Interactive Fly, Drosophila



Interaction of YY1 with the nuclear matrix

YY1 is a zinc finger-containing transcription factor that can both repress and activate transcription. YY1 appears to use multiple mechanisms to carry out its diverse functions. Recently, it was observed that YY1 can exist in multiple nuclear compartments. In addition to being present in the nuclear extract fraction, YY1 is also a component of the nuclear matrix. YY1 can be sequestered in vivo into a high-molecular-weight complex and can be dislodged from this complex either by treatment with formamide or by incubation with an oligonucleotide containing the YY1 DNA binding site sequence. By transfecting plasmids expressing various YY1 deletion constructs and subsequent nuclear fractionation, sequences necessary for association with the nuclear matrix have been identified. These sequences (residues 256-340) co-localize with those necessary for in vivo sequestration of YY1 into the high-molecular-weight complex. YY1 sequences necessary for repression of activated transcription (residues 333-371) have been characterized, as well as those necessary for masking of the YY1 transactivation domain (residues 371-397). Sequences that repress activated transcription partially overlap YY1 sequences necessary for association with the nuclear matrix. However, these sequences are distinct from those that appear to mask the YY1 transactivation domain. The potential role of nuclear matrix association in controlling YY1 function is discussed (Bushmeyer, 1998).

The multifunctional transcription factor YY1 is associated with the nuclear matrix. In osteoblasts, the interaction of several nuclear matrix-associated transcription factors with the bone specific osteocalcin gene contributes to tissue-specific and steroid hormone-mediated transcription. A canonical nuclear matrix targeting signal (NMTS) is present in all members of the AML/CBFbeta transcription factor family, but not in other transcription factors. Therefore, sequences that direct YY1 (414 amino acids) to the nuclear matrix have been defined. A series of epitope tagged deletion constructs were expressed in HeLa S3 and in human Saos-2 osteosarcoma cells. Subcellular distribution was determined in whole cells and nuclear matrices in situ by immunofluorescence. Amino acids 257-341 in the C-terminal domain of YY1 are necessary for nuclear matrix association. Sequences within the N-terminal domain of YY1 permit weak nuclear matrix binding. These data further suggest that the Gal4 epitope tag contains sequences that affect subcellular localization, but not targeting to the nuclear matrix. The targeted association of YY1 with the nuclear matrix provides an additional level of functional regulation for this transcription factor, which exhibits positive and negative control (McNeil, 1998).

The subnuclear location of transcription factors may functionally contribute to the regulation of gene expression. Several classes of gene regulators associate with the nuclear matrix in a cell type, cell growth, or cell cycle related-manner. To understand control of nuclear matrix-transcription factor interactions during tissue development, the subnuclear partitioning of a panel of transcription factors (including NMP-1/YY-1, NMP-2/AML, AP-1, and SP-1) were systematically analyzed during osteoblast differentiation using biochemical fractionation and gel shift analyses. Nuclear matrix association of the tissue-specific AML transcription factor NMP-2, but not the ubiquitous transcription factor YY1, is developmentally upregulated during osteoblast differentiation. There are multiple AML isoforms in mature osteoblasts, consistent with the multiplicity of AML factors that are derived from different genes and alternatively spliced cDNAs. These AML isoforms include proteins derived from the AML-3 gene and partition between distinct subcellular compartments. It is concluded that the selective partitioning of the YY1 and AML transcription factors with the nuclear matrix involves a discriminatory mechanism that targets different classes and specific isoforms of gene regulatory factors to the nuclear matrix at distinct developmental stages. These results are consistent with a role for the nuclear matrix in regulating the expression of bone-tissue specific genes during development of the mature osteocytic phenotype (Lindenmuth, 1997).

Other YY1 protein interactions

YY1 is a zinc finger transcription factor with unusual structural and functional features. In a yeast two-hybrid screen, two cellular proteins, cyclophilin A (CyPA) and FK506-binding protein 12 (FKBP12), interacted with YY1. These interactions are specific and also occur in mammalian cells. Cyclosporin A and FK506 efficiently disrupt the YY1-CyPA and YY1-FKBP12 interactions. Overexpression of human CyPA and FKBP12 have different effects on YY1-regulated transcription: these effects are promoter-dependent. These results suggest that immunophilins may be mediators in the functional role of YY1 (Yang, 1995).

Regulated proteolysis has been postulated to be critical for proper control of cell functions. Muscle development, in particular, involves a great deal of structural adaptation and remodeling mediated by proteases. The transcription factor YY1 represses muscle-restricted expression of the sarcomeric alpha-actin genes. Consistent with this repressor function of YY1, the nuclear regulator is down-regulated at the protein level during skeletal as well as cardiac muscle cell differentiation. However, the YY1 message remains relatively unaltered throughout the myoblast-myotube transition, implicating a post-translational regulatory mechanism. YY1 can be a substrate for cleavage by the calcium-activated neutral protease calpain II (m-calpain) and the 26 S proteasome. The calcium ionophore A23187 destabilizes YY1 in cultured myoblasts, and the decrease in YY1 protein levels can be prevented by calpain inhibitor II and calpeptin. Treatment with the proteasome inhibitors MG132 and lactacystin results in the stabilization of YY1 protein, which is consistent with the finding that YY1 is readily polyubiquitinated in reticulocyte lysates. Proteolytic targeting by calpain II and the proteasome involves different structural elements of YY1. Thus, this study illustrates two proteolytic pathways through which the transcriptional regulator can be differentially targeted under different cell growth conditions (Walowitz, 1998).

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD+ as well as the transfer of these polymers onto acceptor proteins. The function of ADPRT is thought to be related to a number of nuclear processes, including DNA repair and transcription. The transcription factor Yin Yang 1 (YY1) is a potent regulator of RNA polymerase II (Pol II)-dependent transcription. In this study Alu-retroposon-associated binding sites for YY1 located in the distal region of the promoter of the human ADPRT gene have been identified suggesting a possible involvement of this protein in the regulation of ADPRT-gene expression. In the presence of the recombinant automodification domain of the ADPRT, the formation of specific YY1 complexes, detected in gel-shift experiments, was strongly inhibited, indicating that this domain of the enzyme may interact directly with YY1. In accordance with this result, YY1 is specifically precipitated from nuclear extracts by ADPRT immobilized on sepharose. These results suggest a direct ADPRT-YY1 interaction, which may be of importance in the regulation of Pol II-dependent transcription. They also indicate that in some human promoters this regulation may be mediated by retroposons of the Alu family (Oei, 1997).

FK506-binding proteins (FKBPs) are cellular receptors for immunosuppressants that belong to a subgroup of proteins, known as immunophilins, with peptidylprolyl cis-trans isomerase (PPIase) activity. Sequence comparison suggested that the HD2-type histone deacetylases and the FKBP-type PPIases may have evolved from a common ancestor enzyme. FKBP25 physically associates with the histone deacetylases HDAC1 and HDAC2 and with the HDAC-binding transcriptional regulator YY1. An FKBP25 immunoprecipitated complex contains deacetylase activity, and this activity is associated with the N-terminus of FKBP25, distinct from the FK506/rapamycin-binding domain. Furthermore, FKBP25 can alter the DNA-binding activity of YY1. Together, these data firmly establish a relationship between histone deacetylases and the FKBP enzymes and provide a novel and critical function for the FKBPs (Yang, 2001).

The early stages of vertebrate development depend heavily on control of maternally transcribed mRNAs that are stored for long periods in complexes termed messenger ribonucleoprotein particles (mRNPs) and utilized selectively following maturation and fertilization. The transcription factor YY1 is associated with cytoplasmic mRNPs in vertebrate oocytes; however, the mechanism by which any of the mRNP proteins associate with mRNA in the oocyte is unknown. This study demonstrates the mechanism by which YY1 associates with mRNPs depends on its direct RNA binding activity. High affinity binding for U-rich single-stranded RNA and A:U RNA duplexes was observed in the nanomolar range, similar to the affinity for the cognate double-stranded DNA-binding element. Similar RNA binding affinity was observed with endogenous YY1 isolated from native mRNP complexes. In vivo expression experiments reveal epitope-tagged YY1 assembled into high molecular mass mRNPs, and assembly was blocked by microinjection of high affinity RNA substrate competitor. These findings present the first clues to how mRNPs assemble during early development (Belak, 2007).

Effects of YY1 mutation

To determine the biological role of YY1 in mammalian development, mice deficient for YY1 were generated by gene targeting. Homozygosity for the mutated YY1 allele results in embryonic lethality in the mouse. YY1 mutants undergo implantation and induce uterine decidualization but rapidly degenerate around the time of implantation. A subset of YY1 heterozygote embryos are developmentally retarded and exhibit neurulation defects, suggesting that YY1 may have additional roles during later stages of mouse embryogenesis. These studies demonstrate an essential function for YY1 in the development of the mouse embryo (Donohoe, 1999).

Transcriptional activation by YY1

YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. YY1 is ubiquitously expressed in growing, differentiated, and growth-arrested cells. The protein is phosphorylated and has a half-life of 3.5 h. To define functional domains, a large panel of YY1 mutant proteins was generated. These were used to define precisely the DNA-binding domain, the region responsible for nuclear localization, and the transactivation domain. The two acidic domains at the N terminus each provide about half of the transcriptional activating activity. The spacer region between the Gly/Ala-rich and zinc finger domains has accessory function in transactivation. YY1 has been shown previously to bind to TAFII55, TATA box-binding protein, transcription factor IIB, and p300. In addition, cAMP-responsive element-binding protein (CBP)-binding protein has been identified as a YY1 binding partner. Surprisingly, these proteins do not bind to the domains involved in transactivation, but rather to the zinc finger and Gly/Ala-rich domains of YY1. Thus, these proteins do not explain the transcriptional activating activity of YY1, but rather may be involved in repression or in initiation (Austen, 1997).

The responsiveness of genes to steroid hormones is principally mediated by functional interactions between DNA-bound hormone receptors and components of the transcriptional initiation machinery, including TATA-binding protein, TFIIB, or other RNA polymerase II associated factors. This interaction can be physiologically modulated by promoter context-specific transcription factors to facilitate optimal responsiveness of gene expression to hormone stimulation. One postulated regulatory mechanism involves the functional antagonism between hormone receptors and nonreceptor transcription factors interacting at the same hormone response element. the multifunctional regulator YY1 represses 1,25-dihydroxyvitamin D3 (vitamin D)-induced transactivation of the bone tissue-specific osteocalcin gene. YY1 recognition sequences have been identified within the vitamin D response element (VDRE) of the osteocalcin gene that are critical for YY1-dependent repression of vitamin D-enhanced promoter activity. YY1 and vitamin D receptor (VDR)/retinoid X receptor heterodimers compete for binding at the osteocalcin VDRE. In addition, YY1 interacts directly with TFIIB, and one of the two tandemly repeated polypeptide regions of TFIIB spanning the basic domain is responsible for this interaction. TFIIB and VDR can also interact directly, and these factors synergize to mediate transactivation. These results suggest that YY1 regulates vitamin D enhancement of osteocalcin gene transcription in vivo by interfering with the interactions of the VDR with both the VDRE and TFIIB (Guo, 1997).

A novel transcription factor binding element in the human p53 gene promoter has been characterized. It lies about 100 bp upstream of the major reported start site for human p53 gene transcription. On the basis of DNase I footprinting studies, electromobility shift assay patterns, sequence specificity of binding, the binding pattern of purified transcription factors, effects of specific antibodies, and methylation interference analysis, the site has been identified as a composite element that can bind both YY1 and nuclear factor 1 (NF1) in an independent and mutually exclusive manner. The site is conserved in the human, rat, and mouse p53 promoters. The occupancy of the site varies in a tissue-specific manner. It binds principally YY1 in nuclear extracts of rat testis and spleen and NF1 in extracts of liver and prostate. This may facilitate tissue-specific control of p53 gene expression. When HeLa cells are transiently transfected with human p53 promoter-chloramphenicol acetyltransferase reporter constructs, a mutation in this composite element which disables YY1 and NF1 binding causes a mean 64% reduction in basal p53 promoter activity. From mutations that selectively impair YY1 or NF1 binding and the overexpression of YY1 or NF1 in HeLa cells it is concluded that both YY1 and NF1 function as activators when bound to this site. In transient cotransfections E1A could induce the activity of the p53 promoter to a high level; 12S E1A is threefold as efficient as 13S E1A in this activity, and YY1 bound to the composite element has been shown to mediate 55% of this induction. Overexpressed YY1 is able to synergistically activate the p53 promoter with E1A, when not specifically bound to DNA. Deletion of an N-terminal domain of E1A, known to be required for direct E1A-YY1 interaction and E1A effects mediated through transcriptional activator p300, blocks the E1A induction of p53 promoter activity (Furlong, 1996).

Regulation of eukaryotic messenger RNA transcription is governed by DNA sequence elements that serve as binding sites for sequence-specific transcription factors. These include upstream and downstream promoter-proximal elements, enhancers, repressors, and silencers, all of which modulate the rate of specific initiation by RNA polymerase II. In addition, the promoter-proximal region between -45 and +30 (relative to the start of initiation) contains two highly conserved motifs, the TATA sequence at around -30 and CA at +1. Although the TATA element-binding factor TFIID has been purified and cloned from several organisms, and has provided invaluable insight into the process of transcription initiation and its regulation, little is known about factors that interact at the +1 region. The adeno-associated virus type 2 P5 promoter +1 region (P5 + 1 element) binds transcription factor YY1. This sequence is necessary and sufficient for accurate basal transcription. Further, partially purified YY1 can restore basal level transcription from a P5 + 1 element in a HeLa extract depleted for YY1 or a Drosophila embryo extract devoid of YY1 activity, whereas a YY1-specific antibody can block the reactivation. Using electrophoretic mobility shift assay, YY1-related factors have been identified that bind to two other transcription initiators in cellular genes (Seto, 1991).

YY1 is required for Ezh2 binding because RNA interference of YY1 abrogates chromatin recruitment of Ezh2 and prevents H3-K27 methylation

The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. Ezh2 expression is developmentally regulated in the myotome compartment of mouse somites and its down-regulation coincides with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibits muscle differentiation, and this property is conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 is associated with the transcriptional regulator YY1. Both Ezh2 and YY1 are detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes: their presence correlates with methylation of K27 of histone H3. YY1 is required for Ezh2 binding because RNA interference of YY1 abrogates chromatin recruitment of Ezh2 and prevents H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociate from muscle loci, H3-K27 becomes hypomethylated and MyoD and SRF are recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation (Cartetti, 2004).

These results indicate that Ezh2 is recruited at the chromatin of selected muscle regulatory regions by the transcriptional regulator YY1. Both can be coimmunoprecipitated from myoblast and not myotube cell extracts, and the proteins colocalize at the same muscle chromatin regions in a developmentally regulated manner. The interaction of endogenous YY1 and Ezh2 is likely to be mediated by the PcG EED protein because recombinant YY1 and Ezh2 do not directly associate. Previous reports have demonstrated a negative role for YY1 in regulating muscle gene expression through interaction with distinct nucleotides within the CarG-box [CC(A+T-rich)6GG], one of the DNA elements required for muscle-specific gene transcription. Transcriptional activation coincides with replacement of YY1 by the serum response factor (SRF), whose interaction with the CarG-box is required for muscle-specific transcription to proceed. These data suggest a two-step activation model of muscle gene expression. In the repressed state, YY1 recruits a complex containing both Ezh2 and HDAC1 that silences transcription through histone methylation (H3-K27) and deacetylation. Transcriptional activation entails the initial removal of the YY1-Ezh2-HDAC1 repressive complex and subsequent recruitment of the activators SRF (which replaces YY1) and the MyoD family of transcription factors and associated acetyltransferases. Since YY1 binding tolerates a substantial nucleotide heterogeneity in its DNA recognition sites, muscle and non-muscle-specific CarG-less regulatory regions may be also occupied and regulated in a similar manner. In contrast, Ezh2 does not appear to promiscuously regulate expression of all muscle-specific genes as indicated by the transient coexpression of Ezh2 and myogenin in the myotome of developing embryos and lack of Ezh2 recruitment and H3-K27 methylation at the myogenin promoter. Distinct histone methyltransferases and deacetylases have been shown to modify histones at the myogenin promoter (Cartetti, 2004 and references therein).

Juxtaposed Polycomb complexes co-regulate vertebral identity

Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 [Drosophila homologs Psc and Su(z)2] and EED (Drosophila homolog; Esc), pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity (Kim, 2006).

At least two PcG complexes with diverse composition and function in chromatin remodeling have been identified in mammals. The Polycomb repressive complex 1 (PRC1) involves the paralogous PcG proteins BMI1/MEL18, M33/PC2, RAE28, and RING1A. Evidence for PRC1-mediated chromatin modification derived from ubiquitylation at lysine 119 of histone H2A (H2A-K119). A second PcG complex, PRC2, encompasses EED, the histone methyltransferase EZH2, the zinc finger protein SUZ12, the histone-binding proteins RBAP46/RBAP48, and the histone deacetylase HDAC1. Several EED isoforms, generated by alternate translation start site usage of eed mRNA, differentially engage in PRC2-related complexes (PRC2/3/4), targeting the histone methyltransferase activity of EZH2 to H3-K27 or H1-K26. PcG complexes bind to cis-acting Polycomb response elements (PREs), which encompass several hundred base pairs and are necessary and sufficient for PcG-mediated repression of target genes. Whereas the function of several PREs has been delineated in Drosophila, similar elements await characterization in mammals (Kim, 2006 and references therein).

An antibody raised against residues 123-140 of the EED amino terminus precipitated three distinct isoforms of approximately 50 and 75 kDA from E12.5 trunk, representing three of the four EED isoforms previously reported in 293 cells. In addition to EZH2 and YY1, dimethylated H3-K27 co-immunoprecipitated with EED. Immunoprecipitation identified three BMI1 isoforms of approximately 39-41 kDA. BMI1 was found in a complex with RING1B, but not dimethylated H3-K27. Similar to the EED complex, the BMI1 complex also contained YY1. It should be emphasized that all (co-)immunoprecipitating bands were detected by at least two antibodies against different epitopes. Strikingly, while dimethylated H3-K27 engaged in the EED complex, trimethylated H3-K27 did not appear to associate with either the EED or the BMI1 complex. Importantly, reciprocal co-immunoprecipitation detected EED and BMI1 in separate protein complexes (Kim, 2006).

Ectopic expression in mutant embryos revealed Hoxc8 and Hoxa5 as downstream targets of EED and BMI1 function. ChIP detected EED and BMI1 binding immediately upstream of the Hoxc8 transcribed region near putative promoter elements. The binding sites could not be separated, indicating close proximity of the complexes. EED and BMI1 binding also clustered within a small fragment 1.5 kb upstream of the Hoxc8 transcription start site, suggesting long-range juxtaposition of heterologous PcG complexes. Similar to EED and BMI1, YY1 localized to both regions. In support of YY1 binding to Hox regulatory regions, inspection of the mouse genome sequence revealed clusters of putative YY1 binding sites in both regions a and b, including TGTCCATTAG and CCCCCATTCC (region a), as well as ACACCATGGC, TTTCCATTAG and TCCCCATAAA (region b). CCAT represents the core of the YY1 consensus binding site, while flanking sequences exhibited significant tolerance for multiple nucleotides. EED, BMI1 and YY1 also co-localized approximately 1.5 kb upstream of the transcription start site of Hoxa5. In addition to PcG binding, ChIP detected trimethylated H3-K27 throughout the regulatory regions of Hoxc8 and Hoxa5. Furthermore, dimethylated H3-K27 localized to region b of Hoxc8 (Kim, 2006).

Spatial regulation of EED and BMI1 binding to Hox regulatory regions was evident from ChIP analysis of dissected anterior and posterior regions of E12.5 trunk. In agreement with transcriptional silencing of Hoxc8 and Hoxa5, EED and BMI1 binding was detected upstream of these loci in anterior regions of the trunk. By contrast, EED and BMI1 binding was absent from posterior regions of the trunk, where Hoxc8 and Hoxa5 are transcribed. These findings implicate PcG complexes in Hox gene repression in anterior regions of the AP axis (Kim, 2006).

The combined interpretation of the co-immunoprecipitation and ChiP results indicates that trimethylated H3-K27 did not form a complex with EED or BMI1, despite co-localization of the three proteins in Hox regulatory regions. By contrast, co-immunoprecipitation demonstrated physical association of the EED complex with dimethylated H3-K27. In aggregate, the results support a model in which EED- and BMI1-containing chromatin remodeling complexes exist as separate, but juxtaposed, biochemical entities at Hox target loci (Kim, 2006).

Transcriptional repression by YY1

Continued: pleiohomeotic Evolutionary homologs
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pleiohomeotic: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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