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Heart failure is associated with alterations in cardiac and skeletal muscle energy metabolism resulting in a generalized myopathy. We investigated the molecular and cellular effects of angiotensin-converting enzyme inhibition (ACEi) on skeletal muscle metabolism in infarcted animals. Myocardial infarction (MI) was obtained by left descending coronary artery ligation. Sham, MI, and MI-treated rats (perindopril, 2 mg.kg(-1).day(-1) given 7 days after MI) were studied 1 and 4 mo after surgery. Oxygen consumption of white gastrocnemius (Gas) muscle was studied in saponin-permeabilized fibers, using the main substrates of mitochondrial respiration. mRNA expression of nuclear factors (PGC-1alpha, NRF-2alpha, and mtTFA), involved in the transcription of mitochondrial proteins, and of MCIP1, a marker of calcineurin activation, were also determined. Echocardiographic left ventricular fractional shortening was reduced in both MI and perindopril group after 1 and 4 mo, whereas systemic blood pressure was reduced by 16% only in the MI group after 4 mo. The capacity of Gas to oxidize glutamate-malate, glycerol-triphosphate, or pyruvate (-30%, P < 0.01; -32%, P < 0.05; -33%, P < 0.01, respectively), was greatly decreased. Furthermore, PGC-1alpha (-54%), NRF-2alpha (-45%), and MCIP1 (-84%) gene expression were significantly downregulated. ACEi improved survival, left ventricular function, and blood pressure. Perindopril protected also totally the Gas mitochondrial function and preserved the mRNAs concentration of the mitochondrial transcriptional factors. Moreover, PGC-1alpha correlated with Gas oxidative capacity (r = 0.48), mitochondrial cytochrome-c oxidase (r = 0.65), citrate synthase (r = 0.45) activities, and MCIP1 expression (r = 0.44). Thus ACEi totally prevented MI-induced alterations of skeletal muscle mitochondrial function and protein expression, halting the development of this metabolic myopathy.

Expression of CD18, the beta chain of the leukocyte integrins, is transcriptionally regulated by retinoic acid (RA) in myeloid cells. Full RA responsiveness of the CD18 gene requires its proximal promoter, which lacks a retinoic acid response element (RARE). Rather, RA responsiveness of the CD18 proximal promoter requires ets sites that are bound by GA-binding protein (GABP). The transcriptional coactivator, p300, further increases CD18 RA responsiveness. We demonstrate that GABPalpha, the ets DNA-binding subunit of GABP, physically interacts with p300 in myeloid cells. This interaction involves the GABPalpha pointed domain (PNT) and identifies p300 as the first known interaction partner of GABPalpha PNT. Expression of the PNT domain, alone, disrupts the GABPalpha-p300 interaction and decreases the RA responsiveness of the CD18 proximal promoter. Chromatin immunoprecipitation and chromosome conformation capture demonstrate that, in the presence of RA, GABPalpha and p300 at the proximal promoter recruit retinoic acid receptor/retinoid X receptor from a distal RARE to form an enhanceosome. A dominant negative p300 construct disrupts enhanceosome formation and reduces the RA responsiveness of CD18. Thus, proteins on the CD18 proximal promoter recruit the distal RARE in the presence of RA. This is the first description of an RA-induced enhanceosome and demonstrates that GABP and p300 are essential components of CD18 RA responsiveness in myeloid cells.

Nuclear respiratory factor-1 and nuclear respiratory factor-2 activate the transcription of several respiratory chain enzymes and are prime candidates for bigenomic coordinated regulation of cytochrome oxidase subunit genes from the two genomes. Peroxisome proliferator-activated receptor gamma coactivator 1 is a proposed coactivator of nuclear respiratory factor-1 and nuclear respiratory factor-2-dependent transcription, but its significance and function in neurons are unknown. Our current study indicates that nuclear respiratory factor-1, nuclear respiratory factor-2, and peroxisome proliferator-activated receptor gamma coactivator-1 are expressed in rat visual cortical neurons, and that neuronal activity directly regulates the protein and mRNA expressions of these factors after functional inactivation in vivo and in vitro. Changes in peroxisome proliferator-activated receptor gamma coactivator-1 preceded those of nuclear respiratory factor-1 and nuclear respiratory factor-2, suggesting that it may be the prime sensor of neuronal activity and its energy demand.

The GA-repeat binding protein (GABP) is a ubiquitous transcription factor involved in transcriptional regulation of genes encoding proteins involved in a variety of cellular processes including adipocyte differentiation, mitochondrial respiration, and neuromuscular signaling. GABP is composed of two subunits; the GABP alpha subunit is a member of the Ets-family of transcription factors, and the unrelated ankyrin repeat containing GABP beta subunit. We previously identified a bidirectional promoter directing the expression of Gabpa (GAA) gene in one direction and ATP Synthase Coupling Factor 6 (Atp5j) (CF6) gene in the other [Chinenov, Y., Coombs, C. and Martin, M. E., 2000a. "Isolation of a bi-directional promoter directing the expression of the mouse GABP alpha and ATP Synthase Coupling Factor 6 genes. Gene 261:311-320.]. In this study we characterize sequence elements and regulatory factors contributing to the promoter activities of the GAA/CF6 bidirectional promoter. The core of the GAA/CF6 bidirectional promoter is retained within a 400 bp sequence and contains four GABP binding sites, a Sp1/3 binding site and an YY1 binding site. Site-directed mutagenesis demonstrated that while no single factor binding site was essential for promoter activity in either direction, the GA1 site located proximal to the previously mapped transcription start sites functioned cooperatively with the other GABP binding sites and with the Sp1/3 and YY1 sites to provide transcriptional activation of the GAA and CF6 promoters. The other GABP sites and the Sp1/3 and YY1 binding sites were functionally redundant for basal promoter activities in both directions. Electrophoretic mobility shift assays identified multiple DNA-protein complexes containing GABP alpha, GABP beta, Sp1, Sp3 or YY1 proteins, including one ternary complex containing GABP alpha, GABP beta and Sp1 proteins. Binding of GABP to the GAA/CF6 bi-directional promoter provides the potential for autoregulation of GABP alpha expression and confirms the importance of GABP in the coordinate expression of respiratory chain components.

Previously, we reported that decreased brain-specific angiogenesis inhibitor 2 (BAI2) induced increased VEGF expression. The regulatory mechanisms for this process are not understood. Here we show that GA-binding protein gamma (GABPgamma) associates with the cytoplasmic domain of BAI2, and GABPalpha/gamma or GABPalpha/beta works as a transcriptional repressor of VEGF in SHSY5Y cells. Transcriptional activity of wild-type VEGF promoter was significantly increased in anti-sense BAI2-transfected cells, but not that of VEGF promoter harboring mutated GABP sites. In in vivo focal cerebral ischemia model, the decrease in BAI2 accompanied by decreased GABPalpha and GABPgamma elicited increased VEGF expression before the onset of HIF-1alpha. Our results point out that BAI2 controls VEGF transcription through GABP under normal conditions and cerebral ischemia.

DNA motifs, or cis-elements, are short nucleotide sequence patterns recognized by various transcription factors (TFs). In promoters, these TFs bind in a complex combinatorial manner in order to regulate the expression of a downstream gene. The combinatorial space is frequently large and difficult to manage since vertebrates have thousands of transcription factors and more than 20,000 genes. We introduce a computer program called CAYCE (Combinatorial AnalYsis of Cis-Elements) that systematically detects statistically overrepresented DNA motif association rules independent of Microarray information. CAYCE is an adaptation of the apriori algorithm traditionally used for association rule mining, but offers three significant advancements. (1) It analyzes multiple occurrences of an item, corresponding to multiple TF binding sites, (2) It compares results with a biologically relevant background, and (3), it provides p-values for straightforward statistical interpretation. CAYCE can be easily applied to any item-set data where the investigator is also interested in multiple occurrences of a single item, and/or overrepresentation of association rules compared with a background. Applying CAYCE to human promoters in 1% of the human genome, we discover that motif clusters containing five repetitions of SP1 are the most statistically significant.

Homeobox genes encode transcription factors that regulate embryonic development and postnatal events. Rhox5 (previously called Pem), the founding member of a homeobox gene cluster that we recently identified on the X chromosome, is selectively expressed in granulosa cells in the ovary and other somatic-cell types in other reproductive organs. In this report, we investigate its regulation in granulosa cells in the rat ovary. We found that Rhox5 expression in the ovary is governed by the Rhox5 distal promoter and is expressed at least as early as Day 5 postpartum. Rhox5 mRNA levels are regulated during the ovarian cycle, peaking before ovulation. Deletion analysis revealed a 25-nt element essential for distal promoter transcription in primary granulosa cells. This distal promoter element contains two ETS and one SP1 transcription-factor family binding sites that mutagenesis analysis indicated were essential for high-level transcription. This element was both necessary and sufficient for transcription, because it activated transcription when placed upstream of a heterologous minimal promoter. Cold competition and electrophoretic mobility shift assay studies demonstrated that SP1, SP3, and the ETS family transcription factor GABP bound this element. Dominant-negative forms of GABP and SP3 repressed distal promoter expression in primary rat granulosa, showing that these factors are crucial for Rhox5 expression. Cotransfection of dominant-negative mutants indicated that Rhox5 expression in granulosa cells is regulated by the c-Jun N-terminal protein kinase (JNK, MAPK8) and RAS pathways, which are known to be upstream of ETS family transcription factors. The discovery that Rhox5 expression in granulosa cells is regulated by MAPK pathways and ETS and SP1 family members provides an opportunity to understand how these regulatory pathways and factors collaborate to regulate gene expression during the ovarian cycle.

Aims/hypothesis: Dysfunction in mitochondrial oxidative phosphorylation plays a central role in insulin resistance and type 2 diabetes. Nuclear respiratory factor 1 (NRF1) is a transcription factor that acts on nuclear genes encoding respiratory subunits and components of the mitochondrial transcription and replication machinery. Thus, we investigated its genetic association with type 2 diabetes., Methods: The NRF1 gene was sequenced to identify polymorphisms in 24 Korean DNA samples and then common variants were genotyped in 766 patients with type 2 diabetes and 303 non-diabetic subjects., Results: Twelve single nucleotide polymorphisms and one insertion/deletion polymorphism were identified. Six common variants among them were genotyped in a larger study. Although three individual polymorphisms appeared to be associated with type 2 diabetes (g.-46350insdel A, g.+141G>T and g.+54529A>G), the effects were only marginal. However, a haplotype (H2) was associated with a decreased risk of type 2 diabetes and another haplotype (H4) was associated with an increased risk of type 2 diabetes (p values for the Haplo. Score test were 0.009 and 0.004, respectively)., Conclusions/interpretation: We demonstrated that two common haplotypes of NRF1 gene are associated with type 2 diabetes in the Korean population.