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PubMed2006

Stem cells primed for action: polycomb repressive complexes restrain the expression of lineage-specific regulators in embryonic stem cells.

Stem cells are characterised by a capacity to self renew and generate progeny capable of differentiating along several defined lineage paths. Embryonic Stem (ES) cells are derived from the inner cell mass (ICM) of early-stage embryos and can contribute to all tissues of the developing embryo. Discovering how ES cell pluripotency and lineage induction is achieved is important for understanding normal development and for successfully applying stem cell-based therapies. A series of recent studies have shown that the chromatin profile of ES cells is unusual and have revealed a critical role for the Polycomb Repressive Complexes (PRCs) in maintaining pluripotency. In human and mouse ES cells many genes that encode transcription factors that are required for lineage specification bind PRC2 and carry bivalent (or opposing) histone signatures, being enriched for conventional indicators of active chromatin such as acetylated H3K9 and methylated H3K4, while lying within domains of repressive trimethylated H3K27. Mutant ES cells that lack H3K27 methylation inappropriately expressed these genes showing that PRC2 represses lineage-specific gene programs in ES cells. Here we discuss the implications of these new discoveries and explore the interdependence of PRC1 and PRC2 in regulating lineage-specific gene expression in ES cells.

Animals↗
PubMed2005

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication.

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.

ATP-Binding Cassette Transporters↗
PubMed2003

A putative coiled-coil domain of prohibitin is sufficient to repress E2F1-mediated transcription and induce apoptosis.

Prohibitin is a potential tumor suppressor protein that can repress E2F-mediated transcription and arrest cell proliferation. We had shown previously that prohibitin could bind to the Rb protein as well as E2F and this binding was necessary to suppress cell proliferation. Here we show that the E2F1 binding domain of prohibitin has the potential to fold into a coiled-coil structure. This coiled-coil domain by itself could physically interact with E2F1 and block its transcriptional activity. Like full-length prohibitin, the coiled-coil domain also recruited histone deacetylase 1 to repress E2F1. The coiled-coil domain also exhibited growth suppressive properties and we observed a 64% reduction in colony numbers when transfected into T47D cells. Interestingly, a synthetic peptide corresponding to the coiled-coil domain induced apoptosis in four different human cell lines. It is possible that agents that can mimic this peptide would be of value in controlling proliferative disorders.

Apoptosis↗
PubMed1999

hnRNP U inhibits carboxy-terminal domain phosphorylation by TFIIH and represses RNA polymerase II elongation.

This study describes a potential new function of hnRNP U as an RNA polymerase (Pol II) elongation inhibitor. We demonstrated that a subfraction of human hnRNP U is associated with the Pol II holoenzyme in vivo and as such recruited to the promoter as part of the preinitiation complex. hnRNP U, however, appears to dissociate from the Pol II complex at the early stage of transcription and is therefore absent from the elongating Pol II complex. When tested in the human immunodeficiency virus type 1 transcription system, hnRNP U inhibits elongation rather than initiation of transcription by Pol II. This inhibition requires the carboxy-terminal domain (CTD) of Pol II. We showed that hnRNP U can bind TFIIH in vivo under certain conditions and inhibit TFIIH-mediated CTD phosphorylation in vitro. We find that the middle domain of hnRNP U is sufficient to mediate its Pol II association and its inhibition of TFIIH-mediated phosphorylation and Pol II elongation. The abilities of hnRNP U to inhibit TFIIH-mediated CTD phosphorylation and its Pol II association are necessary for hnRNP U to mediate the repression of Pol II elongation. Based on these observations, we suggest that a subfraction of hnRNP U, as a component of the Pol II holoenzyme, may downregulate TFIIH-mediated CTD phosphorylation in the basal transcription machinery and repress Pol II elongation. With such functions, hnRNP U might provide one of the mechanisms by which the CTD is maintained in an unphosphorylated state in the Pol II holoenzyme.

Heterogeneous-Nuclear Ribonucleoprotein U↗
PubMed2005

SIRT1 deacetylation and repression of p300 involves lysine residues 1020/1024 within the cell cycle regulatory domain 1.

The SIR2 family of nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases modulates diverse biological functions in different species, including longevity, apoptosis, cell cycle exit, and cellular differentiation. SIRT1, the closest mammalian ortholog of the yeast SIR2 (silent information regulator 2) gene, represses several transcription factors, including p53, NFkappaB and forkhead proteins. The p300 protein serves as a rate-limiting transcriptional cointegrator of diverse transcription factors either to activate or to repress transcription through modular subdomains. Herein, SIRT1 physically interacted with and repressed p300 transactivation, requiring the NAD-dependent deacetylase activity of SIRT1. SIRT1 repression involved the CRD1 transcriptional repression domain of p300. Two residues within the CRD1 domain (Lys-1020 and Lys-1024) were required for SIRT1 repression and served as substrates for SIRT1 deacetylation. These residues also serve as acceptor lysines for modification by the ubiquitin-like SUMO protein. The SUMO-specific protease SSP3 relieved SIRT1 repression of p300. SSP3 antagonism of SIRT1 required the SUMO-deconjugating function of SSP3. Thus, p300 serves as a deacetylase substrate for SIRT1 through a conserved SUMO consensus motif. Because p300 is a limiting transcriptional cofactor, deacetylation and repression of p300 by SIRT1 may serve an important integration point during metabolism and cellular differentiation.

Amino Acid Motifs↗
PubMed1999

CDR1, a multidrug resistance gene from Candida albicans, contains multiple regulatory domains in its promoter and the distal AP-1 element mediates its induction by miconazole.

We previously demonstrated that the CDR1 gene, encoding a multidrug transporter in Candida albicans, is differentially upregulated by various drugs and steroids. In order to get an insight into the molecular basis of the induction of this gene we analyzed its promoter region. The transcription start site was mapped to 63 nucleotides upstream of the initiating ATG. Reporter assays revealed the presence of four upstream activating and four upstream repressing sequence domains along the entire promoter. Like the native gene, promoter-luciferase recombinants showed enhanced activity in response to various stresses like drugs, human steroid hormones and heavy metals. Mutational analysis demonstrated that while the proximal promoter (-345/+1) contains all the regulatory domains required for its induction by various other stresses, the miconazole response is mediated via the distal promoter (-857/-1147), harboring an AP-1 site. The involvement of the AP-1 element in mediating the latter effect was evident by an increase in AP-1 binding activity following miconazole treatment.

Antifungal Agents↗
PubMed1993

Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells.

Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT. The poor activation suggested that the NC or PR domains could repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Amino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.

Animals↗
PubMed2007

Structure/function analysis of Xenopus cryptochromes 1 and 2 reveals differential nuclear localization mechanisms and functional domains important for interaction with and repression of CLOCK-BMAL1.

Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral processes within an organism and also synchronize these processes with the external environment. A cell autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops, drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a central part in the negative feedback loop of the molecular clock by translocating to the nucleus and repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mechanisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the second half of the photolyase homology region (PHR) of CRY is important for repression through facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the molecular clock.

ARNTL Transcription Factors↗
PubMed2005

RGG-boxes of the EWS oncoprotein repress a range of transcriptional activation domains.

The Ewings Sarcoma Oncoprotein (EWS) interacts with several components of the mammalian transcriptional and pre-mRNA splicing machinery and is also found in the cytoplasm and even on the cell surface. The apparently diverse cellular functions of EWS are, however, not well characterized. EWS harbours a potent N-terminal transcriptional activation domain (the EAD) that is revealed in the context of oncogenic EWS-fusion proteins (EFPs) and a C-terminal RNA-binding domain (RBD) that recruits pre-mRNA splicing factors and may couple transcription and splicing. In contrast to EFPs, the presumed transcriptional role of normal EWS remains enigmatic. Here, we report that multiple RGG-boxes within the RBD are necessary and sufficient for cis-repression of the EAD and that RGG-boxes can also repress in-trans, within dimeric partners. Lys can functionally substitute for Arg, indicating that the basic nature of the Arg side chain is the critical determinant of RGG-box-mediated repression. In addition to the EAD, RGG-boxes can repress a broad range of activation domains (including those of VP16, E1a and CREB), but repression can be alleviated by the simultaneous presence of more than one activation domain. We therefore propose that a key function of RGG boxes within native EWS is to restrict promiscuous activation by the EAD while still allowing EWS to enter functional transcription complexes and participate in other transactions involving pre-mRNAs.

Amino Acid Motifs↗
PubMed1999

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.

Animals↗
PubMed1995

Transcriptional repression by the C-terminal domain of p53.

We have previously shown that monomeric p53 can transactivate target genes in vivo and that C-terminal fragments of p53 are oncogenic. To further elaborate these findings a series of C-terminal truncations of p53 was generated. The transactivation capacity and the ability of the truncated p53 to suppress oncogene-mediated transformation were studied. We found that p53 truncated at amino acid 303 (p53wtdl303) can still function in both assays, though less efficiently than full length wild type (wt) p53. Transforming C-terminal fragments inhibited transactivation induced by full length wt p53. Surprisingly, they also inhibited transactivation by wtdl303, with which they do not share any overlapping sequences. Furthermore, the C-terminal fragments repressed the transactivation domains of several viral and cellular transcriptional activators. These data raise the possibility that the C-terminal domain of p53 may compete with the p53 transactivation domain for a common basal transcription factor.

Animals↗
PubMed2001

Intracellular localization of the Ret finger protein depends on a functional nuclear export signal and protein kinase C activation.

The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain. RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type. Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP. Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells. In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells. Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Calpha (PKCalpha) abrogated masking, leading to the cytoplasmic localization of RFP. Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP. Thus, the nuclear export of RFP is regulated positively by PKC activation. However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP.

Active Transport, Cell Nucleus↗
PubMed1999

M31, a murine homolog of Drosophila HP1, is concentrated in the XY body during spermatogenesis.

The formation of the sex vesicle, or XY body, during male meiosis and pairing of the sex chromosomes are thought to be essential for successful spermatogenesis. Despite its cytological discovery a century ago, the mechanism of XY body formation, particularly heterochromatinization of the sex chromosomes, has remained unclear. The HP1 class of chromobox genes are thought to encode proteins involved in the packaging of chromosomal DNA into repressive heterochromatin domains, as seen, for example, in position-effect variegation. Study of the distribution of a murine HP1-like chromodomain protein, M31, during spermatogenesis revealed spreading from the tip of the XY body in mid-stage pachytene spermatocytes to include the whole of the XY body in late-pachytene spermatocytes. We also demonstrate that the formation of the XY body during spermatogenic progression in neonatal mice coincides with the expression of a novel nuclear isoform of M31, M31(p21). These results support the view that a common mechanistic basis exists for heterochromatin-induced repression, homeotic gene silencing, and sex-chromosome inactivation during mammalian spermatogenesis.

Animals↗
PubMed1992

Functional diversity of E1A gene autoregulation among human adenoviruses.

Autoregulation of the adenovirus E1A gene involves its constitutive expression and positively and negatively regulated transcription. Dissection of this process will identify basal-level cis elements and autoregulatory targets of the E1A promoter and functional domains within the trans-acting E1A gene products. In this report, the DNA sequence of the human subgroup B adenovirus type 3 (Ad3) E1A gene is presented and compared with that of the E1A genes of similar and distantly related human adenoviruses. The cDNA forms of the Ad3 E1A gene, corresponding to two major early mRNA species, are cloned, sequenced, and subcloned into plasmid expression vectors. Cotransfections of cell cultures are performed with Ad5 or Ad3 E1A gene expression plasmids and a reporter gene under control of the Ad5 or Ad3 E1A promoter. The Ad5 and Ad3 E1A promoters are similarly repressed by either serotype's 12S cDNA gene products. The Ad3 E1A promoter responds much more strongly than the Ad5 E1A promoter to transactivation by 13S cDNA gene products. In contrast, the 13S cDNA gene of Ad5 has greater transactivation activity than that of Ad3. Experiments with missense mutations of the Ad5 E1A gene indicate that transactivation of the Ad5 E1A promoter is weak, just reversing or balancing negative autorepression. Single amino acid substitutions in the conserved, repressive functional domain 2 of the E1A gene modulate transactivating activity that is usually associated with the separate and distal conserved functional domain 3. These results suggest a strong structure-function relationship influenced by the variable sequences separating these conserved domains.

Adenovirus Early Proteins↗
PubMed1999

Unique forms of human and mouse nuclear receptor corepressor SMRT.

Nuclear hormone receptors have been shown to repress transcription in the absence of ligand. This repression is mediated by a corepressor complex that contains the Sin3A protein and histone deacetylases (HDAC1 and 2). Studies by several groups demonstrate that this complex is recruited to nuclear receptors through the highly related corepressors SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor). We describe here the cloning, characterization, and chromosomal mapping of forms of human and mouse SMRT that includes a 1,000-aa extension, which reveals striking homology to the amino terminus of N-CoR. Structure and function studies of wild-type and natural splicing variants suggest the presence of 3-4 amino terminal domains that repress in a cooperative as well as mechanistically distinct fashion.

Amino Acid Sequence↗
PubMed2005

DNA recognition properties of the LHX3b LIM homeodomain transcription factor.

LHX3 is a LIM homeodomain transcription factor with established roles in pituitary and nervous system development. Mutations in the human LHX3 gene are associated with severe hormone deficiency diseases. Previous studies have shown that the human LHX3 gene produces at least three protein isoforms: LHX3a, LHX3b, and M2-LHX3. In gene activation assays, LHX3a and M2-LHX3 are significantly more active than LHX3b because the actions of LHX3b are repressed by an inhibitory domain in its amino terminus. In this report, we investigate the molecular characteristics that result in reduced transcriptional capacity of LHX3b by determining the optimal DNA binding preference of LHX3b. Site selection experiments using purified human LHX3b reveal that it selects AT-rich sequences that contain ATTA/TAAT motifs. The pool of sequences selected by LHX3b is similar to that selected by LHX3a but does not conform to as strict a consensus. Further, the LHX3b-selected sites are bound more avidly by LHX3a and M2-LHX3 suggesting that LHX3b does not act by recognizing LHX3b-specific binding sites in target genes. We conclude that the amino terminal repression domain of LHX3b mostly acts to reduce the transcriptional potency of LHX3 by inhibiting the DNA binding affinity of the homeodomain, with some reduction in DNA binding specificity.

DNA↗
PubMed2001

Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3.

The covalent modification of histone tails has regulatory roles in various nuclear processes, such as control of transcription and mitotic chromosome condensation. Among the different groups of enzymes known to catalyze the covalent modification, the most recent additions are the histone methyltransferases (HMTases), whose functions are now being characterized. Here we show that a SET domain-containing protein, G9a, is a novel mammalian lysine-preferring HMTase. Like Suv39 h1, the first identified lysine-preferring mammalian HMTase, G9a transfers methyl groups to the lysine residues of histone H3, but with a 10-20-fold higher activity. It was reported that lysines 4, 9, and 27 in H3 are methylated in mammalian cells. G9a was able to add methyl groups to lysine 27 as well as 9 in H3, compared with Suv39 h1, which was only able to methylate lysine 9. Our data clearly demonstrated that G9a has an enzymatic nature distinct from Suv39 h1 and its homologue h2. Finally, fluorescent protein-labeled G9a was shown to be localized in the nucleus but not in the repressive chromatin domains of centromeric loci, in which Suv39 h1 family proteins were localized. This finding indicates that G9a may contribute to the organization of the higher order chromatin structure of non-centromeric loci.

Amino Acid Sequence↗
PubMed2000

The human T cell leukemia/lymphotropic virus type 1 Tax protein represses MyoD-dependent transcription by inhibiting MyoD-binding to the KIX domain of p300. A potential mechanism for Tax-mediated repression of the transcriptional activity of basic helix-loop-helix factors.

The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral and cellular gene transcription. It mainly functions by interacting with cellular transcription factors and the KIX domain of the p300/CBP coactivators. Tax can also repress the transcription of cellular genes through the basic helix-loop-helix (bHLH) protein family. To investigate the molecular mechanisms of this Tax-mediated inhibition, we analyzed its effect on the transcriptional activity of the myogenic MyoD protein, which was used as a paradigm of bHLH factors. In this study, we show that overexpression of the p300 coactivator in transient transfection assays was sufficient to rescue MyoD repression by Tax. Furthermore, an N-terminal domain of p300 (amino acids 379-654) containing the region of KIX serving as the Tax binding site was found, when overexpressed, to potentiate Tax-mediated transactivation of HTLV-1 proviral as well as MyoD-dependent transcription, and to antagonize the inhibition by Tax of the transcriptional activity of MyoD. These results revealing the presence of an N-terminal MyoD binding site were confirmed by in vitro protein-protein interaction assays that demonstrate that MyoD binds to the KIX domain of p300 and that Tax competes with MyoD binding in a nonreciprocal manner. These observations provide evidence that Tax binding to the KIX domain of p300 prevents bHLH proteins from contacting this N-terminal domain of the coactivator, thus resulting in their transcriptional repression. As bHLH proteins are implicated in many developmental fate decisions, especially during thymopoiesis, Tax-mediated inhibition of their transcriptional activity may contribute to the induction of HTLV-1-linked leukemogenesis.

Animals↗