Showing papers on "Corepressor published in 2000"

The Myc/Max/Mad Network and the Transcriptional Control of Cell Behavior

Abstract: The Myc/Max/Mad network comprises a group of transcription factors whose distinct interactions result in gene-specific transcriptional activation or repression. A great deal of research indicates that the functions of the network play roles in cell proliferation, differentiation, and death. In this review we focus on the Myc and Mad protein families and attempt to relate their biological functions to their transcriptional activities and gene targets. Both Myc and Mad, as well as the more recently described Mnt and Mga proteins, form heterodimers with Max, permitting binding to specific DNA sequences. These DNA-bound heterodimers recruit coactivator or corepressor complexes that generate alterations in chromatin structure, which in turn modulate transcription. Initial identification of target genes suggests that the network regulates genes involved in the cell cycle, growth, life span, and morphology. Because Myc and Mad proteins are expressed in response to diverse signaling pathways, the network can be viewed as a functional module which acts to convert environmental signals into specific gene-regulatory programs.

Both corepressor proteins SMRT and N-CoR exist in large protein complexes containing HDAC3.

Abstract: We present evidence that both corepressors SMRT and N‐CoR exist in large protein complexes with estimated sizes of 1.5–2 MDa in HeLa nuclear extracts. Using a combination of conventional and immunoaffinity chromatography, we have successfully isolated a SMRT complex and identified histone deacetylase 3 (HDAC3) and transducin (β)‐like I (TBL1), a WD‐40 repeat‐containing protein, as the subunits of the purified SMRT complex. We show that the HDAC3‐containing SMRT and N‐CoR complexes can bind to unliganded thyroid hormone receptors (TRs) in vitro . We demonstrate further that in Xenopus oocytes, both SMRT and N‐CoR also associate with HDAC3 in large protein complexes and that injection of antibodies against HDAC3 or SMRT/N‐CoR led to a partial relief of repression by unliganded TR/RXR. These findings thus establish both SMRT and N‐CoR complexes as bona fide HDAC‐containing complexes and shed new light on the molecular pathways by which N‐CoR and SMRT function in transcriptional repression.

A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness.

Abstract: The corepressor SMRT mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. Here we report the isolation of a novel SMRT-containing complex from HeLa cells. This complex contains transducin beta-like protein 1 (TBL1), whose gene is mutated in human sensorineural deafness. It also contains HDAC3, a histone deacetylase not previously thought to interact with SMRT. TBL1 displays structural and functional similarities to Tup1 and Groucho corepressors, sharing their ability to interact with histone H3. In vivo, TBL1 is bridged to HDAC3 through SMRT and can potentiate repression by TR. Intriguingly, loss-of-function TRbeta mutations cause deafness in mice and humans. These results define a new TR corepressor complex with a physical link to histone structure and a potential biological link to deafness.

Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression

Abstract: The transcriptional corepressor SMRT functions by mediating the repressive effect of transcription factors involved in diverse signaling pathways. The mechanism by which SMRT represses basal transcription has been proposed to involve the indirect recruitment of histone deacetylase HDAC1 via the adaptor mSin3A. In contrast to this model, a two-hybrid screen on SMRT-interacting proteins resulted in the isolation of the recently described HDAC5 and a new family member termed HDAC7. Molecular and biochemical results indicate that this interaction is direct and in vivo evidence colocalizes SMRT, mHDAC5, and mHDAC7 to a distinct nuclear compartment. Surprisingly, HDAC7 can interact with mSin3A in yeast and in mammalian cells, suggesting association of multiple repression complexes. Taken together, our results provide the first evidence that SMRT-mediated repression is promoted by class I and class II histone deacetylases and that SMRT can recruit class II histone deacetylases in a mSin3A-independent fashion.

BCoR, a novel corepressor involved in BCL-6 repression

Abstract: BCL-6 encodes a POZ/zinc finger transcriptional repressor that is required for germinal center formation and may influence apoptosis. Aberrant expression of BCL-6 due to chromosomal translocations is implicated in certain subtypes of non-Hodgkin's lymphoma. The POZ domains of BCL-6 and several other POZ proteins interact with corepressors N-CoR and SMRT. Here we identify and characterize a novel corepressor BCoR (BCL-6 interacting corepressor), which is expressed ubiquitously in human tissues. BCoR can function as a corepressor when tethered to DNA and, when overexpressed, can potentiate BCL-6 repression. Specific class I and II histone deacetylases (HDACs) interact in vivo with BCoR, suggesting that BCoR may functionally link these two classes of HDACs. Strikingly, BCoR interacts selectively with the POZ domain of BCL-6 but not with eight other POZ proteins tested, including PLZF. Additionally, interactions between the BCL-6 POZ domain and SMRT, N-CoR, and BCoR are mutually exclusive. The specificity of the BCL-6/BCoR interaction suggests that BCoR may have a role in BCL-6-associated lymphomas.

The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding.

Abstract: The human progesterone receptor (PR) exists as two functionally distinct isoforms, hPRA and hPRB. hPRB functions as a transcriptional activator in most cell and promoter contexts, while hPRA is transcriptionally inactive and functions as a strong ligand-dependent transdominant repressor of steroid hormone receptor transcriptional activity. Although the precise mechanism of hPRA-mediated transrepression is not fully understood, an inhibitory domain (ID) within human PR, which is necessary for transrepression by hPRA, has been identified. Interestingly, although ID is present within both hPR isoforms, it is functionally active only in the context of hPRA, suggesting that the two receptors adopt distinct conformations within the cell which allow hPRA to interact with a set of cofactors that are different from those recognized by hPRB. In support of this hypothesis, we identified, using phage display technology, hPRA-selective peptides which differentially modulate hPRA and hPRB transcriptional activity. Furthermore, using a combination of in vitro and in vivo methodologies, we demonstrate that the two receptors exhibit different cofactor interactions. Specifically, it was determined that hPRA has a higher affinity for the corepressor SMRT than hPRB and that this interaction is facilitated by ID. Interestingly, inhibition of SMRT activity, by either a dominant negative mutant (C'SMRT) or histone deacetylase inhibitors, reverses hPRA-mediated transrepression but does not convert hPRA to a transcriptional activator. Together, these data indicate that the ability of hPRA to transrepress steroid hormone receptor transcriptional activity and its inability to activate progesterone-responsive promoters occur by distinct mechanisms. To this effect, we observed that hPRA, unlike hPRB, was unable to efficiently recruit the transcriptional coactivators GRIP1 and SRC-1 upon agonist binding. Thus, although both receptors contain sequences within their ligand-binding domains known to be required for coactivator binding, the ability of PR to interact with cofactors in a productive manner is regulated by sequences contained within the amino terminus of the receptors. We propose, therefore, that hPRA is transcriptionally inactive due to its inability to efficiently recruit coactivators. Furthermore, our experiments indicate that hPRA interacts efficiently with the corepressor SMRT and that this activity permits it to function as a transdominant repressor.

Sequestration and inhibition of Daxx-mediated transcriptional repression by PML

Abstract: Acute promyelocytic leukemia (APL) arises as a result of chromosomal translocation involving the retinoic acid (RA) receptor α (RARα) gene on chromosome 17 fused with either the promyelocytic leukemia gene (PML) on chromosome 15, the promyelocytic leukemia zinc finger gene (PLZF) on chromosome 11, the nucleophosmin/B23 (NPM) gene on chromosome 5, or the nuclear mitotic apparatus gene (NuMA) on chromosome 11 (30, 39). The t(15;17) translocation between PML and RARα accounts for nearly all APL cases. This translocation creates an oncogenic fusion protein, PML-RARα, which contains both the DNA-binding domain (DBD) and ligand-binding domains of RARα and the N terminus of PML. Transgenic mice that overexpress PML-RARα or PLZF-RARα developed an APL-like phenotype (9, 21, 26), suggesting that these fusion proteins are directly involved in APL pathogenesis. Recent studies have focused on analyzing the functional properties of PML-RARα and PLZF-RARα (20, 22, 25, 40) in order to understand the molecular basis of leukemogenesis. Both fusion proteins form homodimers that bind to RA response elements and interact with the nuclear receptor corepressors SMRT (silencing mediator for retinoid and thyroid hormone action) and N-CoR (nuclear receptor corepressor), which in turn recruit a histone deacetylase complex (1, 27, 40, 46). Pharmacological concentrations of all-trans-RA (atRA) induce dissociation of the corepressors from PML-RARα, but not PLZF-RARα, due to the presence of an additional, RA-insensitive corepressor-interacting surface on PLZF. This differential degree of dissociation of corepressors induced by atRA correlates with the ability of histone deacetylase inhibitors and atRA to induce terminal differentiation of these two subtypes of APL cells. These findings indicate that abnormalities in transcriptional repression by the oncogenic fusion proteins may be involved in leukemogenesis. PML belongs to a family of proteins characterized by the presence of a RING finger domain (8). RING finger proteins are implicated in transcriptional regulation, and some members of the RING family are associated directly with chromatin (53). Ablation and overexpression experiments suggest an important role of PML in the regulation of cell growth, hematopoietic cell differentiation, tumorigenesis, apoptosis, and RA signaling (44, 63). In normal cells, PML is concentrated within 10 to 20 nuclear structures known as nuclear domains 10 (ND10), Kruppel bodies, nuclear bodies, or PML-oncogenic domains (PODs) (2, 17, 33, 59, 65). The POD structure is disrupted in the t(15;17) translocated APL cells (17, 33, 65), presumably through interaction of wild-type PML with PML-RARα. Interestingly, the POD structure reorganizes upon treatment with atRA or arsenic trioxide (As2O3), a process that correlates with differentiation of APL cells, indicating that the POD structure might affect promyelocyte differentiation. In addition to PML, the POD contains several other proteins, including the 100-kDa nuclear protein antigen (Sp100) (2), the small ubiquitin-related modifier (SUMO-1 [41], also known as PML-interacting clone 1 [PIC1] [7], ubiquitin-like 1 [UBL1] [57], or sentrin [48]), and the 140-kDa protein (Sp140) (6). Sp100 is a nuclear antigen recognized by autoantibodies from patients with primary biliary cirrhosis (62). Expression of both PML and Sp100 are upregulated by interferon (23). SUMO-1 was recently identified as a ubiquitin-like protein that forms covalent conjugates with PML and Sp100 (7, 58). In addition, the CREB-binding protein (CBP) and the retinoblastoma tumor suppressor (pRB) have been found in the PODs (35, 61). Also, the PODs are targets of several viral proteins, which alter POD structure (11, 14, 18). Although there is evidence for POD's role in transcriptional activation (15, 35), DNA replication (19), apoptosis (51, 64), and viral infection (14, 42), the precise function of PODs in these processes remains unclear. We have sought to understand the function of PODs through identification of PML-interacting proteins that also localize in the PODs. By using the yeast two-hybrid system, we identified SUMO-1 and the Fas-binding protein Daxx (68) (J. D. Chen and R. M. Evans, unpublished data). Daxx has been shown to promote Fas-mediated apoptosis through activation of the Jun NH2-terminal kinase (JNK) and JNK kinase kinase ASK1 (apoptosis signal-regulating kinase 1) (12). Recent data suggest that Daxx is not sufficient for Fas-mediated apoptosis, since a Fas mutant that selectively binds to Daxx but not the Fas-adaptor death domain-containing protein (FADD/MORT1) failed to induce apoptosis (13). Other evidence suggests that Daxx may interact with the centromeric protein-c (CENP-C) and may bind to a steroidogenic factor 1 (SF-1)-like DNA element (32, 50). Therefore, the exact mechanism by which Daxx regulates Fas-mediated apoptosis may involve nuclear processes. In the present study, we have characterized both biochemical and functional interactions between Daxx and PML. Daxx resides primarily in the cell nucleus, where it forms a complex with PML. Confocal immunofluorescence data demonstrate that Daxx colocalizes with PML in the PODs, and such colocalization persists in NB4 APL cells (36) before and after treatment with atRA and As2O3. Daxx possesses strong transcriptional repressor activity and appears to interact directly with histone deacetylases. Intriguingly, overexpression of PML inhibits Daxx-mediated transcriptional repression and, in cells that lack PML, Daxx is preferentially associated with condensed chromatin. Our data reveal a new role for Daxx in transcriptional repression and suggest a novel function of PML and the POD structure in the suppression of transcriptional repression.

The histone deacetylase-3 complex contains nuclear receptor corepressors

Abstract: Acetylation and deacetylation of nucleosomal histones have profound effects on gene transcription in all eukaryotes. In humans, three highly homologous class I and four class II histone deacetylase (HDAC) enzymes have been identified to date. The class I deacetylases HDAC1 and HDAC2 are components of multisubunit complexes, one of which could associate with the nuclear hormone receptor corepressor, N-CoR. N-CoR also interacts with class II deacetylases HDAC4, HDAC5, and HDAC7. In comparison with HDAC1 and HDAC2, HDAC3 remains relatively uncharacterized, and very few proteins have been shown to interact with HDAC3. Using an affinity purification approach, we isolated an enzymatically active HDAC3 complex that contained members of the nuclear receptor corepressor family. Deletion analysis of N-CoR revealed that HDAC3 binds multiple N-CoR regions in vitro and that all of these regions are required for maximal binding in vivo. The N-CoR domains that interact with HDAC3 are distinct from those that bind other HDACs. Transient overexpression of HDAC3 and microinjection of Abs against HDAC3 showed that a component of transcriptional repression mediated by N-CoR depends on HDAC3. Interestingly, data suggest that interaction with a region of N-CoR augments the deacetylase activity of HDAC3. These results provide a possible molecular mechanism for HDAC3 regulation and argue that N-CoR is a platform in which distinct domains can interact with most of the known HDACs.

Nuclear receptor corepressors partner with class II histone deacetylases in a Sin3-independent repression pathway

Abstract: Transcriptional repression mediated by corepressors N-CoR and SMRT is a critical function of nuclear hormone receptors, and is dysregulated in human myeloid leukemias. At the present time, these corepressors are thought to act exclusively through an mSin3/HDAC1 complex. Surprisingly, however, numerous biochemical studies have not detected N-CoR or SMRT in mSin3- and HDAC1-containing complexes. Each corepressor contains multiple repression domains (RDs), the significance of which is unknown. Here we show that these RDs are nonredundant, and that one RD, which is conserved in N-CoR and SMRT, represses transcription by interacting directly with class II HDAC4 and HDAC5. Endogenous N-CoR and SMRT each associate with HDAC4 in a complex that does not contain mSin3A or HDAC1. This is the first example of a single corepressor utilizing distinct domains to engage multiple HDAC complexes. The alternative HDAC complexes may mediate specific repression pathways in normal as well as leukemic cells.

The Yin-Yang of TCF/beta-catenin signaling.

Abstract: Publisher Summary Wingless/Wnt signaling directs cell-fate choices during embryonic development. Upon Wingless/Wnt signaling, a cascade is initiated that results in the accumulation of cytoplasmic β-catenin. In the absence of Wingless/Wnt signals, a key negative regulator of the pathway, GSK3β, is activated that mediates the downregulation of cytoplasmic β-catenin/Armadillo via the ubiquitin-proteasome pathway. In the absence of nuclear β-catenin, the Tcfs recruit the corepressor protein Groucho to the target gene enhancers and actively repress their transcription. An additional corepressor protein, CREB-binding protein (CBP), is also involved in repression of Tcf target gene activity. In APC-deficient colon carcinoma cell lines, β-catenin accumulates and is complexed with nuclear Tcf-4. A proportion of APC wild-type colon carcinomas and melanomas also contains constitutive nuclear Tcf-4/β-catenin complexes as a result of dominant mutations in the N terminus of β-catenin that render it insensitive to downregulation by APC, GSK3β, and Axin/Conductin. This results in the unregulated expression of Tcf-4 target genes such as c-myc . Based on the established role for Tcf-4 in maintaining intestinal stem cells it is likely that deregulation of c-myc expression because of constitutive Tcf-4/ β-catenin activity promotes uncontrolled intestinal cell proliferation.

Transcriptional repression by nuclear hormone receptors.

Abstract: Repression by nuclear receptors plays important roles in acute promyelocytic leukemia and other diseases. Nuclear receptor corepressor (N-CoR) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) are corepressor proteins whose modular structure facilitates receptor interaction as well as transduction of repression signals involving histone deacetylation, alterations in chromatin structure and direct interactions with the basal transcription machinery. Interactions between nuclear receptors and corepressor complexes have multiple determinants. This allows regulation, and potentially therapeutic manipulation, of receptor, corepressor, cell-type and target-gene specificity.

Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family

Abstract: Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and lymphomagenesis. Here we demonstrate that Pax5 exerts its repression function by recruiting members of the Groucho corepressor family. In a yeast two-hybrid screen, the groucho-related gene product Grg4 was identified as a Pax5 partner protein. Both proteins interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and C-terminal transactivation domain of Pax5. The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding. Moreover, Grg4 efficiently represses the transcriptional activity of Pax5 in an octapeptide-dependent manner. Similar protein interactions resulting in transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families. In agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos. These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein family.

Allosteric Effects of Pit-1 DNA Sites on Long-Term Repression in Cell Type Specification

Abstract: Reciprocal gene activation and restriction during cell type differentiation from a common lineage is a hallmark of mammalian organogenesis. A key question, then, is whether a critical transcriptional activator of cell type-specific gene targets can also restrict expression of the same genes in other cell types. Here, we show that whereas the pituitary-specific POU domain factor Pit-1 activates growth hormone gene expression in one cell type, the somatotrope, it restricts its expression from a second cell type, the lactotrope. This distinction depends on a two-base pair spacing in accommodation of the bipartite POU domains on a conserved growth hormone promoter site. The allosteric effect on Pit-1, in combination with other DNA binding factors, results in the recruitment of a corepressor complex, including nuclear receptor corepressor N-CoR, which, unexpectedly, is required for active long-term repression of the growth hormone gene in lactotropes.

Relief of gene repression by torso RTK signaling: role of capicua in Drosophila terminal and dorsoventral patterning.

Abstract: Differentiation of the embryonic termini in Drosophila depends on signaling by the Tor RTK, which induces terminal gene expression by inactivating at the embryonic poles a uniformly distributed repressor activity that involves the Gro corepressor. Here, we identify a new gene, cic, that acts as a repressor of terminal genes regulated by the Tor pathway. cic also mediates repression along the dorsoventral axis, a process that requires the Dorsal morphogen and Gro, and which is also inhibited by Tor signaling at the termini. cic encodes an HMG-box transcription factor that interacts with Gro in vitro. We present evidence that Tor signaling regulates terminal patterning by inactivating Cic at the embryo poles. cic has been evolutionarily conserved, suggesting that Cic-like proteins may act as repressors regulated by RTK signaling in other organisms.

Gene Regulation by Thyroid Hormone

Abstract: Regulation of gene expression by thyroid hormones (T3, T4) is mediated via thyroid hormone receptors (TRs). TRs are DNA-binding transcription factors that function as molecular switches in response to ligand. TRs can activate or repress gene transcription depending on the promoter context and ligand-binding status. In most cases, in the absence of ligand, TRs interact with a corepressor complex containing histone deacetylase activity, which actively inhibits transcription. The binding of ligand triggers a conformational change in the TR that results in the replacement of the corepressor complex by a coactivator complex containing histone acetyltransferase activity, through which the chromatin structure is remodeled, thereby leading to activation of transcription. In addition, the finding that several TR-interacting coregulators act more directly on the basal transcriptional machinery suggests that mechanisms independent of histone acetylation and deacetylation also are involved in TR action.

Molecular Determinants for Targeting Heterochromatin Protein 1-Mediated Gene Silencing: Direct Chromoshadow Domain–KAP-1 Corepressor Interaction Is Essential

Abstract: It is clear that many of the effector domains in eukaryotic transcription factors act as protein-protein interfaces which allow the assembly of macromolecular complexes at various sites in the nucleus. The constellation of transcription factors that are arranged at a gene promoter integrate different complexes via coactivators and corepressors, collectively termed cofactors. Cofactors can act directly upon the process of transcription by regulating components of the RNA polymerase complex or through components of chromatin, often leading to changes in gene expression which are stable through cell division. Core histone modification by histone deacetylases and histone acetyltransferases, which can be targeted, respectively, by corepressors and coactivators, has emerged as a common mechanism for influencing gene expression by altering chromatin (58). Another example is the methylation of CpG islands, which is associated with epigenetically imprinted alleles or repressed genes (8). More recent evidence showing that the methyl-CpG-binding protein is present in a complex with histone deacetylase activity (26, 44) suggests a link between these two chromatin modifications that promote gene silencing. Thus, by acting as links between modifiers of chromatin and site-specific transcription factors, corepressors and coactivators can alter the gene expression profile of a cell in a heritable manner. To investigate the mechanistic steps through which transcription repressors down-regulate gene expression, we have focused on the KRAB (Kruppel-associated box) domain as a model. The KRAB domain is a potent repression domain present in nearly one-third of the members of a family of zinc finger transcription factors for which there are an estimated 300 to 700 human genes (7, 36). The family is characterized by an amino-terminal KRAB repression domain linked to multiple arrays of Cys2His2-type zinc fingers, which are responsible for DNA binding (29). Like many other repression domains, the KRAB repression domain retains repressor activity when transferred to a heterologous DNA binding domain (35, 62, 65). KAP-1 is a universal corepressor for the KRAB domain and is the founding member of a small family of cofactors collectively designated the transcriptional intermediary factor 1 (TIF1) family in humans and mice (61). KAP-1 was isolated by affinity chromatography (19) and subsequently by yeast two-hybrid screening (27, 40). It is a 97-kDa nuclear phosphoprotein and contains a number of domains common to the family but also found in other types of transcriptional regulators (see Fig. ​Fig.1A).1A). At the amino terminus is the RBCC multidomain unit, comprised of a RING finger, two B boxes, and a coiled-coil domain. The RBCC domain is essential for binding to the KRAB domain and participates in multimerization of KAP-1 (19, 48). A plant-like homeodomain (PHD) and a bromodomain are tandemly arranged at the carboxy terminus. The central region of KAP-1 is the least conserved among the family members and is generally rich in prolines, glycines, and serines. KAP-1 itself possesses potent repression activity, and this function is contributed by the carboxy terminus, including the PHD and the bromodomain (19, 40, 61). FIG. 1 Schematic representations of the KAP-1 protein and the HP1BD. (A) The KAP-1 protein is shown in linear format with the conserved domains noted by shaded boxes and with amino acid positions indicated. A RING finger, two B boxes, and a coiled-coil region ... The basis for KAP-1 function in transcription repression is not yet fully understood, but the ability of KAP-1 to bind heterochromatin protein 1 (HP1) is very likely to have a role. The interaction between these families of proteins was first uncovered in a yeast two-hybrid screen using TIF1α (32). There are three different HP1 proteins in humans and mice: HP1α, HP1β, and HP1γ (mouse HP1β and HP1γ are also referred to as M31 or MOD1 and M32 or MOD2, respectively [25]). All the proteins share a basic structure of an amino-terminal chromodomain (CD) and a carboxy-terminal chromoshadow domain (CSD) linked by a hinge region (see Fig. ​Fig.2A).2A). The CD is present in numerous proteins, but the CSD has been found only in the HP1 proteins and is thus considered the signature motif for this family (2, 47, 56). FIG. 2 Schematic representations of the HP1 protein, the CD, and the CSD. (A) Diagram of human HP1α depicting the prototypical structure of the HP1 proteins. The relevant domains are noted; NLS, nuclear localization signal. The CSD polypeptide fragment ... Studies of HP1 in Drosophila melanogaster provide one of the best examples of epigenetic mechanisms of gene regulation. Position effect variegation (PEV) in D. melanogaster refers to the process of silencing euchromatic genes that have integrated adjacent to heterochromatin (63). Gene silencing occurs in only a subset of cells, and this state is faithfully inherited by their progeny, leading to variegated or mosaic patterns of expression. The stochastic nature of PEV is hypothesized to be due to the variable spreading of heterochromatin into adjacent regions (55). Genetic screens for modifiers of PEV led to the discovery of the suppressor allele Su(var)2-5, which encoded HP1, a nonhistone chromosomal protein first identified by its localization to heterochromatin in polytene nuclei (14, 23). D. melanogaster HP1 plays a dose-dependent role in PEV and likely contributes to the formation and/or stabilization of heterochromatin (15). More recent studies showing that there is a correlation between overexpression of the CD protein Swi6 and mat locus imprinting in fission yeast and increased expression of an M31 transgene and changes in variegation gene expression in mice indicate that HP1 function has been strongly conserved (17, 43). Position effects are known to play a role in both human and murine genetic defects (6, 28). In many cases, it appears that positively acting elements are removed and/or that negatively acting (silencing) regions are repositioned near genes. Indeed, boundary elements and locus control regions may function as barriers that prevent inappropriate spreading of heterochromatin or association with heterochromatic regions in nuclei (18, 38, 60). Moreover, enhancer function has been postulated to work by keeping genes out of heterochromatic environs (10). Emerging studies of gene regulation by Ikaros have revealed a colocalization of inactive genes with centromeric heterochromatin and M31 in normal cycling lymphocytes (9). Thus, local compartmentalization of genes in the nucleus is emerging as a common mechanism for repressing transcription in a stable manner. However, the mechanisms responsible for sequence-specific targeting of this repression are unclear. Heterochromatin, as defined classically by cytological appearance, is interspersed throughout chromosomes but is abundant near centromeres and telomeres and is frequently composed of repetitive sequences (64). The mammalian HP1 proteins have distinct euchromatic or heterochromatic staining patterns in nuclei, suggesting that their roles have become specialized or that targeting to chromatin has been regulated uniquely (39, 52). KAP-1 is dynamically associated with the euchromatic and heterochromatic regions, suggesting that it links heterochromatin-mediated gene regulation to localization in a specific chromosomal territory (52). This model proposes that KRAB-zinc finger proteins recruit the KAP-1 corepressor to DNA; this complex, in turn, binds to HP1, which may then nucleate local heterochromatin formation, resulting in gene silencing. In order to understand the interaction between KAP-1 and HP1 in more detail, we have reconstituted the complex using recombinant proteins and have comprehensively defined its biochemical properties. We have found that the CSD in HP1 is required for direct binding to KAP-1 and that a stretch of 15 residues in the middle of KAP-1, the HP1BD, is necessary and sufficient for association. We present evidence that the CSD dimerizes and that it binds the KAP-1 HP1BD in a 2:1 stoichiometry, with an apparent Kd of approximately 60 nM. Mutational analysis with the human HP1α CSD and KAP-1 has pinpointed specific residues that are essential for both binding and transcription repression activity in either protein. We also show that interactions with three other HP1 binding proteins are disrupted by the same CSD mutations as those which disrupt KAP-1 binding; however, these domains are not equally able to repress transcription or inhibit HP1-mediated transcription repression.

Mice lacking the transcriptional corepressor TIF1beta are defective in early postimplantation development.

Abstract: TIF1beta, a member of the transcriptional intermediary factor 1 family, has been reported to function as a corepressor for the large class of KRAB domain-containing zinc finger proteins of the Kruppel type. To address the biological function of TIF1beta, we have generated TIF1beta-deficient mice by gene disruption. TIF1beta protein was detected in wild-type but not TIF1beta(-/-) blastocysts. Homozygous mutant embryos, which developed normally until the blastocyst stage and underwent uterine implantation, were arrested in their development at the early egg-cylinder stage at about embryonic day (E) 5.5 and were completely resorbed by E8.5. Taken together, these results provide genetic evidence that TIF1beta is a developmental regulatory protein that exerts function(s) essential for early postimplantation development.

Cell signaling switches HOX-PBX complexes from repressors to activators of transcription mediated by histone deacetylases and histone acetyltransferases.

Abstract: The Hoxb1 autoregulatory element comprises three HOX-PBX binding sites. Despite the presence of HOXB1 and PBX1, this enhancer fails to activate reporter gene expression in retinoic acid-treated P19 cell monolayers. Activation requires cell aggregation in addition to RA. This suggests that HOX-PBX complexes may repress transcription under some conditions. Consistent with this, multimerized HOX-PBX binding sites repress reporter gene expression in HEK293 cells. We provide a mechanistic basis for repressor function by demonstrating that a corepressor complex, including histone deacetylases (HDACs) 1 and 3, mSIN3B, and N-CoR/SMRT, interacts with PBX1A. We map a site of interaction with HDAC1 to the PBX1 N terminus and show that the PBX partner is required for repression by the HOX-PBX complex. Treatment with the deacetylase inhibitor trichostatin A not only relieves repression but also converts the HOX-PBX complex to a net activator of transcription. We show that this activation function is mediated by the recruitment of the coactivator CREB-binding protein by the HOX partner. Interestingly, HOX-PBX complexes are switched from transcriptional repressors to activators in response to protein kinase A signaling or cell aggregation. Together, our results suggest a model whereby the HOX-PBX complex can act as a repressor or activator of transcription via association with corepressors and coactivators. The model implies that cell signaling is a direct determinant of HOX-PBX function in the patterning of the animal embryo.

The SMRT Corepressor Is Regulated by a MEK-1 Kinase Pathway: Inhibition of Corepressor Function Is Associated with SMRT Phosphorylation and Nuclear Export

Abstract: The SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor participates in the repression of target gene expression by a variety of transcription factors, including the nuclear hormone receptors, promyelocytic leukemia zinc finger protein, and B-cell leukemia protein 6. The ability of SMRT to associate with these transcription factors and thereby to mediate repression is strongly inhibited by activation of tyrosine kinase signaling pathways, such as that represented by the epidermal growth factor receptor. We report here that SMRT function is potently inhibited by a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) cascade that operates downstream of this growth factor receptor. Intriguingly, the SMRT protein is a substrate for phosphorylation by protein kinases operating at multiple levels in this MAPKKK pathway, including the MAPKs, MAPK–extracellular signal-regulated kinase 1 (MEK-1), and MEK-1 kinase (MEKK-1). Phosphorylation of SMRT by MEKK-1 and, to a lesser extent, MEK-1 inhibits the ability of SMRT to physically tether to its transcription factor partners. Notably, activation of MEKK-1 or MEK-1 signaling in transfected cells also leads to a redistribution of the SMRT protein from a nuclear compartment to a more perinuclear or cytoplasmic compartment. We suggest that SMRT-mediated repression is regulated by the MAPKKK cascade and that changes both in the affinity of SMRT for its transcription factors and in the subcellular distribution of SMRT contribute to the loss of SMRT function that is observed in response to kinase signal transduction.

Human Herpesvirus 8 LANA Interacts with Proteins of the mSin3 Corepressor Complex and Negatively Regulates Epstein-Barr Virus Gene Expression in Dually Infected PEL Cells

Abstract: The human herpesvirus 8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in all latently HHV-8 infected cells and in HHV-8-associated tumors, including primary effusion lymphoma (PEL). To better understand the contribution of LANA to tumorigenesis and to the PEL phenotype, we performed a yeast two-hybrid screen which identified the corepressor protein SAP30 as a LANA binding protein. SAP30 is a constituent of a large multicomponent complex that brings histone deacetylases to the promoter. Glutathione S-transferase affinity assays confirmed interaction between LANA and SAP30 and also demonstrated interactions between LANA and two other members of the corepressor complex, mSin3A and CIR. The corepressors bound to the amino-terminal 340-amino-acid domain of LANA. In transient expression assays, this same domain of LANA mediated repression when targeted to a 5xGal4tk-CAT reporter as a GAL4-LANA fusion. PEL cells have the unusual feature that they are frequently dually infected with both HHV-8 and Epstein-Barr virus (EBV). We found that EBV EBNA-1 expression is downregulated in PEL cells at both the RNA and protein levels. In transient expression assays, LANA repressed activated expression from the EBV Qp and Cp latency promoters. Reduction of endogenous Qp activity could also be demonstrated in EBV-infected Rael cells transfected with a LANA expression plasmid. In contrast to the effect of LANA on EBV latency promoters, LANA activated expression from its own promoter. The data indicate that LANA can mediate transcriptional repression through recruitment of an mSin3 corepressor complex and further that LANA-mediated repression is likely to contribute to the low level of EBV latency gene expression seen in dually infected PEL cells.

VDR-Alien: a novel, DNA-selective vitamin D(3) receptor-corepressor partnership.

Abstract: The vitamin D receptor (VDR) is a transcription factor that transmits incoming 1,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) signaling via combined contact with coactivator proteins and specific DNA binding sites (VDREs), which ultimately results in activation of transcription. In contrast, the mechanisms of transcriptional repression via the VDR are less well understood. This study documents VDR-dependent transcriptional repression largely via histone deacetylase (HDAC) activity. Direct, ligand-sensitive protein-protein interaction of the VDR with the nuclear receptor corepressor (NCoR) and a novel corepressor, called Alien, was demonstrated to be comparable but independent of the VDR AF-2 trans-activation domain. Functional assays indicated that Alien, but not NCoR, displays selectivity for different VDRE structures for transferring these repressive effects into gene regulatory activities. Moreover, superrepression via Alien was found to be affected only in part by HDAC inhibitors such as trichostatin A. Finally, for a dissociation of VDR-Alien complexes in vitro and in vivo, higher ligand concentrations were needed than for a dissociation of VDR-NCoR complexes. This suggests that Alien and NCoR are using different interfaces for interaction with the VDR and different pathways for mediating superrepression, which in turn characterizes Alien as a representative of a new class of corepressors. Taken together, association of the VDR with corepressor proteins provides a further level of transcriptional regulation, which is emerging as a complex network of protein-protein interaction-mediated control.

Ssn6–Tup1 interacts with class I histone deacetylases required for repression

Abstract: Ssn6–Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions. Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6–Tup1-mediated repression. In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo. Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6–Tup1 repression. Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs interact physically with Ssn6–Tup1. These findings suggest that Ssn6–Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1–histone interactions.