Thyroid hormone regulation of alpha-myosin heavy chain promoter activity assessed by in vivo DNA transfer in rat heart

We have studied the thyroid-hormone responsiveness of the alpha-myosin heavy chain (MHC) gene in vivo by directly injecting an expression vector containing the alpha-MHC 5' regulatory sequences (-613 to +421 base pairs) into the rat heart. In the expression vector pAM1Luc the alpha-MHC promoter elements direct the synthesis of firefly luciferase. Although thyroxine administration of both euthyroid and thyroidectomized rats for 5 days increased alpha-MHC promoter activity, the pAM1Luc gene construct did not mimic expression of the endogenous gene. These studies of direct gene transfer into mammalian myocardium suggest that additional cis-acting elements necessary for the in vivo response to thyroid hormone reside outside the -613 to +421 region.     

An anti-sense c-erbA clone inhibits thyroid hormone-induced expression from the alpha-myosin heavy chain promoter.

The effects of thyroid hormone on expression of cardiac myosin heavy chain genes generally are thought to be mediated by nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors that have been identified as the products of the protooncogene, c-erbA. This hypothesis has been tested by transfection of cardiomyocytes in primary culture with a plasmid, pRSVhEACAT-, expressing anti-sense c-erbA mRNA. Because only a low percentage of cells (20%) could be transfected in primary culture an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct was used as a reporter gene. The results indicate that the anti-sense plasmid almost completely blocks T3-induced activity of the reporter gene (less than 1% control) while transfection of a similar amount of the sense construct, pRSVhEACAT+, has no effect. When the c-erbA plasmids were cotransfected with constructs containing T3-independent promoters, no effects on expression were observed. The combined use of an anti-sense construct and a report gene provides a means of studying the role of c-erbA products in intracellular signal transduction even in differentiated, nondividing cells like those of the heart.     

Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.     

Characterization of the allelic differences in the mouse cardiac alpha-myosin heavy chain coding sequence.

We have obtained and sequenced the coding sequence of the mouse cardiac alpha-myosin heavy chain (Myhc alpha) from the A/J, BALB/cByJ, C57BL/6J, and DBA/2J inbred mouse strains. Overlapping cDNA sequences were obtained using RNA-PCR and anchor-PCR techniques for these studies. In the A/J mouse strain, the full-length message is 5989 bp long and encodes for a protein consisting of 1938 amino acids (Mr 223,689). The protein deduced sequence of the A/J Myhc alpha was compared with corresponding sequences of human and rat Myhc alpha and beta. These results demonstrated that the mouse Myhc alpha is highly conserved and has maintained the alpha-isoform-specific divergent cluster observed in other Myhc alpha proteins. One difference was the loss of a glutamine at residue 1932, which is due to a change in an RNA splicing site sequence. Allelic variability was observed in both nucleotide and amino acid sequences among the four different inbred mouse strains and generally appears to be random in nature. Three of the nucleotide changes resulted in a different amino acid, while the remaining 46 were silent substitutions.     

Identification of Yin Yang 1-interacting partners at −1026C/A in the human iNOS promoter

Previously we demonstrated the association between human iNOS −1026C/A variant and susceptibility to hypertension, and found that −1026C/A altered the Yin Yang 1 (YY1)-binding pattern. In the current study, we verified that −1026C/A was located in a vital regulatory region of the iNOS promoter, wherein existed a DNA-binding complex composed of YY1, nuclear factor I (NFI) and activator protein-1 (AP-1). We also observed that YY1 bound dominantly to −1026C, and NFI bound dominantly to −1026A. Furthermore, the repressive effect of YY1 was more evident than NFI on the iNOS promoter activity, resulting in a more marked reduction of iNOS expression via YY1/AP-1 than via NFI/AP-1 under the stimulation of cytomix. In conclusion, diverse binding affinities of YY1 and its interacting partners to iNOS −1026C/A resulted in differential promoter activity, and potent inhibition of iNOS expression by YY1/AP-1 complex with −1026C may contribute to an enhanced risk for hypertension.