Transcriptional Autoregulatory Loops Are Highly Conserved in Vertebrate Evolution

BACKGROUND: Feedback loops are the simplest building blocks of transcriptional regulatory networks and therefore their behavior in the course of evolution is of prime interest. METHODOLOGY: We address the question of enrichment of the number of autoregulatory feedback loops in higher organisms. First, based on predicted autoregulatory binding sites we count the number of autoregulatory loops. We compare it to estimates obtained either by assuming that each (conserved) gene has the same chance to be a target of a given factor or by assuming that each conserved sequence position has an equal chance to be a binding site of the factor. CONCLUSIONS: We demonstrate that the numbers of putative autoregulatory loops conserved between human and fugu, danio or chicken are significantly higher than expected. Moreover we show, that conserved autoregulatory binding sites cluster close to the factors' starts of transcription. We conclude, that transcriptional autoregulatory feedback loops constitute a core transcriptional network motif and their conservation has been maintained in higher vertebrate organism evolution

Co-Activation of Nuclear Factor-kappaB and Myocardin/Serum Response Factor Conveys the Hypertrophy Signal of High Insulin Levels in Cardiac Myoblasts

Background and Objective: Hyperinsulinemia contributes to cardiac hypertrophy and heart failure in patients with the metabolic syndrome and type 2 diabetes. Here high circulating levels of tumor necrosis factor (TNF)-alpha may synergize with insulin in signaling inflammation and cardiac hypertrophy. We tested whether high insulin affects activation of TNF-alpha-induced NF-kappaB and myocardin/serum response factor (SRF) to convey hypertrophy signaling in cardiac myoblasts. Methods and Results: In canine cardiac myoblasts, treatment with high insulin (10-8-10-7 M) for 0-24 h increased insulin receptor substrate (IRS)-1 phosphorylation at Ser307, decreased protein levels of chaperone-associated ubiquitin (Ub) E3 ligase C terminus of heat shock protein 70-interacting protein (CHIP), increased SRF activity, as well as beta-myosin heavy chain (MHC) and myocardin expressions. Here siRNAs to myocardin or NF-kappaB, as well as CHIP overexpression prevented - while siRNA-mediated CHIP disruption potentiated - high insulin-induced SR element (SRE) activation and beta-MHC expression. Insulin markedly potentiated TNF-alpha-induced NF-kappaB activation Compared with insulin alone, insulin+TNF-alpha increased SRF/SRE binding and beta-MHC expression, which was reversed by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and by NF-kappaB silencing. In the hearts of db/db diabetic mice, in which Akt phosphorylation was decreased, p38MAPK, Akt1 and IRS-1 phosphorylation at Ser307 were increased, together with myocardin expression as well as SRE and NF-kappaB activities. Conclusion: In response to high insulin, cardiac myoblasts increase the expression or the promyogenic transcription factors myocardin/SRF in a CHIP-dependent manner. Insulin potentiates TNF-alpha in inducing NF-kappaB and SRF/SRE activities. In hyperinsulinemic states, myocardin may act as a nuclear effector of insulin, promoting cardiac hypertrophy

Striated muscle activator of Rho signalling (STARS) is reduced in ageing human skeletal muscle and targeted by miR-628-5p

Aim: The striated muscle activator of Rho signalling (STARS) is a muscle-specific actin-binding protein. The STARS signalling pathway is activated by resistance exercise and is anticipated to play a role in signal mechan-otransduction. Animal studies have reported a negative regulation of STARS signalling with age, but such regulation has not been investigated in humans. Methods: Ten young (18–30 years) and 10 older (60–75 years) subjects completed an acute bout of resistance exercise. Gene and protein expres-sion of members of the STARS signalling pathway and miRNA expression of a subset of miRNAs, predicted or known to target members of STARS signalling pathway, were measured in muscle biopsies collected pre-exer-cise and 2 h post-exercise. Results: For the first time, we report a significant downregulation of the STARS protein in older subjects. However, there was no effect of age on the magnitude of STARS activation in response to an acute bout of exer-cise. Finally, we established that miR-628-5p, a miRNA regulated by age and exercise, binds to the STARS 3’UTR to directly downregulate its tran-scription. Conclusion: This study describes for the first time the resistance exercise-induced regulation of STARS signalling in skeletal muscle from older humans and identifies a new miRNA involved in the transcriptional con-trol of STARS

GATA4 is essential for bone mineralization via ERα and TGFβ/BMP pathways

Osteoporosis is a disease characterized by low bone mass, leading to an increased risk of fragility fractures. GATA4 is a zinc-finger transcription factor that is important in several tissues, such as the heart and intestines, and has recently been shown to be a pioneer factor for estrogen receptor alpha (ERα) in osteoblast-like cells. Herein, we demonstrate that GATA4 is necessary for estrogen-mediated transcription and estrogen-independent mineralization in vitro. In vivo deletion of GATA4, driven by Cre-recombinase in osteoblasts, results in perinatal lethality, decreased trabecular bone properties, and abnormal bone development. Microarray analysis revealed GATA4 suppression of TGFβ signaling, necessary for osteoblast progenitor maintenance, and concomitant activation of BMP signaling, necessary for mineralization. Indeed, pSMAD1/5/8 signaling, downstream of BMP signaling, is decreased in the trabecular region of conditional knockout femurs, and pSMAD2/3, downstream of TGFβ signaling, is increased in the same region. Together, these experiments demonstrate the necessity of GATA4 in osteoblasts. Understanding the role of GATA4 to regulate the tissue specificity of estrogen-mediated osteoblast gene regulation and estrogen-independent bone differentiation may help to develop therapies for postmenopausal osteoporosis

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth.

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue we have focused our studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, we recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. We found the differences between Snail and Serpent to correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorki target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a ‘multi hit’ effect may be achieved through the aberrant activation of just a single gene

AMP-Activated Protein Kinase-Regulated Activation of the PGC-1α Promoter in Skeletal Muscle Cells

The mechanisms by which PGC-1α gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1α using AICAR, an activator of AMPK, that is known to increase PGC-1α expression. A 2.2 kb fragment of the human PGC-1α promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-κB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1α promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at −495 within the PGC-1α promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1α promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1α promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1α promoter activity. The USF-1-mediated increase in PGC-1α promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1α gene expression. This could represent a potential therapeutic target to control PGC-1α expression in skeletal muscle

Posttranscriptional regulation of transformation by human papillomavirus type 16 E7 and expression of this oncogene

Many epidemiological and experimental studies have strongly implicated human papillomavirus type 16 (IIPV-16) in cervical neoplasia. The oncogenic potential of this virus can be demonstrated by transformation of primary baby rat kidney (BRK) cells by cotransfection of the viral genome with the activated EJ-ras oncogene. I performed a mutational analysis of the viral genome to map the regions essential for its transforming activity. For the SV40-bascd early region expression plasmids, the disruption of the E6, E2, E5 and the 3' region of the El open reading frames (ORFs) did not affect the transforming activity of mutated plasmids, whereas the insertion of translation termination linkers within the E7 ORE abolished transformation. Additional sequences present in the 3'-flanking region of the E7 ORF were also required for efficient transformation. -- The 3' flanking region sequences were analyzed in detail in SV40-based E7 expression plasmids by progressive deletion analysis and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within this 3'-flanking region abolished transformation. Regeneration of the wild- type sequence in a previously transformation incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in higher levels of transformation, whereas mutating the + 2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the transformation-deficient splice site mutants accumulated lower levels of E7 RNA, primarily because of rapid destabilization of E7 RNA. -- The splice sites present within the E6 ORF were examined for their ability to substitute for the loss of nt 880 splice donor site function. The wild-type E6 splice sites, as well as the heterologous splice sites of the SV40 intron, were able to partially substitute for the nt 880 splice site function. These results indicated that the efficient accumulation of HPV-16 E7 RNA and transformation of BRK cells depend on the presence of an intron in the transcription unit. -- Recent studies have indicated the presence of naturally occurring HPV-16 antisense (AS) RNA in cervical carcinomas. I have detected AS E7 and E7/E1 RNA in Cos-1 cells transiently transfected with SV40-based HPV-16 early region expression plasmids. By deletion mutation analysis, the AS promoter was localized to nt 4031-4338 of HPV-16. Primer extension analysis and RNase protection assays indicated that the AS RNA was initiated from multiple sites in an AT-rich region around nt 4100. The AS promoter was active when cloned downstream of the chloramphenicol acetyl transferase (Cat) gene, giving rise to AS Cat RNA. -- AS E7 RNA was detected in both the nucleus and cytoplasm. The AS RNA formed a duplex with the sense (S) E7 RNA. The presence of AS RNA was correlated with reduced splicing of E7 RNA from the nt 880 splice site and the synthesis of E7 protein