Biomedical subjects

P K Bhavsar

Publications and source records attributed to P K Bhavsar.

6 recordsLinked to original sources

PubMed2001

Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter.

OBJECTIVES: The expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative binding elements within the proximal promoter, a CACC-box sequence overlapping a consensus Sp1 element has been identified. The aim of this study was to characterise the factors binding to this element and to determine their importance in the transcriptional activity of the promoter. METHODS: A combination of supershift and competition electrophoretic mobility shift assays (EMSA) were used to identify the binding of factors to the overlapping CACC-box/Sp1 consensus element. The functional importance of this element was tested by transient transfection into primary neonatal rat cardiac myocytes in culture. RESULTS: At least four factors were able to interact with this region including the zinc finger proteins Sp1, Sp3 and two potentially novel factors. Whereas both Sp1 and Sp3 bound to the consensus Sp1 element, and to a lesser extent the CACC-box, two of the complexes required the intact CACC-box for binding. Site-directed mutagenesis of this region showed that the CACC-box is essential for hTnIc promoter-reporter activity. Further characterisation using EMSA indicated that the factors binding the hTnIc CACC-box are unlikely to be zinc finger proteins as they are insensitive to the addition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human and rat cardiac muscle but absent from a number of cell lines including several derived from skeletal muscle. CONCLUSION: The human cardiac troponin I gene promoter requires an upstream CACC-box element for full activity. This element binds at least two complexes which represent novel, tissue-restricted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors.

Animals↗

PubMed2000

Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter.

The human cardiac troponin I (TnIc) gene exhibits both cardiac-specific and developmentally regulated expression. The structure and expression of this gene as well as the identification of putative regulatory elements have been described previously. This study shows that a minimal promoter containing 98 bp of sequence is sufficient to drive transcription in neonatal rat cardiac myocytes. This region contains several putative cis -regulatory elements including an Initiator element surrounding the start site of transcription, an A/T-rich (TATA/MEF-2) element, two GATA elements and a cytosine-rich region containing overlapping CACC box and Sp1 elements. Using electrophoretic mobility shift assays (EMSAs) this study demonstrates the binding of MEF-2, Oct-1, and recombinant TBP to the A/T-rich element and of GATA-4 to both GATA elements. The CACC/Sp element binds the zinc finger transcription factors Sp1 and Sp3 in addition to an unidentified complex present in neonatal rat cardiac myocytes. Mutation of each of these sites has a deleterious effect on promoter activity as assayed by transient transfection into cardiac myocytes. The data suggest that transcriptional activity of the human TnIc gene can be driven by a compact promoter region and that within this region GATA, MEF-2 Sp1 and CACC box-binding factors are required for optimal activity. Furthermore, a comparison with data obtained for identical elements in the promoters of rodent TnIc genes identifies differences between species which may be of consequence for species-specific promoter function.

Animals↗

PubMed1999

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T.

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.

Blotting, Northern↗

PubMed1996

Isolation and characterization of the human cardiac troponin I gene (TNNI3).

Troponin I (TnI) is a constituent protein of the troponin complex located on the thin filament of striated muscle that provides a calcium-sensitive switch for striated muscle contraction. Unlike other contractile proteins, the cardiac isoform of troponin I (TnIc) is expressed only in cardiac muscle and therefore offers a model for cardiac-specific expression. It is also subject to developmental regulation with increased expression occurring at the time of birth. Here we describe the isolation and characterization of the human TnIc gene (HGMW-approved symbol TNNI3) and its promoter. The gene comprises eight exons contained within 6.2 kb of genomic DNA. The proximal promoter and 1.1-kb 5'-flanking region were sequenced, and several putative cis-acting elements that are conserved between the human and the mouse TnIc genes were identified. In addition, multiple copies of a 37-bp chromosome 19-specific mini-satellite sequence were identified within this region. Following transfection, 2300 bp of 5' sequence is active in both cardiac myocytes and skeletal muscle cells but is inactive in fibroblasts, indicating that it can drive expression but is insufficient to confer cardiac specificity.

Animals↗

PubMed1992

Gene expression during cardiac development.

The vertebrate heart forms as two concentric epithelial cylinders of myocardium and endocardium separated by an extended basement membrane matrix commonly referred to as cardiac jelly. Subsequent maturation involves a complex series of events including asymmetric changes in cell shape and division which contribute to bending and the formation of the bulboventricular loop, the formation of specialised tissues including endocardial cushion tissue of the atrioventricular (AV) and outflow tract regions, the development of conductive tissue and myocyte maturation leading to the overall pattern of expression characteristic of mature heart muscle. These processes depend on a precise spatial and temporal control of gene expression both of genes encoding regulatory molecules and those encoding structural components of the heart. In this chapter we address three aspects of cardiac development, namely, the determination of cell fate during formation of endocardial cushion tissue in the embryonic heart, transitions in troponin gene expression during fetal myocyte maturation, and the use of cloning techniques based on the polymerase chain reaction for identifying transcription factors present in the heart.

Animals↗

PubMed1991

Developmental expression of troponin I isoforms in fetal human heart.

We have used antibodies specific for troponin I proteins to examine human cardiac development and have detected a transiently expressed developmental isoform. This isoform is distinct from adult cardiac troponin I (TnIc) but is indistinguishable, on the basis of electrophoretic mobility and antibody reactivity, from the isoform found in slow skeletal muscle (TnIs). Furthermore, we show that mRNA for TnIs is present in fetal, but not adult, heart. Analysis of a developmental series of fetal samples indicates that there is a transition in expression from TnIs to TnIc which occurs between 20 weeks fetal and 9 months postnatal development.

Base Sequence↗