Interactive Fly, Drosophila

pleiohomeotic


EVOLUTIONARY HOMOLOGS part 3/3

Transcriptional repression by YY1

The four C-terminal GLI-Kruppel type zinc fingers of YY1 have been identified as a transcriptional repression domain. Previous reports have proposed DNA-bending and activator-quenching mechanisms for this zinc finger-mediated repression. Previous work has indicated that p300 and CBP might be involved in YY1-mediated repression. These possible models have been examined for the zinc finger-mediated repression. The role of each zinc finger in the repression and DNA-binding functions was determined by using a structure-and-function approach. Zinc finger 2 of YY1 is here shown to plays a central role in both DNA binding and transcriptional repression. However, a survey of a panel of YY1 mutants indicates that these two functions can be separated, which argues against the DNA-bending model for repression. The physical interaction between YY1 and p300, a coactivator for CREB, is shown not to be sufficient for repression of CREB-mediated transcription. These studies indicate that YY1 functions as an activator-specific repressor. Repression of CTF-1-directed transcription may be accomplished through direct physical interaction between YY1 and this activator. In contrast, physical interaction is not necessary for YY1 to repress Sp1- and CREB-mediated transcription. Rather, the repression likely reflects an ability of YY1 to interfere with communication between these activators and their targets within the general transcription machinery. Taken together, these results suggest that YY1 employs multiple mechanisms to achieve activator-specific repression (Galvin, 1997).

YY1 is a mammalian zinc-finger transcription factor with unusual structural and functional features. It has been implicated as a positive and a negative regulatory factor that binds to the CCATNTT consensus DNA element located in promoters of many cellular and viral genes. A mammalian cDNA that encodes a YY1-binding protein and possesses sequence homology with the yeast transcriptional factor RPD3 (see Drosophila Rpd3) has been identified. RPD3 is a histone deacetylase that mediates repression. A Gal4 DNA binding domain-mammalian RPD3 fusion protein strongly represses transcription from a promoter containing Gal4 binding sites. Association between YY1 and mammalian RPD3 requires a glycine-rich region on YY1. Mutations in this region abolish the interaction with mammalian RPD3 and eliminate transcriptional repression by YY1. These data suggest that YY1 negatively regulates transcription by tethering RPD3 to DNA as a cofactor and that this transcriptional mechanism is highly conserved from yeast to human (Yang, 1996).

YY1 represses transcription when bound upstream of transcriptional initiation sites. This repression can be relieved by adenovirus E1A. Genetic evidence is presented that the ability of E1A to relieve YY1 repression is impaired by mutations that affect E1A binding to its associated protein p300. This suggests that E1A may modulate the repressor activity of YY1 by binding to p300, which may be physically complexed with YY1. A YY1/p300 protein complex in vivo was demonstrate by several independent approaches, and the YY1-interacting domain was mapped to the carboxy-terminal region of p300, distinct from the E1A-binding site. Unlike E2F/RB, the YY1/p300 complex is not disrupted by E1A. Functional studies using recombinant p300 demonstrated unequivocally that p300 is capable of mediating E1A-induced transcriptional activation through YY1. Taken together, these results reveal, for the first time, a YY1/p300 complex that is targeted by E1A and demonstrate a function for p300 in mediating interactions between YY1 and E1A. These data thus identify YY1 as a partner protein for p300 and uncover a molecular mechanism for the relief of YY1-mediated repression by E1A (Lee, 1995).

In the promoters of many immediate early genes, including c-fos, CArG DNA regulatory elements mediate basal constitutive expression and rapid and transient serum induction. CArG boxes also occur in the promoters of muscle-specific genes, including skeletal alpha-actin, where it confers muscle-specific expression. These elements are regulated, at least in part, by the ubiquitous transcription factors serum response factor (SRF) and YY1. The homeobox transcription factor Phox1/MHox has also been implicated in regulation of the c-fos CArG element and is thought to function by facilitating SRF binding to DNA. In vitro and in vivo evidence is provided that the mechanism of YY1 repression of CArG elements results from competition with SRF for overlapping binding sites. The binding sites of YY1 and SRF are described in detail through serial point mutations of the skeletal alpha-actin proximal CArG element and a mutation is identified that dramatically reduces YY1 binding but retains normal SRF binding. YY1 competes with SRF for binding to wild-type CArG elements, but not to this point mutant in vitro. This mutant is sufficient for muscle-specific expression in vivo but is much less sensitive to repression by YY1 overexpression. The YY1/SRF competition was examined to address the role of Phox1 at these elements. Phox1 overexpression does not diminish YY1-mediated repression, suggesting that transcriptional activation by Phox1 does not result from enhanced SRF binding to these elements. These methods may prove to be useful for assessing interactions between other CArG element regulatory factors (Martin, 1997).

The hormone combination of insulin, dexamethasone and prolactin induce accumulation of preproepidermal growth factor (EGF) mRNA in HC11 mouse mammary epithelial cells 16-24 h after the hormones are added to the cultures. Individual hormones or combinations of two of the hormones have no effect on EGF mRNA concentrations. The same hormone combination is capable of inducing expression of a reporter gene construct containing -888 to +25 bp of the EGF gene fused with luciferase. Deletions of the promoter between -888 and -271 bp has no detectable effect on basal or hormone-induced reporter gene expression. However, further deletion from -270 to -74 bp increases baseline to approximately equal hormone-induced reporter gene expression. This deletion also abolishes the hormone-induced increase in reporter gene expression. Sequence analysis suggested that this region contains a binding site for Yin-Yang-1 (YY1), which was confirmed by gelshift analysis. Mutation of the YY1 binding site increases baseline reporter gene expression to the same level as induced by insulin, dexamethasone and prolactin in the wild-type promoter. These results indicate that expression of the EGF gene in mammary epithelium is repressed by the YY1 site, and that removal of repression may play a part in regulating EGF gene expression in lactating mammary tissue (Fang, 1998).

The proto-oncoprotein c-Myc and the multifunctional transcriptional regulator YY1 have been shown to interact directly in a manner that excludes Max from the complex. Since binding to Max is necessary for all known c-Myc activities, the influence of YY1 on c-Myc function has been analyzed. YY1 is shown to be a potent inhibitor of c-Myc transforming activity. The region in YY1 required for inhibition corresponds to a functional DNA-binding domain and is distinct from the domains necessary for direct binding to c-Myc. Furthermore the transactivation domain of YY1 was not necessary, suggesting that gene regulation by YY1, for example, through DNA bending or displacement of regulators from DNA, could be the cause for the negative regulation of c-Myc. This model of indirect regulation of c-Myc by YY1 is supported by the finding that although YY1 does not bind to the c-Myc transactivation domain (TAD) in vitro it is able to inhibit transactivation by Gal4-MycTAD fusion proteins in transient transfections. As for the inhibition of transformation, an intact DNA-binding domain of YY1 was necessary and sufficient for this effect. In addition, YY1 does not alter c-Myc/Max DNA binding, further supporting an indirect mode of action. These findings point to a role of YY1 as a negative regulator of cell growth with a possible involvement in tumor suppression (Austen, 1998).

Transcription of sarcomeric alpha-actin genes is developmentally regulated during skeletal and cardiac muscle development through fine-tuned control mechanisms involving multiple cooperative and antagonistic transcription factors. Among the cis-acting DNA elements recognized by these factors is the sequence CC(A/T)6GG of the serum response element (SRE), which is present in a number of growth factor-inducible and myogenic specified genes. The cardiogenic homeodomain factor, Nkx-2.5, has been shown to serve as a positive acting accessory factor for serum response factor (SRF); together, they provide strong transcriptional activation of the cardiac alpha-actin promoter. In addition, Nkx-2.5 and SRF collaborate to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts. This is accomplished by a mechanism that involves coassociation of SRF and Nkx-2.5 on intact SREs of the alpha-actin promoter. The second SRE of the avian cardiac alpha-actin promoter serves as a binding site for Nkx-2.5, SRF, and zinc finger containing GLI-Kruppel-like factor, YY1. Expression of YY1 inhibits cardiac alpha-actin promoter activity, whereas coexpression of Nkx-2.5 and SRF is able to partially reverse YY1 repression. Displacement of YY1 binding by Nkx-2.5/SRF complex occurs through mutually exclusive binding across the CaSRE2. The interplay and functional antagonism between YY1 and Nkx-2.5/SRF might constitute a developmental as well as a physiologically regulated mechanism for the modulation of modulates cardiac alpha-actin gene expression during cardiogenesis (Chen, 1997).

Muscle-restricted transcription of sarcomeric actin genes is negatively controlled by the zinc finger protein YY1, which is down-regulated at the protein level during myogenic differentiation. To identify cellular proteins that might mediate the function/stability of YY1 in muscle cells, an adult human muscle cDNA library was screened using the yeast two-hybrid cloning system. A novel protein termed YAF2 (YY1- associated factor 2) that interacts with YY1 was isolated and characterized. The YAF2 cDNA encodes a 180 amino acid basic protein containing a single N-terminal C2-X10-C2 zinc finger. Lysine clusters are present that may function as a nuclear localization signal. Domain mapping analysis shows that the first and second zinc fingers of YY1 are targeted for YAF2 protein interaction. In contrast to the down-regulation of YY1, YAF2 message levels increase during in vitro differentiation of both rat skeletal and cardiac muscle cells. YAF2 appears to have a promyogenic regulatory role, since overexpression of YAF2 in C2 myoblasts stimulates myogenic promoter activity normally restricted by YY1. Co-transfection of YY1 reverses the stimulatory effect of YAF2. YAF2 also greatly potentiates proteolytic cleavage of YY1 by the calcium- activated protease m-calpain. The isolation of YAF2 may help in understanding the mechanisms through which inhibitors of myogenic transcription may be antagonized or eliminated by proteolysis during muscle development (Kalenik, 1997).

The nuclear factor Yin-Yang 1 (YY1), a ubiquitous DNA-binding protein, is able to interact with a silencer element in the gamma interferon (IFN-gamma) promoter region. YY1 can directly inhibit the activity of the IFN-gamma promoter by interacting with multiple sites in the promoter. In cotransfection assays, a YY1 expression vector significantly inhibits IFN-gamma promoter activity. Mutation of the YY1 binding site in the native IFN-gamma promoter is associated with an increase in the IFN-gamma promoter activity. Analysis of the DNA sequences of the IFN-gamma promoter reveals a second functional YY1 binding site that overlaps with an AP1 binding site. In this element, AP1 enhancer activity is suppressed by YY1. Since the nuclear level of YY1 does not change upon cell activation, these data support a model that the nuclear factor YY1 acts to suppress basal IFN-gamma transcription by interacting with the promoter at multiple DNA binding sites. This repression can occur through two mechanisms: (1) cooperation with an as-yet-unidentified AP2-like repressor protein, and (2) competition for DNA binding with the transactivating factor AP1 (Ye, 1996).

YY1 is a multifunctional transcription factor that has been shown to regulate the expression of a number of cellular and viral genes, including the human papillomavirus (HPV) oncogenes E6 and E7. In this study, the YY1-mediated repression of the HPV type 16 (HPV-16) E6-E7 promoter was analyzed. A systematic analysis to identify YY1 sites present in the HPV-16 long control region showed that of 30 potential YY1 binding motifs, 24 bind purified recombinant YY1 protein, but only 10 of these are able to bind YY1 when nuclear extracts of HeLa cells are used. Of these, only a cluster of five sites, located in the vicinity of an AP-1 motif, are found to be responsible for repressing the HPV-16 P97 promoter. All five sites are required for repression, the mutation of any one site giving rise to a four- to six-fold increase in transcriptional activity. The target for YY1-mediated repression has been identified as being a highly conserved AP-1 site, and it is proposed that AP-1 may represent a common target for YY1 repression. Data demonstrating that YY1 can bind the transcriptional coactivator CREB-binding protein is provided and a potentially novel mechanism by which YY1 represses AP-1 activity as a result of this YY1-CREB-binding protein interaction is proposed (O'Connor, 1996).

A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals. A new Zn finger protein, RYBP, has been identified that interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex. Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding. RYBP also interacts directly with YY1. In addition, RYBP acts as a transcriptional repressor in transiently transfected cells. Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous. Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals (Garcia, 1999).

A sequence within the transcription control region of the adeno-associated virus P5 promoter has been shown to mediate transcriptional activation by the adenovirus E1A protein. This same element mediates transcriptional repression in the absence of E1A. Two cellular proteins have been found to bind to overlapping regions within this sequence element. One of these proteins, YY1, is responsible for the repression. E1A relieves repression exerted by YY1 and further activates transcription through its binding site. A YY1-specific cDNA has been isolated. Its sequence reveals YY1 to be a zinc finger protein that belongs to the GLI-Kruppel gene family. The product of the cDNA binds to YY1 sites. When fused to the GAL4 DNA-binding domain, it is capable of repressing transcription directed by a promoter that contains GAL4-binding sites, and E1A proteins can relieve the repression and activate transcription through the fusion protein (Shi, 1991).

After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. Endogenous transcription factor Ying Yang 1 (YY1) is associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 are essential for the association of N1IC and YY1 according to the pull-down assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC are present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulates the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. The association between endogenous N1IC and intrinsic YY1 has also been demonstrated in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex (Yeh, 2003).

Expression of CCR5, a major coreceptor for human immunodeficiency virus type 1 (HIV-1), is regulated by a number of transcription factors. The YY1 transcription factor down-regulates CCR5 promoter activity and overexpression of YY1 reduces cell surface CCR5 expression and infectibility by R5-HIV-1. Because YY1 also down-regulates promoter activities of CXCR4, another major coreceptor for HIV-1 and HIV-1 long terminal repeat, this transcription factor may play a critical role in the pathogenesis of HIV-1 disease (Moriuchi, 2003).

Action of YY1 on Hox gene promoters

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. A detailed analysis of the mouse Hoxb4 intron enhancer has been performed to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. Since these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site (Gilthorpe, 2002).

A region of the human HOXD cluster that confers polycomb-group responsiveness

Polycomb group (PcG) proteins are essential for accurate axial body patterning during embryonic development. PcG-mediated repression is conserved in metazoans and is targeted in Drosophila by Polycomb response elements (PREs). However, targeting sequences in humans have not been described. While analyzing chromatin architecture in the context of human embryonic stem cell (hESC) differentiation, a 1.8kb region between HOXD11 and HOXD12 (D11.12) was duscivered that is associated with PcG proteins, becomes nuclease hypersensitive, and then shows alteration in nuclease sensitivity as hESCs differentiate. The D11.12 element repressed luciferase expression from a reporter construct and full repression required a highly conserved region and YY1 binding sites. Furthermore, repression was dependent on the PcG proteins BMI1 and EED and a YY1-interacting partner, RYBP. It is concluded that D11.12 is a Polycomb-dependent regulatory region with similarities to Drosophila PREs, indicating conservation in the mechanisms that target PcG function in mammals and flies (Woo, 2010).

The D11.12 element has several characteristics of a Drosophila PRE, indicating that there is conservation of the mechanisms that target PcG function. The multiple components that combine to make a functional PRE in Drosophila are diverse and still not fully understood. While the study of mammalian PREs is in its infancy, there is reason to think that, like Drosophila, multiple components might contribute to function. Roles in D11.12 were observed for a hyperconserved region, for YY1 and the interacting protein RYBP, and it is suggested that nucleosome free region is also central to function (Woo, 2010).

Focused was placed on D11.12 as playing a potential regulatory role due to its depletion in nucleosome occupancy in mesenchymal stem cells, a level of depletion that changes during differentiation. It is intriguing and somewhat counter-intuitive that sequences associated with recruiting the PcG system are nucleosome depleted. Most characterized activities of the PRC1 and PRC2 families in vitro, including histone methylation, histone ubiquitylation, and chromatin compaction, involve nucleosomes. However, several studies have directly examined depletion of nucleosomes on Drosophila PREs and their association with PcG proteins. Dynamic accessibility of protein-binding sequences might be important for recruiting PcG complexes in vivo (Woo, 2010).

Recent studies suggest that in addition to nucleosome depletion, high levels of histone replacement could be observed where PcG and trxG binding sites exist. This suggests that PRE sequences in flies might be open and dynamic, consistent also with proposals that RNA production from these regions might be important for function. It was found that D11.12 is nuclease-sensitive and associated with the PcG proteins BMI1 and SUZ12. Nucleosome depletion might therefore play a key role mechanistically in establishing the ability to recruit PcG function to a region of the genome, explaining the apparent conservation of this feature between Drosophila and humans (Woo, 2010).

To date, there is only one known human DNA-binding protein, YY1, which has homology to one of the Drosophila proteins which functions to recruit PcG proteins at PREs. Several lines of evidence suggest that YY1 is important to D11.12 function, consistent with previous proposals based upon both functional studies and homology to PHO. It is important to note that while YY1 appears central to D11.12 function, it is unlikely that this protein (or any protein) is generally required for mammalian PRE function. In mice, the PRE-kr has a single YY1 binding site as determined by sequence analysis, however this YY1 binding site is not conserved in the homologous human sequence and no other apparent YY1 binding sites are present. The contribution of the YY1 binding site at the PRE-kr was not examined. It is noted note that in reporter constructs containing D11.12, mutation of the YY1 binding sites impacts binding of BMI1, a PRC1 component, but has little impact on binding of SUZ12, a PRC2 component. This is consistent with models in which PRC2 is recruited prior to PRC1, and suggests that different components of D11.12 might be involved differentially in recruitment of these two complexes. YY1 interacts with RYBP, which in turn interacts with three PRC1 proteins, RING1A, RING1B and CBX2. Thus, at D11.12, YY1 might be involved primarily in PRC1 recruitment (Woo, 2010).

A highly conserved region within D11.12, which shares sequence homologies to organisms as evolutionarily different as zebrafish, is essential for repressive function. This 237 bp conserved region was required for the recruitment of both PRC1 and PRC2 components and for full repression of the reporter gene. In a search for potential regulatory sequences in the Hoxd cluster, Duboule and colleagues made knockout mice deleted of highly conserved sequences, among them the conserved sequence in D11.12. Transgenic studies determined that deletion of this conserved region impacted hoxd11 and hoxd12 expression, however knockout mice with this region deleted displayed no gross phenotype. This lack of gross phenotype might reflect redundancy in either Hox protein function or in regulatory elements with the entire Hoxd cluster. These previous data are consistent with this conserved region having the potential to contribute to regulation in mice; further analysis is needed to determine whether there are contributions of the other nearby elements to function of D11.12 in the genomic context. The mouse PRE-kr element contains a conserved 450 bp sequence within the functionally defined 3kb fragment. Comparison of the conserved regions of D11.12 and PRE-kr using the TRANSFAC database revealed only conserved GAGA factor binding sites, a site defined in Drosophila that has no known binding protein in mammals. Interestingly both conserved region sequences were predicted to form NFRs when analyzed by the nucleosome occupancy feature at the UCSC Genome Browser (Woo, 2010).

The D11.12 element also contains a CpG island. It has not been tested whether this is important to D11.12 function, in part because it is surrounded by key functional elements (namely, the YY1 binding sites and the conserved element), making interpretation of any deletion effect problematic. This element might contribute to the nucleosome-free nature of D11.12, as CpG islands in other areas have been shown to form nucleosomes poorly thereby generating low nucleosome occupancy. It has previously been noted that there is a high correlation of PcG binding sites with CpG islands, leading to the proposal that these elements might be a key determinant of PRE function in mammals (Woo, 2010).

The D11.12 sequence behaves as a strong activating sequence in cells when PcG proteins are knocked down. These knockdowns therefore change the expression from the D11.12 reporter construct by several orders of magnitude in MSCs. A loss of association of the PcG proteins with the D11.12 construct in these cells might allow for the recruitment of activating factors. In Drosophila there is precedent for the same sequence being involved in repression and activation, as PRE elements overlap with Trithorax response elements involved in maintaining activation. It is possible that there is association of trxG components with D11.12 when PcG components have been removed (Woo, 2010).

A key aspect of PcG function is to maintain repression of genes as cells differentiate. It is not clear to what extent PRE sequences, as opposed to other aspects of PcG function, are required for this heritable repression. Repression of an integrated reporter is maintained when MSCs are differentiated into adipocytes. In its natural location, D11.12 remains associated with PcG proteins in adipocytes, although to a lesser degree than in MSCs. In Drosophila, it is known that PcG association can be plastic during differentiation and can be impacted by local activators. A test for whether D11.12 is required for embryonic development will require that the homologous mouse sequence function in this manner, as this type of experiment would require a genetically tractable model system (Woo, 2010).

Action of YY1 on viral promoters

Human papillomavirus type 8 (HPV-8) is a strictly cutaneous oncogenic virus known to induce malignant skin lesions in epidermodysplasia verruciformis patients. Sequences surrounding transcription start sites of the HPV-8 oncogene E6 (nucleotides 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 was 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 on the activity of the HPV-8 E6 promoter; the binding of YY1 to a site upstream of the promoter's cap-position (BS1) activates transcription, whereas the downstream site (BS2) mediates repression. The second downstream YY1 binding site (BS3) seems 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 (Pajunk, 1997).

A subpopulation of stably infected CD4+ cells capable of producing virus upon stimulation has been identified in human immunodeficiency virus (HIV)-positive individuals. Few host factors that directly limit HIV-1 transcription and could support this state of nonproductive HIV-1 infection have been described. YY1, a widely distributed human transcription factor, is known to inhibit HIV-1 long terminal repeat (LTR) transcription and virus production. LSF (also known as LBP-1, UBP, and CP-2) has been shown to repress LTR transcription in vitro, but transient expression of LSF has no effect on LTR activity in vivo. Both YY1 and LSF participate in the formation of a complex that recognizes the initiation region of the HIV-1 LTR. Further, these factors cooperate in the repression of LTR expression and viral replication. This cooperative function may account for the divergent effects of LSF previously observed in vitro and in vivo. Thus, the cooperation of two general cellular transcription factors may allow for the selective downregulation of HIV transcription. Through this mechanism of gene regulation, YY1 and LSF could contribute to the establishment and maintenance of a population of cells stably but nonproductively infected with HIV-1 (Romerio, 1997).

Subcellular distribution of YY1 is linked to the cell cycle

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. Therefore the abundance, DNA-binding activity and localization of the YY1 protein throughout the cell cycle was examined in unperturbed, shake-off-synchronized Chinese hamster ovary and HeLa cells. Whereas the DNA-binding activity of YY1 increases dramatically early in S phase, the YY1 mRNA and protein levels does not. YY1 changes 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 coincides 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 causes a nearly complete loss of nuclear YY1, whereas addition of caffeine or 2-aminopurine to aphidicolin-treated cells restores 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, these 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 (Palko, 2004).

Developmental functions of YY1

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. Expression, activity, and subcellular localization profiles of YY1 were studied during Xenopus laevis development. Abundant levels of YY1 mRNA and protein are 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 is detected only in early oocytes (stages I and II) and in embryos after the midblastula transition (MBT); this suggests that the potential of YY1 to modulate gene expression may be specifically repressed in the intervening period of development. Experiments to determine transcriptional activity show that addition of YY1 recognition sites upstream of the thymidine kinase promoter has 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 show 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 (Ficzycz, 2001).

A series of biochemical analyses were used to explore the potential function of YY1 in the oocyte cytoplasm. YY1 was isolated from oocyte lysates by oligo(dT)-cellulose chromatography, suggesting that it associates with maternally expressed mRNA in vivo. RNA mobility shift assays demonstrate that endogenous YY1 binds to labeled histone mRNA. Size exclusion chromatography of oocyte lysates reveals that YY1 exists in high molecular mass complexes in the range of 480 kDa. Destruction of endogenous RNA by RNase treatment of lysates, abolishes the binding of YY1 to oligo(dT)-cellulose and results in redistribution from 480-kDa complexes to the monomeric form. Microinjection of RNase directly into the cytoplasm releases YY1 from 480-kDa complexes and unmasks its DNA-binding activity, but does not promote translocation to the nucleus. These results provide evidence that YY1 is a component of ribonucleoprotein (mRNP) complexes in the Xenopus oocyte, indicating a novel function for YY1 in the storage or metabolism of maternal transcripts (Ficzycz, 2002).

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, particularly the 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 results in embryos with antero-posterior axial patterning defects and reduction of head structures. XYY1 MO also reduces the expression of En2, a midbrain/hindbrain junction marker; expression is rescued by co-injection of XYY1 mRNA. However, XYY1 MO-injection does 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 (Kwon, 2003).

The progression of progenitors to oligodendrocytes requires proliferative arrest and the activation of a transcriptional program of differentiation. While regulation of cell cycle exit has been extensively characterized, the molecular mechanisms responsible for the initiation of differentiation remain ill-defined. YY1 has been identified a critical regulator of oligodendrocyte progenitor differentiation. Conditional ablation of yy1 in the oligodendrocyte lineage in vivo induces a phenotype characterized by defective myelination, ataxia, and tremor. At the cellular level, lack of yy1 arrests differentiation of oligodendrocyte progenitors after they exit from the cell cycle. At the molecular level, YY1 acts as a lineage-specific repressor of transcriptional inhibitors of myelin gene expression (Tcf4 and Id4), by recruiting histone deacetylase-1 to their promoters during oligodendrocyte differentiation. Thus, YY1 is an essential component of the transcriptional network regulating the transition of oligodendrocyte progenitors from cell cycle exit to differentiation (He, 2007).


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