1: J Biol Chem. 2005 Sep 16;280(37):32531-8. Epub 2005 May 31. Conditional mutagenesis of the murine serum response factor gene blocks cardiogenesis and the transcription of downstream gene targets. Niu Z, Yu W, Zhang SX, Barron M, Belaguli NS, Schneider MD, Parmacek M, Nordheim A, Schwartz RJ. Center for Cardiovascular Development, Division of Cardiovascular Sciences, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, 77030, USA. Serum response factor (SRF) homozygous-null embryos from our backcross of SRF(LacZ/)(+) "knock-in" mice failed to gastrulate and form mesoderm, similar to the findings of an earlier study (Arsenian, S., Weinhold, B., Oelgeschlager, M., Ruther, U., and Nordheim, A. (1998) EMBO J. 17, 6289-6299). Our use of embryonic stem cells provided a model system that could be used to investigate the specification of multiple embryonic lineages, including cardiac myocytes. We observed the absence of myogenic alpha-actins, SM22alpha, and myocardin expression and the failure to form beating cardiac myocytes in aggregated SRF null embryonic stem cells, whereas the appearance of transcription factors Nkx2-5 and GATA4 were unaffected. To study the role of SRF during heart organogenesis, we then performed cardiac-specific ablation of SRF by crossing the transgenic alpha-myosin heavy chain Cre recombinase line with SRF LoxP-engineered mice. Cardiac-specific ablation of SRF resulted in embryonic lethality due to cardiac insufficiency during chamber maturation. Conditional ablation of SRF also reduced cell survival concomitant with increased apoptosis and reduced cellularity. Significant reductions in SRF (> or =95%), atrial naturetic factor (> or =80%), and cardiac (> or =60%), skeletal (> or =90%), and smooth muscle (> or =75%) alpha-actin transcripts were also observed in the cardiac-conditional knock-out heart. This was consistent with the idea that SRF directs de novo cardiac and smooth muscle gene activities. Finally, quantitation of the knock-in LacZ reporter gene transcripts in the hearts of cardiac-conditional knock-out embryos revealed an approximately 30% reduction in gene activity, indicating SRF gene autoregulation during cardiogenesis. PMID: 15929941 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Biol Chem. 2004 Dec 31;279(53):55626-32. Epub 2004 Oct 18. Identification of a novel serum response factor cofactor in cardiac gene regulation. Zhang X, Azhar G, Zhong Y, Wei JY. Donald W. Reynolds Department of Geriatrics, University of Arkansas for Medical Sciences and Geriatric Research, 4301 W. Markham #748, Little Rock, AR 72205, USA. The transcription factor serum response factor (SRF) plays an important role in the regulation of a variety of cardiac genes during development and during adult aging. A novel SRF cofactor, herein called p49/STRAP, for SRF-dependent transcription regulation-associated protein, was recently identified in our laboratory. This protein interacted mainly with the transcriptional activation domain of the SRF protein and was found to bind to SRF or to the complex of SRF and another cofactor, such as myocardin or Nkx2.5. The expression of p49/STRAP affected the promoter activity of SRF target genes in a non-uniform manner. For example, p49 activated MLC2v and cardiac actin promoters when it was co-transfected with SRF, but it repressed atrial natriuretic factor promoter activity, which was strongly induced by myocardin. The p49/STRAP mRNA was observed to be highly expressed in fetal, adult, and senescent human hearts, and also in hearts of young adult and old mice, suggesting that p49/STRAP may be an important SRF cofactor in the transcriptional regulation of mammalian cardiac muscle genes throughout the life span. PMID: 15492011 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Mol Cell Biol. 2004 Jun;24(12):5281-9. Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality. Parlakian A, Tuil D, Hamard G, Tavernier G, Hentzen D, Concordet JP, Paulin D, Li Z, Daegelen D. Laboratoire de Biologie Moleculaire de la Differenciation, Universite Paris 7, 75005 Paris, France. Serum response factor (SRF) is at the confluence of multiple signaling pathways controlling the transcription of immediate-early response genes and muscle-specific genes. There are active SRF target sequences in more than 50 genes expressed in the three muscle lineages including normal and diseased hearts. However, the role of SRF in heart formation has not been addressed in vivo thus far due to the early requirement of SRF for mesoderm formation. We have generated a conditional mutant of SRF by using Cre-LoxP strategy that will be extremely useful to study the role of SRF in embryonic and postnatal cardiac functions, as well as in other tissues. This report shows that heart-specific deletion of SRF in the embryo by using a new beta MHC-Cre transgenic mouse line results in lethal cardiac defects between embryonic day 10.5 (E10.5) and E13.5, as evidenced by abnormally thin myocardium, dilated cardiac chambers, poor trabeculation, and a disorganized interventricular septum. At E9.5, we found a marked reduction in the expression of essential regulators of heart development, including Nkx2.5, GATA4, myocardin, and the SRF target gene c-fos prior to overt maldevelopment. We conclude that SRF is crucial for cardiac differentiation and maturation, acting as a global regulator of multiple developmental genes. PMID: 15169892 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Dev Biol. 2003 Sep 1;261(1):116-31. Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF. Small EM, Krieg PA. Department of Cell Biology and Anatomy, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA. The atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. Mouse knockout and mammalian cell culture studies have shown that the ANF gene is regulated by combinatorial interactions between Nkx2-5, GATA-4, Tbx5, and SRF; however, the molecular mechanisms leading to chamber-specific expression are currently unknown. We have isolated the Xenopus ANF promoter in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, a T-box binding site (TBE), and two SRF binding sites (SREs). Our transgenic studies show that mutation of either SRE, the TBE or the distal GATA element, strongly reduces expression from the ANF promoter. However, mutations of the NKE, the proximal GATA, or both elements together, result in relatively minor reductions in transgene expression within the myocardium. Surprisingly, mutation of these elements results in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery-associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. We propose that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex that is required for atrial-specific expression of the ANF gene. PMID: 12941624 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Cell. 2002 Sep 20;110(6):725-35. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Shin CH, Liu ZP, Passier R, Zhang CL, Wang DZ, Harris TM, Yamagishi H, Richardson JA, Childs G, Olson EN. Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA. We have discovered an unusual homeodomain protein, called HOP, which is comprised simply of a homeodomain. HOP is highly expressed in the developing heart where its expression is dependent on the cardiac-restricted homeodomain protein Nkx2.5. HOP does not bind DNA and acts as an antagonist of serum response factor (SRF), which regulates the opposing processes of proliferation and myogenesis. Mice homozygous for a HOP null allele segregate into two phenotypic classes characterized by an excess or deficiency of cardiac myocytes. We propose that HOP modulates SRF activity during heart development; its absence results in an imbalance between cardiomyocyte proliferation and differentiation with consequent abnormalities in cardiac morphogenesis. PMID: 12297046 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Cell. 2002 Sep 20;110(6):713-23. Hop is an unusual homeobox gene that modulates cardiac development. Chen F, Kook H, Milewski R, Gitler AD, Lu MM, Li J, Nazarian R, Schnepp R, Jen K, Biben C, Runke G, Mackay JP, Novotny J, Schwartz RJ, Harvey RP, Mullins MC, Epstein JA. Department of Medicine, University of Pennsylvania Health System, Philadelphia, PA 19104, USA. Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development. PMID: 12297045 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Mol Cell Cardiol. 2002 Jul;34(7):807-21. Differential regulation of the cardiac sodium calcium exchanger promoter in adult and neonatal cardiomyocytes by Nkx2.5 and serum response factor. Muller JG, Thompson JT, Edmonson AM, Rackley MS, Kasahara H, Izumo S, McQuinn TC, Menick DR, O'Brien TX. Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA. Nkx2.5 and serum response factor (SRF) are critically important transcription factors in cardiac morphogenesis. They are also widely expressed in adult cardiomyocytes, but there is little data to indicate their possible role in adult cardiac cells. In this paper we demonstrate that the interaction of Nkx2.5 and SRF in cardiac-specific gene regulation is different between neonatal and adult cardiomyocytes. Our experimental model utilizes transient transfection and adenovirus mediated gene transfer of the proximal promoter fragment of the cardiac isoform of the sodium-calcium exchanger gene (NCX1). This promoter construct (NCX184) contains a single Nkx2.5-response element (NKE) and a single serum response element (CArG). In rat neonatal cardiomyocytes NCX184 activity is substantially induced with Nkx2.5 or SRF and additively with both. Mutagenesis of these NKE and CArG elements demonstrated the specificity of the interactions, which was confirmed with gel retardation analysis of cardiac ventricular tissue. In contrast, in adult cardiomyocytes, co-infection of Nkx2.5 and SRF adenovirus vectors showed Nkx2.5 induction but SRF did not have additive effects on NCX1 promoter regulation. As opposed to NCX1, the proximal atrial natriuretic factor (ANF) promoter was regulated identically in response to SRF and Nkx2.5 in both adult and neonatal cardiomyocytes. These results show that Nkx2.5-SRF interactions are capable of producing different transcriptional responses in adult versus neonatal cardiomyocytes, implying important differences in NCX1 promoter tertiary complex formation dependent on developmental stage. Copyright 2002 Elsevier Science Ltd. All rights reserved. PMID: 12099720 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Biol Chem. 2002 Jul 12;277(28):25775-82. Epub 2002 Apr 30. Combinatorial expression of GATA4, Nkx2-5, and serum response factor directs early cardiac gene activity. Sepulveda JL, Vlahopoulos S, Iyer D, Belaguli N, Schwartz RJ. Department of Pathology, University of Pittsburgh Medical Center, Pennsylvania 15213, USA. Herein, the restricted expression of serum response factors (SRF) closely overlapped with Nkx2-5 and GATA4 transcripts in early chick embryos coinciding with the earliest appearance of cardiac alpha-actin (alphaCA) transcripts and nascent myocardial cells. The combinatorial expression of SRF, a MADS box factor Nkx2-5 (a NK4 homeodomain), and/or GATA4, a dual C4 zinc finger protein, in heterologous CV1 fibroblasts and Schneider 2 insect cells demonstrated synergistic induction of alphaCA promoter activity. These three factors induced endogenous alphaCA mRNA over a 100-fold in murine embryonic stem cells. In addition, the DNA-binding defective mutant Nkx2-5pm efficiently coactivated the alphaCA promoter in the presence of SRF and GATA4 in the presence of all four SREs and was substantially weakened when individual SREs were mutated and or serially deleted. In contrast, the introduction of SRFpm, a SRF DNA-binding mutant, blocked the activation with all of the alphaCA promoter constructions. These assays indicated a dependence upon cooperative SRF binding for facilitating the recruitment of Nkx2-5 and GATA4 to the alphaCA promoter. Furthermore, the recruitment of Nkx2-5 and GATA4 by SRF was observed to strongly enhance SRF DNA binding affinity. This mechanism allowed for the formation of higher ordered alphaCA promoter DNA binding complexes, led to a model of SRF physical association with Nkx2-5 and GATA4. PMID: 11983708 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Biol Chem. 2001 Jan 12;276(2):1026-33. GATA-4 and serum response factor regulate transcription of the muscle-specific carnitine palmitoyltransferase I beta in rat heart. Moore ML, Wang GL, Belaguli NS, Schwartz RJ, McMillin JB. Department of Pathology and Laboratory Medicine, Medical School, University of Texas-Houston Health Science Center, Baylor College of Medicine, Houston, Texas 77030, USA. Transcriptional regulation of nuclear encoded mitochondrial proteins is dependent on nuclear transcription factors that act on genes encoding key components of mitochondrial transcription, replication, and heme biosynthetic machinery. Cellular factors that target expression of proteins to the heart have been well characterized with respect to excitation-contraction coupling. No information currently exists that examines whether parallel transcriptional mechanisms regulate nuclear encoded expression of heart-specific mitochondrial isoforms. The muscle CPT-Ibeta isoform in heart is a TATA-less gene that uses Sp-1 proteins to support basal expression. The rat cardiac fatty acid response element (-301/-289), previously characterized in the human gene, is responsive to oleic acid following serum deprivation. Deletion and mutational analysis of the 5'-flanking sequence of the carnitine palmitoyltransferase Ibeta (CPT-Ibeta) gene defines regulatory regions in the -391/+80 promoter luciferase construct. When deleted or mutated constructs were individually transfected into cardiac myocytes, CPT-I/luciferase reporter gene expression was significantly depressed at sites involving a putative MEF2 sequence downstream from the fatty acid response element and a cluster of heart-specific regulatory regions flanked by two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and competition specificity (or supershifts) with oligonucleotides and antibodies. Individual expression vectors for Nkx2.5, serum response factor (SRF), and GATA4 enhanced CPT-I reporter gene expression 4-36-fold in CV-1 cells. Although cotransfection of Nkx and SRF produced additive luciferase expression, the combination of SRF and GATA-4 cotransfection resulted in synergistic activation of CPT-Ibeta. The results demonstrate that SRF and the tissue-restricted isoform, GATA-4, drive robust gene transcription of a mitochondrial protein highly expressed in heart. PMID: 11038368 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Mech Dev. 2000 Mar 1;91(1-2):369-73. Transient cardiac expression of the tinman-family homeobox gene, XNkx2-10. Newman CS, Reecy J, Grow MW, Ni K, Boettger T, Kessel M, Schwartz RJ, Krieg PA. Division of Molecular Cell and Developmental Biology, School of Biological Sciences, University of Texas at Austin, Austin 78712, USA. In Drosophila, the tinman homeobox gene is absolutely required for heart development. In the vertebrates, a small family of tinman-related genes, the cardiac NK-2 genes, appear to play a similar role in the formation of the vertebrate heart. However, targeted gene ablation of one of these genes, Nkx2-5, results in defects in only the late stages of cardiac development suggesting the presence of a rescuing gene function early in development. Here, we report the characterization of a novel tinman-related gene, XNkx2-10, which is expressed during early heart development in Xenopus. Using in vitro assays, we show that XNkx2-10 is capable of transactivating expression from promoters previously shown to be activated by other tinman-related genes, including Nkx2-5. Furthermore, Xenopus Nkx2-10 can synergize with the GATA-4 and SRF transcription factors to activate reporter gene expression. PMID: 10704867 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Circ Res. 1998 Mar 23;82(5):566-75. Evolutionarily conserved promoter region containing CArG*-like elements is crucial for smooth muscle myosin heavy chain gene expression. Zilberman A, Dave V, Miano J, Olson EN, Periasamy M. Division of Cardiology and Cardiovascular Research Center, University of Cincinnati, Ohio 45267, USA. In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5' flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between -967 and -1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation. PMID: 9529161 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------