Project description:The peptide hormone Urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Transcriptome data from Ucn3 null islets compared to wild type controls reveals selective depletion for delta cell markers and supports a role for Ucn3 as a trigger for somatostatin-mediated negative feedback.
Project description:The peptide hormone Urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Transcriptome data from Ucn3 null islets compared to wild type controls reveals selective depletion for delta cell markers and supports a role for Ucn3 as a trigger for somatostatin-mediated negative feedback. Isolated islets from Ucn3 knockout animals compared to wild type littermate controls. Islets from three indivual males were used for each genotype, lysed in Trizol for RNA isolation and library construction.
Project description:Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we explore the endogenous composition of the Sin3a-Hdac complex in pluripotent embryonic stem (ES) and differentiated cells. To do this, we established an endogenous double immunoprecipitation strategy coupled with quantitative mass spectrometry (ENDIP-MS) allowing us to define the precise composition of the Sin3a complex in multiple cell types. We identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, but not in differentiated cells. Fam60a co-occupies H3K4me3 positive promoters with Sin3a and is essential to maintain it on chromatin. Consistent with this, Fam60a depletion phenocopies the loss of Sin3a, leading to decreased proliferation, an extended G1-phase and the deregulation of genes associated with differentiation. Taken together, our data characterise Fam60a as an essential core subunit of a variant Sin3a complex in ES cells required to promote rapid proliferation and to prevent unscheduled differentiation.
Project description:mRNA expression analysis of arrested HCT116 cells (wild-type or SIN3B-/-) transfected with either non-targeting control siRNA or one of three SIN3A siRNAs and treated with Idasanulin to activate p53.
Project description:Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through multiple transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Gene expression in the whole testes of age-matched Ddx4(Vasa/Mvh)-cre mediated conditional Sin3a gene-targeted(VSKO) mice, germ cell-specific Sin3a hemizygous (HEMI) mice, and wild type (WT) mice was measured at postnatal day 0. Two biological replicate animals were used for each condition.
Project description:Circulating cell-free unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of β-cell death for the presymptomatic detection of diabetes. However, given the variability of CpG methylation in the INS gene in different cell types, this gene alone may not yield sufficiently specific information to unambiguously report β-cell damage. We employed an unbiased approach using data from a human DNA methylation gene array to identify the CHTOP gene as a candidate biomarker whose CpGs show a greater frequency of unmethylation in human islets. When tested across an array of non-islet human tissues by digital PCR, both INS and CHTOP contain unmethylated CpG sites in several of these tissues, but in a non-overlapping pattern: INS showed a slightly higher frequency of unmethylation in adipose tissue, whereas CHTOP appeared to be unmethylated in pancreas, brain, and skeletal muscle. Notably, INS and CHTOP genes are both unmethylated in human β and α cells, suggesting that each species at best would represent markers of islet cell death in general, and together might distinguish death arising from islets vs. other tissues. To validate unmethylated CHTOP as a biomarker for islet cell damage, we used digital PCR to measure cell-free circulating DNA in human populations. Compared to healthy controls, we observed that levels of differentially methylated CHTOP and INS were higher in youth with new onset type 1 diabetes and in healthy youth who have first-degree relatives with T1D. When tested in youth across a spectrum of metabolic dysfunction, increased levels of unmethylated INS and CHTOP were observed in obese individuals compared to lean controls. Together, these data suggest that simultaneous measurement of both circulating differentially methylated INS and CHTOP is likely to detect islet death in T1D, and raise new questions about β-cell health in populations at risk for both T1D and T2D. Importantly, our data support the use of multiple parameters to increase the accuracy of biomarkers of β-cell health in youth with diabetes.
Project description:Tyrosine phosphorylation is a hallmark for activation of Signal Transducer and Activator of Transcription (STAT) proteins, but their transcriptional activity also depends on other secondary modifications. Type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the Sin3a complex as an important mediator of this STAT3 transcriptional repression. Sin3a directly interacts with the DNA-binding domain of STAT3 and alters its acetylation status. SIN3A silencing enhances recruitment of STAT3 and enhanceosome components to the SOCS3 promoter, resulting in histone hyperacetylation and enhanced transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against Influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent STAT1/3 transcriptional switch.
Project description:SIN3A is the central scaffold protein of the SIN3/HDAC transcriptional repressor complex. We previously found that SIN3A participates in the mouse preimplantation development by finetuning HDAC1 expression. However, it remains unresolved if this functional significance of SIN3A was conserved in other mammals. The objective of this work was thus to characterize the expression profiles and the functional role of SIN3A in preimplantation development using non-rodent animal models. RNA-seq results show a large amount of SIN3A mRNA is present in oocytes and early embryos prior to embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in all species examined. Interestingly, immunofluorescence data show that SIN3A protein level peaks at 4-cell stage in pigs compared with morula stage in cattle, suggesting a differential role of SIN3A among species. Indeed, SIN3A depletion in early embryos causes a developmental arrest at 2-cell stage in pigs but does not affect bovine early embryonic development. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause 2-cell block, however, leads to a reduced blastocyst rate, suggesting the effect of SIN3A depletion on porcine early embryos is independent of HDAC1. By using un-biased RNA-seq approach, we found that CCNB1 transcript level is dramatically reduced. Moreover, CCNB1 knockdown results in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 into SIN3A-depleted embryos could partly rescue embryonic development beyond 2-cell stage. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, which probably involving the regulation of CCNB1 expression.