Expression of Rod-Derived Cone Viability Factor: Dual Role of CRX in Regulating Promoter Activity and Cell-Type Specificity

International audienceBACKGROUND: RdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina. METHODOLOGY/PRINCIPAL FINDINGS: In order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 5'-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 5'-deletion analysis identified the human -205/+57 bp and murine -351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human -393/+27 bp and murine -195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (-393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (-77 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role. CONCLUSIONS/SIGNIFICANCE: The regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity

Nontelomeric TRF2-REST Interaction Modulates Neuronal Gene Silencing and Fate of Tumor and Stem Cells

SummaryRemoval of TRF2, a telomere shelterin protein, recapitulates key aspects of telomere attrition including the DNA-damage response and cell-cycle arrest [1]. Distinct from the response of proliferating cells to loss of TRF2 [2, 3], in rodent noncycling cells, TRF2 inhibition promotes differentiation and growth [4, 5]. However, the mechanism that couples telomere gene-silencing features [6–8] to differentiation programs has yet to be elucidated. Here we describe an extratelomeric function of TRF2 in the regulation of neuronal genes mediated by the interaction of TRF2 with repressor element 1-silencing transcription factor (REST), a master repressor of gene networks devoted to neuronal functions [9–12]. TRF2-REST complexes are readily detected by coimmunoprecipitation assays and are localized to aggregated PML-nuclear bodies in undifferentiated pluripotent human NTera2 stem cells. Inhibition of TRF2, either by a dominant-negative mutant or by RNA interference, dissociates TRF2-REST complexes resulting in ubiquitin-proteasomal degradation of REST. Consequentially, REST-targeted neural genes (L1CAM, β3-tubulin, synaptophysin, and others) are derepressed, resulting in acquisition of neuronal phenotypes. Notably, selective damage to telomeres without affecting TRF2 levels causes neither REST degradation nor cell differentiation. Thus, in addition to protecting telomeres, TRF2 possesses a novel role in stabilization of REST thereby controlling neural tumor and stem cell fate

Identification of SOX9 Interaction Sites in the Genome of Chondrocytes

Our previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a "master switch" in this differentiation. Heterozygous mutations in SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies.The ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9.Our results provide a broad understanding of the strategies used by a "master" transcription factor of differentiation in control of the genetic program of chondrocytes

Myocardial ultrasonic tissue characterization in patients with thyroid dysfunction

<p>Abstract</p> <p>Background</p> <p>Structural myocardial abnormalities have been extensively documented in hypothyroidism. Experimental studies in animal models have also shown involvement of thyroid hormones in gene expression of myocardial collagen. This study was planned to investigate the ability of ultrasonic tissue characterization, as evaluated by integrated backscatter (IBS), to early identify myocardial involvement in thyroid dysfunction.</p> <p>Patients and Methods</p> <p>We studied 15 patients with hyperthyroidism (HYPER), 8 patients with hypothyroidism (HYPO), 14 patients with subclinical hypothyroidism (SCH) and 19 normal (N) subjects, who had normal LV systolic function. After treatment, 10 HYPER, 6 HYPO, and 8 SCH patients were reevaluated. IBS images were obtained and analyzed in parasternal short axis (papillary muscle level) view, at left ventricular (LV) posterior wall. The following IBS variables were analyzed: 1) the corrected coefficient (CC) of IBS, obtained by dividing IBS intensity by IBS intensity measured in a rubber phantom, using the same equipment adjustments, at the same depth; 2) cardiac cyclic variation (CV) of IBS - peak-to-peak difference between maximal and minimal values of IBS during cardiac cycle; 3) cardiac cyclic variation index (CVI) of IBS - percentual relationship between the cyclic variation (CV) and the mean value of IBS intensity.</p> <p>Results</p> <p>CC of IBS was significantly larger (p < 0.05) in HYPER (1.57 ± 0.6) and HYPO (1.53 ± 0.3) as compared to SCH (1.32 ± 0.3) or N (1.15 ± 0.27). The CV (dB) (HYPO: 7.5 ± 2.4; SCH: 8.2 ± 3.1; HYPER: 8.2 ± 2.0) and the CVI (HYPO: 35.6 ± 19.7%; SCH: 34.7 ± 17.5%; HYPER: 37.8 ± 11.6%) were not significantly different in patients with thyroid dysfunction as compared to N (7.0 ± 2.0 and 44.5 ± 15.1%).</p> <p>Conclusions</p> <p>CC of IBS was able to differentiate cardiac involvement in patients with overt HYPO and HYPER who had normal LV systolic function. These early myocardial structural abnormalities were partially reversed by drug therapy in HYPER group. On the other hand, although mean IBS intensity tended to be slightly larger in patients with SCH as compared to N, this difference was not statistical significant.</p

HbA1c as a risk factor for heart failure in persons with diabetes: the Atherosclerosis Risk in Communities (ARIC) study

Heart failure (HF) incidence in diabetes in both the presence and absence of CHD is rising. Prospective population-based studies can help describe the relationship between HbA1c, a measure of glycaemia control, and HF risk

Ethylene receptor ETR2 controls trichome branching by regulating microtubule assembly in Arabidopsis thaliana

The single-celled trichome of Arabidopsis thaliana is a widely used model system for studying cell development. While the pathways that control the later stages of trichome development are well characterized, the early signalling events that co-ordinate these pathways are less well understood. Hormones such as gibberellic acid, salicylic acid, cytokinins, and ethylene are known to affect trichome initiation and development. To understand the role of the plant hormone ethylene in trichome development, an Arabidopsis loss-of-function ethylene receptor mutant, etr2-3, which has completely unbranched trichomes, is analysed in this study. It was hypothesized that ETR2 might affect the assembly of the microtubule cytoskeleton based on analysis of the cytoskeleton in developing trichomes, and exposures to paclitaxol and oryzalin, which respectively act either to stabilize or depolymerize the cytoskeleton. Through epistatic and gene expression analyses it is shown that ETR2 is positioned upstream of CHROMATIN ASSEMBLY FACTOR1 and TRYPTICHON and is independent of the GLABRA2 and GLABRA3 pathways. These results help extend understanding of the early events that control trichome development and identify a signalling pathway through which ethylene affects trichome branching

Defining functional DNA elements in the human genome

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease