Transcriptional Regulation Of Heart And Muscle-specific Gene Smyd1 And Other Development-related Genes

The recognition and association of transcriptional factors tothe target regulatory element of downstream gene are the firstand pivotal step of gene transcription which is highlyregulated by both cis-and trans-acting factors. Although genetranscripts are synthesized by RNA polymerase, the special andtemporal expression pattern of each gene is distinct, which isdue to the combinatorial regulation of multipletranscriptional factors. During my doctoral thesis research,the projects including the studies of transcriptionalregulations of a cardiac and muscle specific gene Smyd1 and apuberty related gene KiSS1. These studies revealed how theexpressions of these two genes are regulated by upstream genesor extracellular signals on the molecular level, which are thedirect molecular evidences to support in vivo studies. At thesame time, functional studies of a HLH transcriptional factor,named GCIP, during myogenesis has been done as wellas some ofcollaborative researches.1. Smyd1 gene, the direct target of SRF and Myogenin, promotes myogenesisSmyd1 is a heart and muscle specific SET-MYND domaincontaining protein functioning as a methyltransferase whichmodifies H3-K4 residues and activates downstream genetranscription. In adult human tissues tested, Smyd1 isrestricted in heart and skeletal muscle. Over-expression ofSmyd1 promotes myoblasts differentiation and myotube formationin C2C12 cells due to up-regulation of muscle specifictranscriptional factors. The expression of Smyd1 is elevatedwhen serum response factor (SRF) and Myogenin is over-expressedin myoblasts respectively. We isolated the Smyd1 gene promoterand identified DNAregulatory elements that might control Smyd1expression during Myogenesis. The proximal promoter of theSmyd1 gene contained binding sites for several factors involvedin myoblasts differentiation including Myogenin, SRF, andmyocyte enhancer factor 2 (MEF2). EMSA. and ChIP assaysdemonstrated that SRF and Myogenin bind to CArG and E-boxelements on Smyd1 promoter region respectively.Co-transfection experiments suggested that binding sites ofboth Myogenin and SRF are necessary for activation of thepromoter. Taken together, these studies indicate that Smyd1 isa key regulator of myogenic differentiation which acts as a downstream target of muscle regulatory factors.2. GCIP/CCNDBP1 regulates myogenic differentiation by forminga functional protein complex with E47 and MyoDDifferentiation of skeletal muscle is a highly orderedmulti-step process called myogenesis, which involves theexpression of muscle-specific genes, withdrawal of cell cycleand formation of multinucleated myotube. It is controlled bya family of muscle-specific basic helix-loop-helix (bHLH)transcription factors, such as MyoD, by heterodimerizationwith ubiquitous bHLH proteins, called E proteins, to activatemuscle genes by binding E-boxes (CANNTG). In this study, weshow that GCIP/CCNDBP1, a recently identified HLH leucine-richprotein without a predicted basic DNAbinding region, regulatesmuscle specific gene expression and E47/MyoDheterodimerization. Both the mRNA and protein expressionlevels of GCIP were up-regulated during myogenicdifferentiation of C2C12 cells. Over-expression of GCIP inC2C12 cells promotes E47/MyoD complex association, activationof muscle specific transcription,and myotube formation duringskeletal muscle cell differentiation while the mutant form ofGCIP reduced the E47/MyoD heterodimerization and inefficient muscle differentiation. These findings identify a novelpro-myogenic role for the recently identified GCIP/CCNDBP1protein in forming a functional protein complex with MyoD/E47heterodimers that are essential for myogenesis.3. Estrogen regulates KiSS1 gene expression through estrogenreceptorαand SP protein complexesKisspeptins, secreting neuropeptides encoded by the KiSS1(KiSS-1) gene, are natural ligands of a newly identified Gprotein-coupled receptor-54, GPR54. Activation ofKiSS1/GPR54 signaling results in potent activation ofhypothalamus-pituitary-gonadal axis and initiates puberty.Recent data has shown that in female mice KiSS1 is positivelyregulated by estradiol (E₂) in the anteroventralperiventricular nucleus (AVPV), an important reproductiveneuroendocrine brain region, but negatively regulated in thearcuate nucleus (Arc). However, little is known about themolecular mechanisms governing E₂ modulated KiSS1 expression.Here, we demonstrate that the expression level of KiSS1 genewas upregulated with the administration of E₂ in estrogenreceptor alpha (ERα) positive hypothalamus GT1-7 cells. Usingtransient transfection of the human KiSS1 gene promoter coupled to luciferase, E₂ increases promoter activity in the presenceof ERα. Deletion analysis of the KiSS1 promoter indicatesthat the E₂-regulated increase in promoter activity dependedon Spl sites located on the proximal promoter region. Usingelectrophoretic mobility shift assays (EMSA), we determinedthat both Spl and Sp3 proteins associate with the four putativeSp1 sites in vitro. Interestingly, Spl transactivates KISS1promoter activity whereas Sp3 functions as a transcriptionalrepressor. Sp1 and ERαform a complex in vivo and the DNAbinding domain of ERαis not necessary to mediate the E₂ inducedactivation of KiSS1 promoter. Chromatin immunoprecipitation(ChIP) analyses demonstrated that both Sp1 and Sp3constitutively bound to GC-rich motif on the KiSS/ promoterwhile the association of ERαwith the KiSS1 promoter isdependent on E₂ exposure. Together, these results demonstratethat E₂ dependent transcriptional activation of the KiSS1 geneis mediated by ERαthrough the interaction of Sp1/Sp3 proteinswith GC-rich motifs of KiSS1 promoter, providinga molecularnechanism in our understanding how steroid hormones feedbackregulate KISS1 expression in the HPG axis.4. Research on other genesThe expression of metastasis suppressor gene KiSS1 is regulated by Sp1/DRIP130 complex in highly metastatic melanoma cells.GCIP/CCNDBP1, a helix-loop-helix protein, suppressestumorigenesis. Identification and characterization of humannovel genes including ZNF394, ZNF480, ZNF411, WDR26 and SNX-L,some of which are expressed in the heart tissue. Through geneknockout method, GPR48/LGR4 has been identified as an orphanGPCR involved in bone and eye development.