No abstract

Math5, a mouse homolog of the Drosophila proneural bHLH transcription factor Atonal, is essential in the developing retina to establish retinal progenitor cell competence for a ganglion cell fate. Elucidating the mechanisms by which Math5 influences progenitor cell competence is crucial for understanding how specification of neuronal cell fate occurs in the retina and it requires knowledge of the downstream target genes that depend on Math5 for their expression. To date, only a handful of genes downstream of Math5 have been identified. To better define the gene network operating downstream of Math5, we used custom-designed microarrays to examine the changes in embryonic retinal gene expression caused by deletion of math5. We identified 270 Math5-dependent genes, including those that were expressed specifically either in progenitor cells or differentiated ganglion cells. The ganglion cell-specific genes included both Brn3b-dependent and Brn3b-independent genes, indicating that Math5 regulates distinct branches of the gene network responsible for retinal ganglion cell differentiation. In math5-null progenitor cells, there was an up-regulation of the proneural genes math3, neuroD, and ngn2, indicating that Math5 suppresses the production of other cell types in addition to promoting retinal ganglion cell formation. The promoter regions of many Math5-dependent genes contained binding sites for REST/NRSF, suggesting that release from general repression in retinal progenitor cells is required for ganglion cell-specific gene activation. The identification of multiple roles for Math5 provides new insights into the gene network that defines progenitor cell competence in the embryonic retina.

Brn3b/Brn-3.2/POU4f2 is a POU domain transcription factor that is essential for retinal ganglion cell (RGC) differentiation, axonal outgrowth and survival. Our goal was to establish a link between Brn3b and the downstream events leading to RGC differentiation. We sought to determine both the number and types of genes that depend on Brn3b for their expression. RNA probes from wild-type and Brn3b-/- E14.5, E16.5 and E18.5 mouse retinas were hybridized to a microarray containing 18,816 retina-expressed cDNAs. At E14.5, we identified 87 genes whose expression was significantly altered in the absence of Brn3b and verified the results by real-time PCR and in situ hybridization. These genes fell into discrete sets that encoded transcription factors, proteins associated with neuron integrity and function,and secreted signaling molecules. We found that Brn3b influenced gene expression in non RGCs of the retina by controlling the expression of secreted signaling molecules such as sonic hedgehog and myostatin/Gdf8. At later developmental stages, additional alterations in gene expression were secondary consequences of aberrant RGC differentiation caused by the absence of Brn3b. Our results demonstrate that a small but crucial fraction of the RGC transcriptome is dependent on Brn3b. The Brn3b-dependent gene sets therefore provide a unique molecular signature for the developing retina.

Introduction Genomic and transcriptomic alterations affecting key cellular processes such us cell proliferation, differentiation and genomic stability are considered crucial for the development and progression of cancer. Most invasive breast carcinomas are known to derive from precursor in situ lesions. It is proposed that major global expression abnormalities occur in the transition from normal to premalignant stages and further progression to invasive stages. Serial analysis of gene expression (SAGE) was employed to generate a comprehensive global gene expression profile of the major changes occurring during breast cancer malignant evolution.

RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV-Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.One of the most important types of nucleic acid structure is the G-quadruplex (G4), which is formed from four guanine bases by stacking of Hoogsteen bonded G-quartets 1 . The G-quadruplexes play a vital role in the human genome and transcriptome 2,3 . The DNA or RNA G-quadruplexes that are formed in cells are associated with many important cellular processes 4-8 . G-quadruplexes have been viewed as emerging therapeutic targets due to their correlation with human diseases [8][9][10][11][12] . Most of the early studies of G-quadruplexes focused on DNA strands, and few studies focused on RNA G-quadruplexes. Recently, RNA G-quadruplexes have been associated with many biological processes, such as telomere maintenance, pre-mRNA splicing and polyadenylation, RNA turnover, mRNA targeting and translation 13 . Therefore, the development of techniques that could efficiently recognize RNA G-quadruplexes structures and investigate their biological functions and impacts is highly desirable. Many techniques including X-ray crystallography and NMR experiments have been utilized to identify high-resolution structures of RNA G-quadruplexes 14,15 . However, these techniques are more suitable for comprehensively studying targeted RNA G-quadruplex structures. Additionally, circular dichroism (CD) has been extensively used to monitor G-quadruplex formation because the positive band at 264 nm and the negative band at 240 nm indicate the formation of parallel-type G-quadruplex structures 16 . However, it is difficult to interpret the G-quadruplex type in the presence of different forms of nucleic acids. Because the probe recognition sites in RNA G-quadruplexes are different from those in other RNA motifs, small molecules that selectively bind to RNA G-quadruplexes and emit fluorescence may be used as RNA G-quadruplex detectors. Our previous study revealed a cyanine dye (CyT) that was able to selectively recognize RNA G-quadruplex structures with ~2000-fold fluorescence enhancement 17 . Due to the important biological functions of RNA G-quadruplex, the development of a multiband probe that can selectively recognize RNA G-quadruplex structures is needed to further expand the application of RNA G-quadruplexes, which have been recognized as significant molecular...