Hairy-related Proteins Research Articles

SCFβTrCP-mediated degradation of SHARP1 in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer associated with metastasis, high recurrence rate, and poor survival. The basic helix-loop-helix transcription factor SHARP1 (Split and Hairy-related Protein 1) has been identified as a suppressor of the metastatic behavior of TNBC. SHARP1 blocks the invasive phenotype of TNBC by inhibiting hypoxia-inducible factors and its loss correlates with poor survival of breast cancer patients. Here, we show that SHARP1 is an unstable protein that is targeted for proteasomal degradation by the E3 ubiquitin ligase complex SCFβTrCP. SHARP1 recruits βTrCP via a phosphodegron encompassing Ser240 and Glu245 which are required for SHARP1 ubiquitylation and degradation. Furthermore, mice injected with TNBC cells expressing the non-degradable SHARP1(S240A/E245A) mutant display reduced tumor growth and increased tumor-free survival. Our study suggests that targeting the βTrCP-dependent degradation of SHARP1 represents a therapeutic strategy in TNBC.

Retinoic acid stimulates transcription of the rat SHARP-2 gene via multiple pathways.

Members of the enhancer of split- and hairy-related protein (SHARP) family, SHARP-1 and SHARP-2, are basic helix-loop-helix transcriptional repressors and belong to the clock genes. In this study, an effect of retinoic acid (RA) on the SHARP family gene expression in the differentiated cells was examined. RA rapidly and temporarily induced the SHARP-2 mRNA expression in hepatic H4IIE cells. Then, whether the SHARP-2 mRNA expression is altered by dexamethasone (Dex), insulin, and the combination of RA and Dex or RA and insulin was examined. Dex had different effects on the expression of SHARP-2 mRNA in the presence or absence of RA. Then, the molecular mechanisms were investigated using inhibitors of various signaling molecules. The RA-induction of SHARP-2 mRNA level was mainly inhibited by LY294002, staurosporine, and actinomycin D, respectively. Finally, whether RA acts on the transcriptional regulatory region of the SHARP-2 gene was analysed using luciferase reporter gene assay. At least two RA-responsive regions were mapped at the nucleotide sequences between -3,700 and -1,600 of the SHARP-2 gene. In addition, this effect was dependent on the RA receptor and retinoid X receptor. Thus, we conclude that RA stimulated transcription of the SHARP-2 gene via multiple pathways.

Maternal serum split and hairy-related protein 1 in preeclampsia versus normal pregnancy

Objective The primary objective was to compare maternal serum split and hairy related protein-1 (SHARP1) in preeclamptic women versus normal pregnant women. The secondary objectives were to compare maternal and neonatal outcomes between preeclampsia and normal pregnant women. Methods This cross-sectional analytic study included 75 preeclamptic pregnant women and 75 normal pregnant women at Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital between March 2018 and May 2019. Blood was collected into nonheparinized tubes. Blood samples were centrifuged and stored at −80 °C until assayed. Maternal serum SHARP1 levels were measured by enzyme-linked immunosorbent assays. Maternal and neonatal outcomes were recorded. Results Median serum SHARP1 level in preeclampsia was significantly higher than in normal pregnancy (5.2 vs. 3.7 ng/mL, p = .007). Preeclamptic women had a higher rate of cesarean section than normal pregnant women (68% vs. 32%, p < .001). Preeclamptic women had lower neonatal birth weight than normal pregnant women (2,546.1 ± 766.3 vs 2,884.5 ± 479.9, p = .001). Neonatal complications were higher in preeclamptic women than normal pregnant women (40% vs. 17.3%, p = .002). Conclusions Serum SHARP1 in preeclampsia was significantly higher than in normal pregnancy.

An insulin-inducible transcription factor, SHARP-1, represses transcription of the SIRT1 longevity gene

The rat enhancer of split- and hairy-related protein (SHARP)-1 genes encode insulin-inducible transcriptional repressors. A longevity gene, sirtuin 1 (SIRT1) encodes protein deacetylase. These play an important role in regulating hepatic glucose metabolism. In this study, to evaluate a correlation with these gene expressions, we examined whether SIRT1 effects on expression of the SHARP-1 gene by a treatment with a SIRT1 inhibitor or activator in rat H4IIE hepatoma cells. Whereas the SIRT1 inhibitor increased the level of SHARP-1 mRNA, the SIRT1 activator decreased it. Next, whether SHARP-1 effect on the transcriptional activity of the human SIRT1 gene using luciferase reporter assays was determined. Promoter activity of the SIRT1 gene was specifically repressed by SHARP-1. Further reporter analysis using 5′- deleted or mutated constructs revealed that an E box sequence (5′-CACGTG-3′) of the SIRT1 gene promoter was required for the inhibitory effect of SHARP-1. Thus, we conclude that expressions between the SHARP-1 and the SIRT1 genes show a negative correlation and that SHARP-1 represses transcription of the SIRT1 gene.

Identification of the MmeHairy gene and expression analysis affected by two SNPs in the 3'-untranslated region in the clam Meretrix meretrix.

As a bHLH transcriptional repressor, Hairy-related proteins can bind to DNA sites in target gene promoters and negatively regulate gene transcription. In the present study, the full-length cDNA of Hairy was obtained from the clam Meretrix meretrix (MmeHairy), and two SNPs in the 3'-untranslated region (UTR) of this gene, SNP1066 and 1067, were identified and characterized. Multiple sequence alignment and phylogenetic analysis revealed that MmeHairy belongs to the Hairy protein subfamily. Analysis of tissue expression patterns showed that the mRNA of MmeHairy had the highest expression level in the hepatopancreas. The expression levels of MmeHairy were up-regulated in the hepatopancreas after Vibrio challenge. Genotyping and quantitative analysis showed that the mRNA levels of MmeHairy were significantly different among individual clams with different genotypes at SNP1066 and 1067 (P<0.05), which indicated that these two SNP loci may affect the expression of MmeHairy and could be used as candidate markers for future selection in M.meretrix breeding programs.

Suppression of the epithelial-mesenchymal transition by SHARP1 is linked to the NOTCH1 signaling pathway in metastasis of endometrial cancer.

BackgroundMechanisms governing the metastasis of endometrial cancer (EC) are poorly defined. Recent data support a role for Enhancer-of-split and hairy-related protein 1 (SHARP1), a basic helix-loop-helix transcription repressor, in regulating invasiveness and angiogenesis of several human cancers. However, the role of SHARP1 in metastasis of EC remains unclear.MethodsHuman EC cell lines (Ishikawa and HEC-1B) were used. SHARP1 was upregulated by lentivirus transduction, while intracellular domain of NOTCH1 (ICN) were upregulated by transient transfection with plasmids. Effects of SHARP1 on cell migration and invasion were evaluated by wound healing assay and transwell invasion assay. Experimental metastasis assay were performed in nude mice. Effects of SHAPR1 on protein levels of target genes were detected by western blotting. Furthermore, the association between SHARP1 and the NOTCH1/EMT pathway was further verified in EC tissue specimens by immunohistochemical analysis.ResultsOverexpression of SHARP1 in EC cells inhibited cell migration, invasion, and metastasis. Exogenous SHARP1 overexpression affected the proteins levels of genes involved in EMT process and NOTCH1 signaling pathway. Upregulation of ICN in SHARP1-overexpressing Ishikawa cells induced cell migration and an EMT phenotype. Additionally, immunohistochemical analysis demonstrated that SHARP1 protein levels were lower in metastatic EC than in primary tumors, and statistical analysis revealed correlations between levels of SHARP1 and markers of EMT and NOTCH1 signaling pathway in human EC tissue specimen.ConclusionsThis work supports a role for SHARP1 in suppressing EMT and metastasis in EC by attenuating NOTCH1 signaling. Therefore, SHARP1 may be a novel marker for lymphatic metastasis in EC patients.

Characterization of hey bHLH genes in teleost fish

Hairy-related basic helix-loop-helix (bHLH) transcription factors are targets of Delta-Notch signaling and represent essential components for a number of cell fate decisions during vertebrate embryogenesis. Hey genes encode a subfamily of hairy-related proteins that have been implicated in processes like somitogenesis, blood vessel and heart development. We have identified and characterized hey genes in three teleost fish lineages using degenerate PCR and database searches. Phylogenetic analysis of Hey proteins suggests a complex pattern of evolution with high divergence of hey2 in Takifugu rubripes (Fugu, Japanese pufferfish) and possibly loss in the related Tetraodon nigroviridis (the freshwater pufferfish). In addition, duplication of hey1 in both pufferfishes, Fugu and Tetraodon, was observed. Conversely, zebrafish (Danio rerio) has the same complement of three hey genes as known from mammals. All three hey genes show much more restricted gene expression profiles in zebrafish when compared to mouse. Importantly, while all three murine Hey genes are expressed in overlapping patterns in the presomitic mesoderm (PSM) and somites, in zebrafish only hey1 shows PSM and somite expression in a highly dynamic fashion. Therefore, while overlapping expression might account for redundancy of hey function in higher vertebrates, this is unlikely to be the case in zebrafish. In deltaD (dlD) deficient after-eight zebrafish mutants, the dynamic expression of hey1 in the PSM is impaired and completely lost in newly formed somitomeres. Overexpression of dlD on the other hand results in the ectopic expression of hey1 in the axial mesoderm. Hence, hey1 represents a target of Delta-Notch signaling dynamically expressed during somite formation in zebrafish.

Gene expression of basic helix-loop-helix transcription factor, SHARP-2, is regulated by gonadotropins in the rat ovary and MA-10 cells.

Basic helix-loop-helix (bHLH) proteins regulate transcription from the E box sequence (5'-CANNTG-3') located in the regulatory region of most gene promoters. The rat enhancer of split- and hairy-related protein 2 (SHARP-2) is a member of the bHLH protein family. To analyze the possible role of SHARP-2 in the rat ovary, the regulation of the expression of the SHARP-2 gene was examined, and the SHARP-2 protein was characterized. Northern blot analysis revealed that the level of SHARP-2 mRNA abruptly and temporarily increases as the result of the action of LH, i.e., eCG or hCG treatment alone or hCG after eCG treatment, in the rat ovary, as indicated by the treatment of primary cultured rat granulosa cells with hCG after FSH treatment or of mouse Leydig MA-10 cells with hCG or 8-bromoadenosine 3',5'-cyclic monophosphate. An in situ hybridization analysis showed that eCG treatment increases the level of the SHARP-2 transcript in theca interna cells and that hCG treatment, after the administration of eCG, increases the level of the SHARP-2 transcript in granulosa cells. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into primary cultured granulosa cells and MA-10 cells revealed that the entire coding sequence of SHARP-2 fused to the GFP is localized in the nucleus. The transcriptional activity of SHARP-2 also was examined using transient DNA transfection experiments. When an expression vector encoding the full length of SHARP-2 was cotransfected with thymidine kinase promoter-luciferase reporter plasmids, with or without E box sequences, into MA-10 cells, the luciferase activity was decreased in an E box-dependent manner. We conclude that the level of SHARP-2 mRNA is regulated by gonadotropins and that SHARP-2 functions as a transcriptional repressor localized in the nucleus.

DEC1 Negatively Regulates the Expression of DEC2 through Binding to the E-box in the Proximal Promoter

Human DEC (differentially expressed in chondrocytes), mouse STRA (stimulated with retinoic acid), and rat SHARP (split and hairy related protein) proteins constitute a new and structurally distinct class of the basic helix-loop-helix proteins. In each species, two members are identified with a sequence identity of >90% in the basic helix-loop-helix region and approximately 40% in the total proteins, respectively. Recently, we have reported that DEC1 is abundantly expressed in colon carcinomas but not in the adjacent normal tissues. The present study was undertaken to extend the expression study of DEC1 and to determine whether DEC1 and DEC2 had similar expression patterns among paired cancer-normal tissues from the colon, lung, and kidney. Without exceptions, DEC1 was markedly higher in the carcinomas, whereas the opposite was true with DEC2. In stable transfectants, tetracycline-induced expression of DEC1 caused proportional decreases in the expression of DEC2. Co-transfection with DEC1 repressed the activity of a DEC2 promoter reporter by as much as 90%. The repression was observed with wild type DEC1 but not its DNA binding-defective mutants. Studies with deletion and site-directed mutants located, in the proximal promoter, an E-box motif that supported the DEC1-mediated repression. Disruption of this E-box markedly abolished the ability of the reporter to respond to DEC1. Our findings assign for DEC1 the first target gene that is regulated through direct DNA binding. DEC/STRA/SHARP proteins are highly identical in the DNA binding domain but much more diverse in other areas. DEC1-mediated repression on the expression of DEC2 provides an important mechanism that these transcription factors regulate the cellular function not only by modulating the expression of their target genes but also the expression of members within the same class.

Abundant expression of Dec1/stra13/sharp2 in colon carcinoma: its antagonizing role in serum deprivation-induced apoptosis and selective inhibition of procaspase activation.

The basic helix-loop-helix (bHLH) proteins are intimately associated with developmental events such as cell differentiation and lineage commitment. The HLH domain in the bHLH motif is responsible for dimerization, whereas the basic region mediates DNA binding. Based on sequence alignment and domain analysis, differentially expressed in chondrocytes/stimulated with retinoic acid/split and hairy-related proteins (DEC/STRA/SHARPs) represent a new class of bHLH proteins. The present study describes the functional characterization of DEC1. Subtractive experiments and blotting analyses demonstrated that DEC1 was highly expressed in colon carcinomas, but not in the adjacent normal tissues. Several cell cycle blockers markedly induced DEC1 expression. Stable transfectants with a tetracycline-inducible construct demonstrated that DEC1 caused proliferation inhibition, antagonized serum deprivation-induced apoptosis and selectively inhibited the activation of procaspases. These activities were highly correlated with the abundance of tetracycline-induced DEC1. Stable transfectants expressing a mutant DEC1 (lacking the DNA-binding domain) exhibited neither proliferation inhibition nor apoptotic antagonism, which suggests that DNA binding is required for these actions. Enzymic assays and immunoblotting analyses demonstrated that induction of DEC1 by tetracycline significantly decreased the activation of procaspases 3, 7 and 9 but not procaspase 8. The selective suppression on the activation of procaspases 3, 7 and 9 over procaspase 8 suggests that DEC1-mediated anti-apoptosis is achieved by blocking apoptotic pathways initiated via the mitochondria. The results functionally distinguish DEC1 from other bHLH proteins and directly link this factor to oncogenesis.

The function of hairy-related bHLH repressor proteins in cell fate decisions.

Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.

TLE, the Human Homolog of Groucho, Interacts with AML1 and Acts as a Repressor of AML1-Induced Transactivation

TheAML1gene encodes DNA-binding proteins that contain the Runt domain and is found at the breakpoints of some translocations associated with leukemias. It has been reported that AML1 plays pivotal roles in myeloid differentiation, probably through the transcriptional regulation of various hematopoietic genes. Here we demonstrate the physical and the functional interaction between AML1 and TLE1 (transducin-like Enhancer of split) human homolog of Groucho that is known to be a corepressor of Hairy-related proteins. TLE1 binds to AML1 through the Runt domain and the C terminus of AML1 that includes the VWRPY motif. The interaction is mainly mediated by the SP domain of TLE1. Moreover, TLE1 inhibits AML1-induced transactivation of the target promoters through the C terminus of AML1. These results suggest that TLE1 acts as a repressor of AML1 and provide important insights into the mechanism of the negative regulation of the AML1 functions in hematopoiesis and leukemogenesis.

Local approach for defining an aid for the design department for optimizing the geometry of fatigue loaded fillet welds : Janosh, J.J., Debiez, S. and Dang Van, K. Welding in the World (1996) 37 (2), 58–71

As a bHLH transcriptional repressor, Hairy-related proteins can bind to DNA sites in target gene promoters and negatively regulate gene transcription. In the present study, the full-length cDNA of Hairy was obtained from the clam Meretrix meretrix (MmeHairy), and two SNPs in the 3′-untranslated region (UTR) of this gene, SNP1066 and 1067, were identified and characterized. Multiple sequence alignment and phylogenetic analysis revealed that MmeHairy belongs to the Hairy protein subfamily. Analysis of tissue expression patterns showed that the mRNA of MmeHairy had the highest expression level in the hepatopancreas. The expression levels of MmeHairy were up-regulated in the hepatopancreas after Vibrio challenge. Genotyping and quantitative analysis showed that the mRNA levels of MmeHairy were significantly different among individual clams with different genotypes at SNP1066 and 1067 (P < 0.05), which indicated that these two SNP loci may affect the expression of MmeHairy and could be used as candidate markers for future selection in M. meretrix breeding programs.

Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression.

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.