Yin Yang 1 Binding Sites Research Articles

Identification of Adjacent Binding Sites for the YY1 and E4BP4 Transcription Factors in the Ovine PrP (Prion) Gene Promoter

The PrP gene encodes the cellular isoform of the prion protein (PrP(c)) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrP(c), exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions -287 to -263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype.

Yin yang 1 directly regulates the transcription of RE‐1 silencing transcription factor

The RE-1 silencing transcription factor (REST) is a master transcription factor that plays a critical role in embryo development, especially during the process of neurogenesis and neural plasticity. However, the mechanism of REST gene transcription regulation is still an open question. Here, by combining bioinformatics analysis and experimental studies, we report that the transcription factor Yin Yang 1 (YY1) bound to a conserved YY1 binding site in the promoter of the mouse REST gene and positively regulated activity of this promoter in SH-SY5Y cells. Furthermore, analysis of microarray data revealed a significant correlation between the expression of YY1 and REST genes. Overall, this study suggests that YY1 directly regulates expression of the REST gene.

Yin Yang 1 enhancescyclooxygenase-2gene expression in macrophages

Expression of cyclooxygenase-2 (COX-2) is associated with the pathogenesis of inflammation and various cancers, including lung cancer. Yin Yang 1 (YY1) is a zinc-finger transcription factor that interacts with histone acetyltransferases and deacetylases for its transcriptional activity and also is involved in inflammation and tumorigenesis. We investigated whether YY1 regulates COX-2 expression. We located a possible YY1 binding site proximal to the transcription initiation site of the COX-2 promoter. Electrophoretic mobility shift assays show that YY1 bound to the putative YY1 site in vitro. To show biological relevance, we performed chromatin immunoprecipitation assays showing that lipopolysaccharide (LPS) treatment induced YY1 binding to the cognate site in the endogenous COX-2 promoter. Overexpression of YY1 in macrophages treated with either LPS or live Pseudomonas aeruginosa increased COX-2 transcriptional activity. Furthermore, YY1 enhanced COX-2 protein expression and prostaglandin D(2) production elicited by LPS treatment. Mechanistically, we observed that LPS treatment resulted in disruption of an interaction between YY1 and p300, a histone acetyltransferase, but did not affect the interaction between YY1 and histone deacetylase 1/2. These data suggest that in response to LPS, YY1 dissociates from p300 and binds to the COX-2 promoter, contributing to COX-2 expression in an inflammatory milieu.

Nucleophosmin/B23 regulates PCNA promoter through YY1

Ectopic over-expression of nucleophosmin/B23 caused a marked up-regulation in the amounts of Yin Yang 1 (YY1) and proliferating cellular nuclear antigen (PCNA) proteins. Transfection with nucleophosmin/B23-targeting siRNA induced a decrease in the intracellular amounts of nucleophosmin/B23, YY1, and PCNA. PCNA expression and its promoter activity were attenuated either by nucleophosmin/B23-siRNA or YY1-siRNA. The ChIP assay showed that positive regulation of PCNA is achieved by binding of YY1 to the initiation site of PCNA promoter. The binding of YY1 to the PCNA promoter and histone H4 acetylation was significantly decreased in nucleophosmin/B23-siRNA-treated cells as compared to control vector-transfected cells. Mutation in YY1 binding site resulted in the loss of PCNA promoter activity and the binding of YY1 to the promoter. The results have indicated that YY1 binds to the initiation site of the PCNA promoter along with histone H4 acetylation, leading to transcriptional activity. We have demonstrated that nucleophosmin/B23 plays an important role in the regulation of PCNA through YY1.

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.

Yin Yang 1 represses α-myosin heavy chain gene expression in pathologic cardiac hypertrophy

In the work presented here, we elucidate a mechanism for the repression of α-myosin heavy chain (MyHC) during pathological cardiac hypertrophy. We demonstrate that the transcription factor Yin Yang 1 (YY1) significantly decreases endogenous α-MyHC mRNA and protein expression in neonatal rat ventricular myocytes. Furthermore, mutation of the YY1 binding sites in the proximal rat α-MyHC promoter increases promoter activity and alleviates YY1-mediated repression of the promoter. Despite the presence of 5 sites that bind YY1, only one site, located at −94 bp of the rat α-MyHC promoter, is both necessary and sufficient for pathological repression of the promoter by phorbol esters, revealing a unique mechanism for the repression of α-MyHC expression during cardiac hypertrophy.

Yin yang 1 negatively regulates the differentiation-specific E1 promoter of human papillomavirus type 6.

Human papillomavirus type 6 (HPV-6) is a low-risk HPV whose replication cycle, like that of all HPVs, is differentiation dependent. We have previously shown that CCAAT displacement protein (CDP) binds the differentiation-induced HPV-6 E1 promoter and negatively regulates its activity in undifferentiated cells (W. Ai, E. Toussaint, and A. Roman, J. Virol. 73:4220-4229, 1999). Using electrophoretic mobility shift assays (EMSAs), we now report that Yin Yang 1 (YY1), a multifunctional protein that can act as a transcriptional activator or repressor and that can also inhibit HPV replication in vitro, binds the HPV-6 E1 promoter. EMSAs, using subfragments of the promoter as competitors, showed that the YY1 binding site is located at the 5' end of the E1 promoter. When a putative YY1 site was mutated, the ability of YY1 to bind was greatly decreased. The activity of the mutated E1 promoter, monitored with the reporter gene luciferase, was threefold greater than that of the wild-type promoter, suggesting that YY1 negatively regulates HPV-6 E1 promoter activity. Nuclear extracts from differentiated keratinocytes showed decreased binding of YY1 to the wild-type promoter. Consistent with this, in differentiated keratinocytes, the activity of the transfected luciferase gene transcribed from the mutated promoter was comparable to that of the wild-type promoter; both promoters were up-regulated in differentiated keratinocytes compared to undifferentiated cells. These data suggest that YY1 functions in undifferentiated keratinocytes but not in differentiated keratinocytes. Both the wild-type and mutated promoters could be negatively regulated by overexpression of a plasmid encoding CDP. Thus, both YY1 and CDP appear to be negative regulators of the differentiation-induced HPV-6 E1 promoter and thereby the HPV life cycle. In contrast, only binding of CDP was detected using the E1 promoter of the high-risk HPV-31.

Yin-yang 1 and Glucocorticoid Receptor Participate in the Stat5-mediated Growth Hormone Response of the Serine Protease Inhibitor 2.1 Gene

A growth hormone-inducible nuclear factor complex (GHINF), affinity-purified using the growth hormone response element (GHRE) from the promoter of rat serine protease inhibitor 2.1, was found to contain Stat5a and -5b, as well as additional components. The ubiquitous transcription factor yin-yang 1 (YY1) is present in GHINF. An antibody to YY1 inhibited the formation of the GHINF.GHRE complex in an electrophoretic mobility shift assay. Furthermore, Stat5 was co-immunoprecipitated from rat hepatic nuclear extracts with antibodies to YY1. An examination of the GHRE shows that, in addition to two gamma-activated sites, it contains a putative YY1 binding site between the two gamma-activated sites, overlapping them both. Mutation of this putative YY1 site results in a decrease of GHINF.GHRE complex formation in an electrophoretic mobility shift assay and a corresponding decrease in growth hormone (GH) response in functional assays. The glucocorticoid receptor was also present in GHINF, and Stat5 co-immunoprecipitates with glucocorticoid receptor in hepatic nuclear extracts from rats treated with GH. GH activation of serine protease inhibitor 2.1 requires the unique sequence of the GHRE encompassing the recognition sites of several transcription factors, and the interaction of these factors enhances the assembly of the transcription complex.

Functional analysis of the promoter region of the human phosphotyrosine phosphatase activator gene: Yin Yang 1 is essential for core promoter activity

The phosphotyrosine phosphatase activator (PTPA) has been isolated as an in vitro regulator of protein phosphatase 2A. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. Here we describe the further isolation, sequencing and functional characterization of the PTPA promoter region. In agreement with its ubiquitous expression, the PTPA promoter displays several characteristics of housekeeping genes: it lacks both a TATA-box and a CAAT-box, it is very GC-rich and it contains an unmethylated CpG island surrounding the transcription initiation site. Transient transfection experiments in different cell types with several truncated chimaeric luciferase reporter gene plasmids revealed the importance of the region between positions -67 and -39 for basal promoter activity. This region coincides remarkably well with the determined CpG island. Further analysis of this region demonstrated the presence of a Yin Yang 1 (YY1) binding motif at positions -52 to -44. Binding of YY1 to this sequence is demonstrated in bandshift and DNase I footprinting experiments. Another YY1 binding motif is found in the 5' untranslated region, at positions +27 to +35. Mutations in either of these sites, abolishing YY1 binding in vitro, have differential effects on promoter activity. Point mutations in both sites completely abolish promoter activity. Moreover, induction of promoter activity by co-transfection with a YY1 expression plasmid is fully dependent upon the presence of both intact YY1 binding sites. Thus YY1 apparently mediates basal transcription of the human PTPA gene through two binding sites within its proximal promoter.

USF/c-Myc Enhances, While Yin-Yang 1 Suppresses, the Promoter Activity of CXCR4, a Coreceptor for HIV-1 Entry

Transcription factors USF1 and USF2 up-regulate gene expression (i.e. , HIV-1 long terminal repeats) via interaction with an E box on their target promoters, which is also a binding site for c-Myc. The c-Myc oncoprotein is important in control of cellular proliferation and differentiation, while Yin-Yang 1 (YY1) has been shown to control the expression of a number of cellular and viral genes. These two proteins physically interact with each other and mutually inhibit their respective biological functions. In this study, we show that USF/c-Myc up-regulates, while YY1 down-regulates the promoter activity of CXCR4, a coreceptor for T cell-tropic HIV-1 entry. We have identified an E box around -260 and a YY1 binding site around -300 relative to the transcription start site. Mutation of the E box abolished USF/c-Myc-mediated up-regulation of CXCR4 promoter activity, and mutation of the YY1 binding site was associated with unresponsiveness to YY1-mediated inhibition. These data suggest that USF/c-Myc and YY1 may play an important role in the HIV-1-replicative cycle, by modulating both the viral fusion/entry process and viral expression.

The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1

Studies on the human glucocorticoid receptor (GR) promoter were carried out so as to understand the regulation of GR expression. A −2738 to +19 fragment of the human GR promoter was used to identify important regulatory elements involved in the control in GR expression in NIH 3T3 cells (mouse fibroblasts) and HeLa cells (human cervical carcinoma cells). Important regulatory domains in the distal region of the human GR promoter were identified by sequential 5′ end deletion analysis. A region between −2490 and −2025 contributed 50% of the measured transcriptional activity to the promoter. Using DNase I footprint analysis, four sites in this region were identified: −2362 to −2339 (mouse footprint, mFP); −2301 to −2293 (distal YY1, dYY1); −2130 to −2122 (middle YY1, mYY1); and, −2086 to −2078 (proximal YY1, pYY1). Three sites contained an identical core sequence, CCAAGATGG and were identified as Yin Yang 1 (YY1) binding sites. The site located at −2362 to −2339 was footprinted in NIH 3T3 cells only. The sequence of this site is a direct repeat with a 2-nucleotide spacer region, and it does not share homology with any known transcription factor binding sites. Computer analysis of the entire promoter sequence revealed an additional YY1 site located at −260 to −249 (initiator YY1, iYY1) with the sequence CTCCTCCATTTTG. Electrophoretic mobility supershift assays, with an anti-YY1 antibody, were used to confirm YY1 binding to these four putative YY1 binding sites. Site-directed deletion of all three upstream YY1 sites but not the iYY1 site, or the iYY1 site alone, showed a ~60% decrease in transcriptional activity of the hGR promoter in HeLa cells but had no effect in NIH 3T3 cells. A similar (50%) decrease in the expression of a full-length hGR/luciferase reporter gene was obtained when HeLa cells were cotransfected with a full-length antisense YY1 expression plasmid. Additionally, a region between −1841 and −1689 contributed to hGR promoter activity in both cell types tested. An Sp1 binding site was identified in this region (−1748 to −1733) by DNase I footprint and mobility supershift analyses. The presence of four YY1 binding sites in the human GR promoter suggests that these sites play a critical role in GR gene regulation.

Regulatory interactions of transcription factor YY1 with control sequences of the E6 promoter of human papillomavirus type 8.

Human papillomavirus type 8 (HPV-8) is a strictly cutaneous oncogenic virus known to induce malignant skin lesions in epidermodysplasia verruciformis patients. Our study shows that sequences surrounding transcription start sites of the HPV-8 oncogene E6 (nt 175-179) and comprising the presumable CCAAC and TATA boxes of the E6 promoter P175 contain a cluster of four motifs similar to the DNA recognition site of the multifunctional cellular transcription factor yin-yang 1 (YY1). Using DNase I footprinting and gel retardation tests it could be demonstrated that three of these motifs indeed act as YY1 binding sites. To test their functional relevance for P175 activity, engineered YY1 binding site mutants were analysed in the context of a P175 test vector using transient expression assays with human keratinocytes. YY1 turned out to exert both positive and negative effects upon the activity of the HPV-8 E6 promoter; binding of YY1 to a site upstream of the promoter's cap-position (BS1) activated transcription, whereas the downstream site (BS2) mediated repression. The second downstream YY1 binding site (BS3) seemed to play an auxiliary role, enhancing the negative effect of YY1 BS2. These observations define YY1 as an important cellular protein involved in the control of E6 oncogene expression of the skin-specific 'high risk' HPV-8 and emphasize the potential regulatory role of sequences located downstream of the transcription start site.

The Complete Sequence of the Host Cell Factor 1 (HCFC1) Gene and Its Promoter: A Role for YY1 Transcription Factor in the Regulation of Its Expression

We report here the complete sequence of the Host Cell Factor (HCFC1) gene, including two kilobases of the 5′-flanking region and 5.9 kb of the first intron. The upstream and 5′-untranslated regions contain several putative transcriptional factor binding sites and a 17-nt-long repeated element (SiSa element) present in six regularly spaced copies, of which five are perfectly identical, while the sixth has a transition substitution (CT for TC) at nucleotides 13 and 14. Four copies are contained in the flanking region, the fifth is at the beginning of the mRNA (position +9), and the sixth is at position 195 of the mRNA. This 17-bp element contains at its 5′ side an octamer sequence known to bind the Yin/Yang 1 (YY1) transcription factor; another YY1 binding octamer is present at the end of the first intron. The promoter also contains several Sp1 binding sites, some of which are located very close to SiSa elements. We demonstrate that YY1 binds to the 5′ half of the SiSa element, whose 3′ region binds in gel shift experiments an additional, as yet unidentified nuclear factor. Therefore the YY1 binding site in HCFC1 overlaps the site of a second factor, as has been described in several YY1-site-containing promoters. This suggests that HCFC1 expression might be regulated by the reciprocal interaction of several transcription factors.