Project description:The histological grade of carcinomas describes the ability of tumor cells to organize differentiated epithelial structures and has prognostic impact. Molecular control of differentiation in normal and cancer cells relies on lineage-determining transcription factors (TFs) that activate the repertoire of cis-regulatory elements controlling cell type-specific transcriptional outputs. TF recruitment to cognate genomic DNA binding sites results in the deposition of histone marks characteristic of enhancers and other cis-regulatory elements. Here we integrated transcriptomics and genome-wide analysis of chromatin marks in human pancreatic ductal adenocarcinoma (PDAC) cells of different grade to identify first, and then experimentally validate the sequence-specific TFs controlling grade-specific gene expression. We identified a core set of TFs with a pervasive binding to the enhancer repertoire characteristic of differentiated PDACs and controlling different modules of the epithelial gene expression program. Defining the regulatory networks that control the maintenance of epithelial differentiation of PDAC cells will help determine the molecular basis of PDAC heterogeneity and progression. Poly(A) fraction of the total RNA from human pancreatic ductal adenocarcinoma cell lines was extracted and subjected to by multiparallel sequencing. Experiments were carried out in unmodified cells in duplicate, genome edited clonal CFPAC1 cells (2 KLF5-deleted CRISPR-Cas9 clones, 3 ELF3-deleted CRISPR-Cas9 clones and 2 wt clones) and CFPAC1 cells ectopically expressing ZEB1 or empty vector control (in duplicate).
Project description:MICU1 is a Ca2+-binding protein that regulates the mitochondrial Ca2+ uniporter channel (mtCU ) and mitochondrial Ca2+ (mCa2+) uptake. MICU1 knockout mice display perinatal lethality and disorganized mitochondrial architecture. These phenotypes are distinct from other mtCU loss-of-function models and thus are not explained by changes in mCa2+ content. Utilizing multiple proteomic approaches, we found that MICU1 localized to mitochondrial complexes lacking MCU, suggesting that MICU1 has cellular functions independent of mCa2+ uptake. The overall aim of the current project is to identify the global and mtCU independent MICU1 interactome to characterize the MCU independent functions of the MICU1.
Project description:SARS-CoV-2 infection causes COVID-19, a severe acute respiratory disease associated with cardiovascular complications including long-term outcomes. The presence of virus in cardiac tissue of patients with COVID-19 suggests this is a direct, rather than secondary, effect of infection.Here, by expressing individual SARS-CoV-2 proteins in the Drosophila heart, we demonstrate interaction of virus Nsp6 with host proteins of the MGA/MAX complex (MGA, PCGF6 and TFDP1).
Project description:We sought understand the molecular mehcanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS through whole transcriptome analysis of strains with phosphorylation mimicking substitutions in key histidine residues of Mga. There were 6 strains analyzed, each in triplicate replicates: 1)wild-type GAS, 2)mga deletion strain 3)mga with alanine at H207 4)mga with aspartate at H207, 5) mga with alanine at H273 6)mga with aspartate at H273
Project description:ELMOD2 cDNA from the IMAGE clone 3897166 was cloned into the pDsRed-Monomer-N1 expression vector. ELMOD2 was overexpressed in the A549(adenomcarcinoma)cell line and cells transfected with empty vector were considered controls. Transcript profiles were compared with the Affymetrix Hgu133plus2 arrays. Three biological replicates of each condition were included.
Project description:Microarray was done on heart tissue from ko and wt; MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the dosage of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals is unknown, and reliable prediction of mRNA targets remains problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets. Experiment Overall Design: Heart tissues from 3 wild type and 3 miR-1-2 knockout mice at postnatal days 10 were used and total RNA was extracted by Trizol. Expression level was compared between wild type and miR-1-2 knockout mice. The affy package from R/Bioconductor was used to generate RMA values.
Project description:Analysis of gene expression (mRNA profiles) from mouse Erythroblasts. Wild-type samples<br>are compared against samples where the miR-451/144 miRNA cluster has been knocked out. <br>Two cell types are analysed, in vitro cultured erythoblasts and ex vivo isolated erythoblasts.