Co-activation of atrial natriuretic factor promoter by Tip60 and serum response factor

Tat-interactive protein 60 (Tip60) is a member of the MYST family of histone acetyltransferases (HATs). In addition to its HAT domain, Tip contains a heterochromatin-associated protein 1-like chromodomain and a zinc finger-like domain. Several alternative splice variants of Tip60 have been characterized, including full-length Tip60alpha, Tip60beta (which lacks exon V encoded by the Tip60 gene), and Tip55 (which encodes a novel 103-amino-acid C terminus). We report here that isoproteins recognized by a pan-Tip60 antibody are strongly and transiently expressed between embryonic days 8 and 11 in the embryonic mouse myocardium. A functional role for Tip60 isoproteins in cardiac myocyte differentiation is suggested by immunoprecipitation experiments showing that Tip60alpha, Tip60beta, and Tip55 can bind serum response factor (SRF) and by transient transfection assessments showing that Tip60 and SRF cooperatively activate the atrial natriuretic factor promoter. Although this combinatorial activity is inhibited by histone deacetylase 7, it was unexpectedly enhanced by point mutation of the HAT domain. Ablation of the chromodomain from Tip60beta caused derepression. These findings suggest that Tip60 modulates expression of SRF-dependent cardiac genes.     

Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF

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.     

SIRT1 negatively regulates the activities, functions, and protein levels of hMOF and TIP60

SIRT1 is a NAD(+)-dependent histone H4K16 deacetylase that controls several different normal physiologic and disease processes. Like most histone deacetylases, SIRT1 also deacetylates nonhistone proteins. Here, we show that two members of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, hMOF and TIP60, are SIRT1 substrates. SIRT1 deacetylation of the enzymatic domains of hMOF and TIP60 inhibits their acetyltransferase activity and promotes ubiquitination-dependent degradation of these proteins. Importantly, immediately following DNA damage, the binding of SIRT1 to hMOF and TIP60 is transiently interrupted, with corresponding hMOF/TIP60 hyperacetylation. Lysine-to-arginine mutations in SIRT1-targeted lysines on hMOF and TIP60 repress DNA double-strand break repair and inhibit the ability of hMOF/TIP60 to induce apoptosis in response to DNA double-strand break. Together, these findings uncover novel pathways in which SIRT1 dynamically interacts with and regulates hMOF and TIP60 through deacetylation and provide additional mechanistic insights by which SIRT1 regulates DNA damage response.     

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus (Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5'-UTR and a 638 bp 3'-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30's. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.     

Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an alpha-actin intronic enhancer

Expression of alpha-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have identified a conserved nuclear factor of activated T cells (NFAT) binding site that overlaps this CArG box and tested the hypothesis that this site plays a previously unrecognized role in regulating alpha-actin expression. A reporter construct prepared using a 56-bp region of the mouse alpha-actin first intron containing SRF, NFAT, and AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal thymidine kinase promoter. Basal reporter activity following expression in SMCs was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin A and FK506. Mutating either the NFAT or SRF binding site essentially abolished reporter activity, suggesting that both NFAT and SRF binding are required. Basal activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-independent and approximately 8-fold lower than that in SMCs. Activation of NFAT in HEK293 cells induced an approximately 4-fold increase in activity that was dependent on the integrity of both NFAT and SRF binding sites. NFATc3.SRF complex formation, demonstrated by co-immunoprecipitation, was facilitated by the presence of SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased alpha-actin expression in cultured SMCs, suggesting that the molecular interaction of NFAT and SRF at SNAP may be physiologically relevant. These data provide the first evidence that NFAT and SRF may interact to cooperatively regulate SMC-specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.