Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity
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ABSTRACT: We analyzed function of DHX38 helicase in RNA splicing. We downregulated DHX38 in HEK293 cells by RNAi and compared the transcriptome of DHX38 knockdown cells with cells treated with negative control siRNA.
Project description:Pericentral retinitis pigmentosa (RP) is an atypical form of RP that affects the near-peripheral retina first and tends to spare the far periphery. This study was performed to further define the genetic basis of this phenotype. We identified a cohort of 43 probands with pericentral RP based on a comprehensive analysis of their retinal phenotype. Genetic analyses of DNA samples from these patients were performed using panel-based next-generation sequencing, copy number variations, and whole exome sequencing (WES). Mutations provisionally responsible for disease were found in 19 of the 43 families (44%) analyzed. These include mutations in RHO (five patients), USH2A (four patients), and PDE6B (two patients). Of 28 putatively pathogenic alleles, 15 (54%) have been previously identified in patients with more common forms of typical RP, while the remaining 13 mutations (46%) were novel. Burden testing of WES data successfully identified HGSNAT as a cause of pericentral RP in at least two patients, suggesting it is also a relatively common cause of pericentral RP. While additional sequencing might uncover new genes specifically associated with pericentral RP, the current results suggest that genetically pericentral RP is not a separate clinical entity, but rather is part of the spectrum of mild RP phenotypes.
Project description:Estrogen Receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1, contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1 co-operating transcription factor mutated in breast tumors, however its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of co-factors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1 binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3 mediated re-distribution of ESR1 binding. The GATA3-mediated re-distributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1 bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen- ESR1 mediated interactions between cis-regulatory elements. Together these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer. GATA and ER binding studied by chromatin immunoprecipitation in breast cancer cell lines, with and without estrogen stimulation and by knocking down GATA
Project description:The X-linked RP3 locus codes for retinitis pigmentosa GTPase regulator (RPGR), a protein of unknown function with sequence homology to the guanine nucleotide exchange factor for Ran GTPase. We created an RPGR-deficient murine model by gene knockout. In the mutant mice, cone photoreceptors exhibit ectopic localization of cone opsins in the cell body and synapses and rod photoreceptors have a reduced level of rhodopsin. Subsequently, both cone and rod photoreceptors degenerate. RPGR was found normally localized to the connecting cilia of rod and cone photoreceptors. These data point to a role for RPGR in maintaining the polarized protein distribution across the connecting cilium by facilitating directional transport or restricting redistribution. The function of RPGR is essential for the long-term maintenance of photoreceptor viability.
Project description:Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RP) is the most common cause of RP in North America. There is no proven cure for the disease, and multiple approaches are being studied. Gene therapy is an evolving field in medicine and ophthalmology. In this review, we will go over the basic concept of gene therapy and the different types of gene therapy that are currently being studied to treat this disease.
Project description:We performed total RNA-seq in the A549 cell line. Cells were treated with either 100 nM dexamethasone or 0.1% ethanol. After 4 hours, cells were washed 2x with PBS and cultured in hormone-free medium for 24 hours, after which cells were treated again for 4 hours with either 100 nM dexamethasone or 0.1% ethanol.
Project description:Expression of SET7/9, a histone methyltransferase, was frequently reduced in cultured and primary gastric cancers. To identify the SET7/9 target genes in gastric cancer, we performed siRNA-based knockdown of SET7/9 in MKN74 and MKN45 cells and then examined significant expression changes of genes by microarray. Transfection of SET7/9 siRNA into MKN74 and MKN45 cells were performed by electroporation. After 48hrs, cells were harvested. Total RNA was used for cDNA microarray.
Project description:Histone H3 lysine 9 tri-methyltransferases (H3K9me3) are related to transcriptional gene silencing. Although SETDB2 has H3K9me3 activity, it is unknown whether SETDB2 is linking to carcinogenesis. Here, we studied alterations and functions of SETDB2 in gastric cancers (GCs). In human clinical samples, overexpression of SETDB2 protein was observed in 30 of 72 (41.7%) primary GC tissues compared with their normal counterparts, and significantly associated with poor prognosis of the patients (P<0.05). SETDB2 protein was significantly detected in late stage of GCs. Moreover, SETDB2 protein was strongly expressed in four (30.8%) of 13 GC cell lines, and knockdown of SETDB2 led to decrease the cell proliferation, migration and invasion. According to the microarray analysis on a GC cell line after knockdown of SETDB2, the expression of WWOX and CADM1 tumor suppressor genes was significantly up-regulated. ChIP analysis showed that the H3K9me3 levels at the promoter regions of WWOX and CADM1 genes were closely regulated by the SETDB2 in GC cells. We also found that SETDB2 bound to the promoter regions after SETDB2 overexpression. Our data suggest that SETDB2 is associated with transcriptional repression of WWOX and CADM1, through H3K9me3, and hence overexpression of SETDB2 may contribute to gastric progression. Transfection of SETDB2 siRNA into MKN74 cells were performed by electroporation. After 48hrs, cells were harvested. Total RNA was used for cDNA microarray.
Project description:Retinitis pigmentosa (RP) is a group of inherited disorders affecting 1 in 3000-7000 people and characterized by abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium of the retina which lead to progressive visual loss. RP can be inherited in an autosomal dominant, autosomal recessive or X-linked manner. While usually limited to the eye, RP may also occur as part of a syndrome as in the Usher syndrome and Bardet-Biedl syndrome. Over 40 genes have been associated with RP so far, with the majority of them expressed in either the photoreceptors or the retinal pigment epithelium. The tremendous heterogeneity of the disease makes the genetics of RP complicated, thus rendering genotype-phenotype correlations not fully applicable yet. In addition to the multiplicity of mutations, in fact, different mutations in the same gene may cause different diseases. We will here review which genes are involved in the genesis of RP and how mutations can lead to retinal degeneration. In the future, a more thorough analysis of genetic and clinical data together with a better understanding of the genotype-phenotype correlation might allow to reveal important information with respect to the likelihood of disease development and choices of therapy.
Project description:Retinitis pigmentosa (RP) is a family of inherited disorders caused by the progressive degeneration of retinal photoreceptors. There is no cure for RP, but recent research advances have provided promising results from many clinical trials. All these therapeutic strategies are focused on preserving existing photoreceptors or substituting light-responsive elements. Vision recovery, however, strongly relies on the anatomical and functional integrity of the visual system beyond photoreceptors. Although the retinal structure and optic pathway are substantially preserved at least in early stages of RP, studies describing the visual cortex status are missing. Using a well-established mouse model of RP, we analyzed the response of visual cortical circuits to the progressive degeneration of photoreceptors. We demonstrated that the visual cortex goes through a transient and previously undescribed alteration in the local excitation/inhibition balance, with a net shift towards increased intracortical inhibition leading to improved filtering and decoding of corrupted visual inputs. These results suggest a compensatory action of the visual cortex that increases the range of residual visual sensitivity in RP.