription

Yin Yang 1 (YY1) plays an indispensable role in embryonic development. YY1 contains an evolutionarily conserved, 22-amino acid segment, the PHO homology region (PHR), which is located within its central domain (spacer) and has been shown previously to participate in the recruitment of Polycomb group of proteins and in YY1-mediated transcription. In this report, we show that the PHR physically interacts with several Abd-B-type Hox proteins. Although ectopic expression of Hoxa11 enhanced target promoter activity, overexpression of YY1 repressed this effect, which was abrogated by YY1 siRNA and the histone deacetylase inhibitor trichostatin A. We have further demonstrated that this suppression effect was the result of YY1-dependent recruitment of HDAC2 to the Hoxa11 target promoter. Taken together, our findings show that YY1 represses Hoxa11-dependent transcription via interactions with the Hox proteins and HDAC recruitment, providing a link between an Abd-type Hox protein and a Polycomb group protein at the level of direct protein-protein interactions. These findings not only provide a novel insight into YY1 function but also identify a new regulation of homeotic protein-mediated transcriptional regulation in general.

Expression of transcription factor Yin Yang 1 in human osteosarcomas

The transcription factor Yin Yang 1 (YY1) is known to be present in some human cancer cell lines and its expression correlates with immune-mediated apoptosis. By using Western blot analysis, we have shown that the YY1 protein is strongly expressed in human osteosarcoma cells and localised mainly in the nucleus. Moreover, by using immunohistochemistry and RT-PCR techniques, we have analysed the expression of YY1 protein in biopsies from human osteosarcomas. The YY1 protein was not detectable by immunohistochemistry in osteoid tissue. However, its expression was restricted to osteosarcoma tissues. These data were confirmed by densitometric analysis of RT-PCR for YY1 expression. Thus, YY1 gene activation appears to be an early event in the process of osteoblastic transformation and its detection may represent, together with the analysis of other established markers, a useful diagnostic tool in human osteosarcomas.

Yin Yang 1 Cooperates with Activator Protein 2 to Stimulate ERBB2 Gene Expression in Mammary Cancer Cells

Overexpression of the ERBB2 oncogene is observed in about 30% of breast cancers and is generally correlated with a poor prognosis. Previous results from our and other laboratories indicated that elevated transcriptional activity contributes significantly to the overexpression of ERBB2 mRNA in mammary adenocarcinoma cell lines. Activator protein 2 (AP-2) transcription factors account for this overexpression through two recognition sequences located 215 and 500 bp upstream from the transcription start site. Furthermore, AP-2 transcription factors are highly expressed in cancer cell lines overexpressing ERBB2. In this report, we examined the cooperative effect of Yin Yang 1 (YY1) on AP-2-induced activation of ERBB2 promoter activity. We detected high levels of YY1 transcription factor in mammary cancer cell lines. Notably, we showed that YY1 enhances AP-2alpha transcriptional activation of the ERBB2 promoter through an AP-2 site both in HepG2 and in HCT116 cells, whereas a carboxyl-terminal-truncated form of YY1 cannot. Moreover, we demonstrated the interaction between endogenous AP-2 and YY1 factors in the BT-474 mammary adenocarcinoma cell line. In addition, inhibition of endogenous YY1 protein by an antisense decreased the transcription of an AP-2-responsive ERBB2 reporter plasmid in BT-474 breast cancer cells. Finally, we detected in vivo AP-2 and YY1 occupancy of the ERBB2 proximal promoter in chromatin immunoprecipitation assays. Our data thus provide evidence that YY1 cooperates with AP-2 to stimulate ERBB2 promoter activity through the AP-2 binding sites.

Cdx Binding Determines the Timing of Enhancer Activation in Postnatal Duodenum

In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.

The Yin Yang-1 (YY1) protein undergoes a DNA-replication-associated switch in localization from the cytoplasm to the nucleus at the onset of S phase.

The essential Yin Yang-1 gene (YY1) encodes a ubiquitous, conserved, multifunctional zinc-finger transcription factor in animals. The YY1 protein regulates initiation, activation, or repression of transcription from a variety of genes required for cell growth, development, differentiation, or tumor suppression, as well as from genes in some retroviruses and DNA viruses. Among the specific functions attributed to YY1 is a role in cell-cycle-specific upregulation of the replication-dependent histone genes. The YY1 protein binds to the histone alpha element, a regulatory sequence found in all replication-dependent histone genes. We therefore examined the abundance, DNA-binding activity and localization of the YY1 protein throughout the cell cycle in unperturbed, shake-off-synchronized Chinese hamster ovary and HeLa cells. We found that, whereas the DNA-binding activity of YY1 increased dramatically early in S phase, the YY1 mRNA and protein levels did not. YY1 changed subcellular distribution patterns during the cell cycle, from mainly cytoplasmic at G1 to mainly nuclear at early and middle S phase, then back to primarily cytoplasmic later in S phase. Nuclear accumulation of YY1 near the G1/S boundary coincided with both an increase in YY1 DNA-binding activity and the coordinate up-regulation of the replication-dependent histone genes. The DNA synthesis inhibitor aphidicolin caused a nearly complete loss of nuclear YY1, whereas addition of caffeine or 2-aminopurine to aphidicolin-treated cells restored both DNA synthesis and YY1 localization in the nucleus. These findings reveal a mechanism by which YY1 localization is coupled to DNA synthesis and responsive to cell-cycle signaling pathways. Taken together, our results provide insight into how YY1 might participate in the cell-cycle control over a variety of nuclear events required for cell division and proliferation.

Yin Yang 1, a vertebrate Polycomb group gene, regulates antero-posterior neural patterning

Polycomb group (PcG) genes are required for the stable repression of the homeotic genes and other developmentally regulated genes. Yin Yang 1 (YY1), a vertebrate homolog of the Drosophila PcG pleiohomeotic (Pho), is a multifunctional protein that can act as a repressor or activator of transcription. Xenopus YY1 (XYY1) protein was localized in the central nervous system (CNS), particularly anterior neural tube of tailbud stage embryos. To elucidate the role of endogenous XYY1, loss-of-function studies were performed using XYY1 antisense morpholino oligonucleotide ( XYY1 MO). Inhibition of XYY1 function resulted in embryos with antero-posterior axial patterning defects and reduction of head structures. XYY1 MO also reduced the expression of En2, a midbrain/hindbrain junction marker, which was rescued by co-injection of XYY1 mRNA. However, XYY1 MO-injection did not affect the expression of HoxB9, a spinal cord marker. These results suggest that YY1 controls antero-posterior patterning of the CNS during Xenopus embryonic development.

The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation

AbstractThe genetic defects underlying the pathogenesis of acute myeloid leukemia (AML) are still largely unknown. Retroviral insertion mutagenesis in mice has become a powerful tool to identify candidate genes involved in the development of leukemia and lymphoma. We have used this strategy with the 1.4 strain of Graffi murine leukemia virus (MuLV), which predominantly causes myeloid leukemias. Here, we report that Graffi-1.4–induced AML frequently harbors virus integrations in the gene encoding the transcription factor Yin Yang 1 (YY1). These integrations occurred in both orientations, and all were located in the 5′ promoter region of the gene, 0.5 to 1.5 kb upstream of the major transcriptional start site. Luciferase reporter assays showed that virus integration in this region increases promoter activity and renders it independent of a functional binding site for Sp1, a major transcriptional regulator of YY1. We used the murine 32D model to study the consequence of perturbed YY1 expression for myelopoiesis. YY1 protein levels were high in 32D parental cells maintained in interleukin-3–containing medium, but they dropped when the cells were induced to differentiate by granulocyte–colony-stimulating factor (G-CSF). Strikingly, G-CSF–induced neutrophilic differentiation was reduced in 32D cell transfectants ectopically expressing YY1. In similar experiments on primary bone marrow cells, enforced YY1 expression blocked the outgrowth of CFU-GM colonies. Increased YY1 expression was seen in some cases of human AML. Collectively, these data imply a possible role of perturbed expression of YY1 in the development of AML through interference with the myeloid differentiation program in the leukemic progenitor cells.

Involvement of YY1 and its correlation with c-myc in NDEA induced hepatocarcinogenesis, its prevention by d-limonene.

Anticarcinogenic activity of d-limonene has been well documented within last few years. We have also reported the anticarcinogenic activity of d-limonene in N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis. The involvement of oncogenes which adds to the mechanisms of d-limonene mediated chemprevention were also suggested in the same model system. The overexpression of c-myc oncoprotein in different durations of NDEA induced hepatrocarcinogenesis is observed which is inhibited completely when d-limonene was treated prior to and along with NDEA. To work further in this direction, an attempt has been made here to know the role of YY1 (Yin Yang 1) transcription factor in N-nitrodiethylamine (NDEA) induced hepatocarcinogenesis and its chemoprevention by d-limonene. Electrophoretic mobility shift assay results have clearly indicated the binding of YY1 in control liver tissue. But this binding is blocked in 60 days and 150 days NDEA treated liver tumors. Thus, it is assumed that there is deregulation of YY1 transcription factor in NDEA induced hepatocarcinogenesis. A similar type of binding to that of control liver tissue has also observed when limonene was given prior to NDEA administration. Western blot analysis has shown inhibition of YY1 protein in NDEA induced liver tumor samples in comparison to normal and both NDEA and limonene treated samples. On the otherhand RT-PCR analysis does not indicate any correlation between YY1 mRNA level and inhibition of YY1 protein. However, along with our earlier information about c-myc with the present study, clearly indicated the involvement of YY1 in NDEA induced hepatocarcinogenesis and d-limonene mediated chemoprevention which might be regulated by c-myc oncoprotein.

Induction of the transcriptional repressor Yin Yang-1 by vascular cell injury. Autocrine/paracrine role of endogenous fibroblast growth factor-2.

Yin Yang-1 (YY1) is a multifunctional transcription factor that can repress the expression of many growth factor, hormone, and cytokine genes implicated in atherogenesis. YY1 expression is activated in rat vascular smooth muscle cells shortly after injury. YY1 DNA binding activity paralleled elevated protein levels in the nucleus. Smooth muscle cell injury triggered the rapid extracellular release of immunoreactive fibroblast growth factor-2 (FGF-2). YY1 induction after injury was blocked by neutralizing antibodies directed against FGF-2. This growth factor increased YY1 mRNA and protein expression and stimulated YY1 binding and transcriptional activity. Overexpression of YY1 inhibited smooth muscle cell replication. Immunohistochemical analysis demonstrated YY1 staining in medial smooth muscle cells, coincident with FGF-2 expression. Proliferating cell nuclear antigen staining, in contrast, was confined mainly to the atherosclerotic intima. This is the first demonstration that YY1 is induced by either injury or FGF-2, is differentially expressed in normal and diseased human arteries, and that its overexpression inhibits vascular smooth muscle but not endothelial cell replication.

Expression, Activity, and Subcellular Localization of the Yin Yang 1 Transcription Factor in Xenopus Oocytes and Embryos

Yin Yang 1 (YY1) is a multifunctional transcription factor that acts as an activator, repressor, or initiator of transcription of numerous cellular and viral genes. Previous studies in tissue culture model systems suggest YY1 plays a role in development and differentiation in multiple cell types, but the biological role of YY1 in vertebrate oocytes and embryos is not well understood. Here we analyzed expression, activity, and subcellular localization profiles of YY1 during Xenopus laevis development. Abundant levels of YY1 mRNA and protein were detected in early stage oocytes and in all subsequent stages of oocyte and embryonic development through to swimming larval stages. The DNA binding activity of YY1 was detected only in early oocytes (stages I and II) and in embryos after the midblastula transition (MBT), which suggested that its potential to modulate gene expression may be specifically repressed in the intervening period of development. Experiments to determine transcriptional activity showed that addition of YY1 recognition sites upstream of the thymidine kinase promoter had no stimulatory or repressive effect on basal transcription in oocytes and post-MBT embryos. Although the apparent transcriptional inactivity of YY1 in oocytes could be explained by the absence of DNA binding activity at this stage of development, the lack of transcriptional activity in post-MBT embryos was not expected given the ability of YY1 to bind its recognition elements. Subsequent Western blot and immunocytochemical analyses showed that YY1 is localized in the cytoplasm in oocytes and in cells of developing embryos well past the MBT. These findings suggest a novel mode of YY1 regulation during early development in which the potential transcriptional function of the maternally expressed factor is repressed by cytoplasmic localization.

The transcriptional regulator Yin Yang 1 activates the myelin PLP gene.

Inauguration of the myelin program in developing oligodendrocytes requires the activation of those genes that encode the myelin proteins and the enzymes responsible for the synthesis and degradation of myelin lipids. An activator of the most abundantly expressed myelin protein, proteolipid protein (PLP), has been identified in a yeast one-hybrid system. The ubiquitously expressed zinc finger protein Yin Yang 1 (YY1) recognizes the myelin PLP promoter in vitro and in vivo. When overexpressed in an oligodendrocyte cell line, YY1 enhances transcription of the PLP promoter. A truncated version of YY1 that includes only the four zinc finger domains has little effect. The binding site for YY1 in the PLP promoter (site 3) fits the YY1 consensus site and DNA-protein complexes containing site 3 can be supershifted with an antibody directed against YY1 protein. Moreover, oligonucleotides with a mutated version of the PLP promoter site 3 are unable to bind to nuclear proteins or to compete for binding in a gel shift system. Finally, mutation of this site greatly reduces the activity of a 1-kb PLP promoter region in transfected glial cells. Our results suggest that PLP is a target gene for the transcriptional regulator YY1. This versatile transcription factor and nuclear matrix protein may boost transcription of the PLP gene to meet the demands of actively myelinating oligodendrocytes.