Posttranslational modification of histones has emerged as a key regulatory signal in eukaryotic gene expression. Recent genetic and biochemical studies link H3-lysine 9 (H3-K9) methylation to HP1-mediated heterochromatin formation and gene silencing. However, the mechanisms that target and coordinate these activities to specific genes is poorly understood. Here we report that the KAP-1 corepressor for the KRAB-ZFP superfamily of transcriptional silencers binds to SETDB1, a novel SET domain protein with histone H3-K9-specific methyltransferase activity. Although acetylation and phosphorylation of the H3 N-terminal tail profoundly affect the efficiency of H3-K9 methylation by SETDB1, we found that methylation of H3-K4 does not affect SETDB1-mediated methylation of H3-K9. In vitro methylation of the N-terminal tail of histone H3 by SETDB1 is sufficient to enhance the binding of HP1 proteins, which requires both an intact chromodomain and chromoshadow domain. Indirect immunofluoresence staining of interphase nuclei localized SETDB1 predominantly in euchromatic regions that overlap with HP1 staining in nonpericentromeric regions of chromatin. Moreover, KAP-1, SETDB1, H3-MeK9, and HP1 are enriched at promoter sequences of a euchromatic gene silenced by the KRAB-KAP-1 repression system. Thus, KAP-1 is a molecular scaffold that is targeted by KRAB-ZFPs to specific loci and coordinates both histone methylation and the deposition of HP1 proteins to silence gene expression. Macromolecular protein complexes containing enzymatic activities that modify the N-terminal tails of the core histones have emerged as key regulators of gene expression in eukaryotes. The constellation of histone modifications, including acetylation, phosphorylation, ubiquination, and methylation, create both synergistic and antagonistic signals that correlate with the transcriptional activity of a gene. This emerging histone code is hypothesized to create an architecture in chromatin that is recognized by nonhistone chromosomal proteins, which then effect the dynamic transition between transcriptionally active versus transcriptionally silent chromatin domains (Jenuwein and Allis 2001). Moreover, the combinatorial nature of these histone modifications and the chromatin-associated proteins that recognize these signals may represent an epigenetic marking system responsible for setting and maintaining heritable programs of gene expression during cellular differentiation and organism development.The role of histone acetylation and phosphorylation in regulation of transcription has been extensively characterized (Cheung et al. 2000;Strahl and Allis 2000;Berger 2001). Although it is well established that arginine and lysine methylation of histones occurs in vivo, the function of these specific modifications remains to be fully described (Strahl et al. 1999). Similar to the discovery of histone acetyltransferases (HATs) and deacetylases (HDACs), the role of histone methylation in the regulation of chromatin structure and gene expression has been greatly facili...

Completed research The recurrent theme that has emerged from the structural study of proteins involved in signal transduction and transcriptional regulation is that multiple independently folded globular domains in these proteins often cooperate in macromolecular recognition. We have used the modularity of highly conserved arnino acid signature motifs as an approach to studying a protein's structure and function. The RING finger motif is a very abundant sequence motif in the protein database. This highly conserved module of approximately 50 amino acids is defined by eight cysteines and/or histidines that coordinate two zinc ions in a unique cross braced fashion. Upon cloning of BRCA1, the most recognizable signature motif in the amino acid sequence was a RING finger at the N-terminus. Moreover, genetic analysis of breast cancer kindreds identified naturally occuring germline mutations that lead to single amino acid substitutions of highly conserved residues in the RING finger and predispose individuals to disease. BAP-1, BRCA1 Associated Protein 1, is a novel, exclusively nuclear ubiquitin carboxy-terminal hydrolase that was identified by the Rauscher lab in a yeast two-hybrid interaction screen for proteins that would interact with the RING finger motif of BRCA1 (Jensen et al. Oncogene 16:1097-1112, 1998). Utilizing a putative coiled-coil structural motif located near the C-terminus of BAP-1, we performed a second, subsequent two-hybrid screen to identify additional proteins that may interact with BAP-1. Sequence from five positive clones, two independent fusions, identically matched nucleotide sequence which encodes the C-terminus of ß-catenin. Quantitative ß-gal assays of yeast extracts revealed the interaction was weak, indicating that either a larger portion of ß-catenin is necessary for optimal interaction or that the interaction might be very transient. To determine if a direct interaction occurred between BAP-1 and ß-catenin, 35 S-met labeled in vitro translates of BAP-1 and ß-catenin were tested for their ability to bind to GST fusions of ß-catenin or the C-terminus of BAP-1 in vitro, respectively, ß-catenin was observed to show some specific association with the C-terminus of BAP-1. In order to verify an in vivo interaction between BAP-1 and ß-catenin, co-immunoprecipitation experiments were employed. In order to verify an in vivo interaction between BAP-1 and ß-catenin, co-immunoprecipitation of 35 S-met labeled protein from transiently transfection of CMV driven expression plasmids containing the entire open reading frames of BAP-1 and ß-catenin into COS1 cells was employed. All attempts to co-immunoprecipitate BAP1 and ß-catenin failed under all experimental conditions (i.e. whole cell lysates vs. nuclear extracts, high salt vs. low salt, different detergents) and washing stringencies. Likewise, immunoprecipitation of endogenous BAP-1 followed by Western analysis with anti-sera against ß-catenin failed to detect in vivo interaction when performed under similar conditions. Identical David C. Schultz,...

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable. A recently emerging paradigm for the epigenetic control and propagation of gene expression states involves the role of chromatin structure. Though historically viewed as a passive packaging structure primarily used to assemble the enormous amount of DNA into a eukaryotic nucleus, the nucleosome with its complement of core histones has emerged as a key target for regulating gene expression (Wolffe and Hansen 2001). The dynamic regulation of chromatin organization appears to be accomplished by macromolecular protein complexes that contain enzymatic activities that modify the tails of the core histones. The constellation of these histone modifications, including acetylation, phosphorylation, ubiquitination, and methylation, create both synergistic and antagonistic signals that correlate with the transcriptional activity of a gene (Wu and Grunstein 2000). This emerging "histone code" is hypothesized to create functionally distinct subdomains in chromatin that define active versus transcriptionally silent genes (Jenuwein and Allis 2001). Histone modifications and the chromatin-associated proteins that interpret these signals may represent an epigenetic marking system responsible for setting and maintaining heritable programs of gene expression during development.The role of histone acetylation/deacetylation [mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively] in modulating gene activity is now well established (Kuo and Allis 1998). The role of histone methylation in the regulation of chromatin structure and gene transcription has been...

The SNAIL transcription factor contains C-terminal tandem zinc finger motifs and an N-terminal SNAG repression domain. The members of the SNAIL family have recently emerged as major contributors to the processes of development and metastasis via the regulation of epithelial-mesenchymal transition events during embryonic development and tumor progression. However, the mechanisms by which SNAIL represses gene expression are largely undefined. Previously we demonstrated that the AJUBA family of LIM proteins function as corepressors for SNAIL and, as such, may serve as a platform for the assembly of chromatin-modifying factors. Here, we describe the identification of the protein arginine methyltransferase 5 (PRMT5) as an effector recruited to SNAIL through an interaction with AJUBA that functions to repress the SNAIL target gene, E-cadherin. PRMT5 binds to the non-LIM region of AJUBA and is translocated into the nucleus in a SNAILand AJUBA-dependent manner. The depletion of PRMT5 in p19 cells stimulates E-cadherin expression, and the SNAIL, AJUBA, and PRMT5 ternary complex can be found at the proximal promoter region of the E-cadherin gene, concomitant with increased arginine methylation of histones at the locus. Together, these data suggest that PRMT5 is an effector of SNAIL-dependent gene repression.The SNAG family of zinc finger transcription factors in vertebrates include GFI-1A, GFI-1B, the insulinoma-associated protein IA-1, the homeobox protein GSH-1, and the SNAIL/SLUG family. These proteins play important roles in the regulation of development, stem cell self-renewal, and tumor progression (5, 22, 49). They share a common set of functional domains: a C-terminal DNA binding domain composed of five to seven Cys2-His2 zinc fingers and a highly conserved N-terminal repression domain designated SNAG. The SNAG motif was first identified from the GFI-1 protein and comprises the first 21 amino acid residues in the N terminus. The SNAG domain is a potent and transferable repression motif (22, 49). However, unlike other repression domains which are associated with zinc finger proteins, such as the KRAB domain and the BTB-POZ domain, whose mechanisms of repression are well established, little is known about the mechanisms of the SNAG domain-mediated repression (9, 15).The SNAIL protein has emerged as a potent regulator of the processes of embryonic development and tumor progression through the regulation of the epithelial-mesenchymal transition (EMT) (5, 36). In mammalian cells, SNAIL induces EMT at least partially through repression of the E-cadherin gene, thereby altering cell adhesion (6). The SNAIL protein has been found in multiprotein complexes containing histone deacetylases (HDACs), mSIN3A, and LOXL2/3 (39, 40). However, the biological significance of these interactions and how SNAIL mediates functional protein complex assembly at specific promoters in the context of chromatin remain undefined.We previously identified novel corepressors that directly bind to the SNAG domains of GFI-1 and SNAIL by using yeast t...

Highlights d 3,000 compounds screened in two cell types against SARS-CoV-2 d Entry pathways are distinct in hepatocyte Huh7.5 and respiratory Calu-3 cells d Only nine compounds that are active in Huh7.5 cells are active in Calu-3 cells d Cyclosporin and cyclophilin inhibitors block SARS-CoV-2 infection in diverse cells

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Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y 3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y 7 , has 352 amino acids and shares 23-30% amino acid identity with the P2Y 1 -P2Y 6 purinoceptors. The P2Y 7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 ؎ 6 nM), much less for UTP and ADP (ϳ1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y 7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y 7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y 7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y 7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y 7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.The widespread occurrence of metabotropic receptors for extracellular ATP has long been inferred from physiological and pharmacological evidence (1). A number of such G proteincoupled ATP receptors have been characterized and a consensus on their nomenclature has termed all of these as P2Y purinoceptors (to be individually named P2Y 1 to P2Y n ), regardless of previous terminology such as P 2U or P 2T for subclasses thereof (2). The first such receptors to be characterized by DNA cloning and expression were the P2Y 1 receptor (where UTP is inactive) (3) and the P2Y 2 receptor (ATP and UTP are equally active) (4). True species homologues (or orthologues) of P2Y 1 have since been obtained, e.g. bovine (5) and human (6), and of P2Y 2 , e.g. from human airway epithelium (7) or human erythroleukemia (HEL) 1 cells (8). Further types identified by cloning have been the P2Y 3 receptor (UDP Ͼ ADP Ͼ ATP) (9) and P2Y 4 (UTP Ͼ Ͼ ATP, and more strongly related to P2Y 2 ) (10, 11). Further novel P2Y receptors have recently been identified from their cDNAs from chicken activated T lymphocytes (12) and rat vascular smooth muscle cells (13) and designated P2Y 5 and P2Y 6 receptors. Previously we have demonstrated at least three P2 purinoceptors on the hematopoietic cell line, HEL cells, by intracellular calcium mobilization and by photoaffinity labeling (8). Here we report the molecular cloning and characterization of one of these, a novel P2 purinergic receptor designated P2Y 7 .

Core Binding Factor (CBF) is required for the development of de®nitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFb-SMMHC are commonly expressed in subsets of acute leukemia. CBFb-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this e ect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is su cient to slow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFb-SMMHC-induced inhibition of proliferation. Representational di erence analysis (RDA) identi®ed cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFb-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16 INK4a , overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These ®ndings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins.