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:Lung cancer is the leading cause of cancer related death in both men and women in the United States. Recently, Smad4 was discovered to be common somatic alteration in human squamous cell lung cancer. Our goal was to delineate the role of Smad4 in lung cancer. We have shown for the first time that the ablation of Pten and Smad4 in the murine airway epithelium harbors a metastatic proximal adeno-squamous lung cancer. knockout group (PTENd/d and SMAD4d/d) and control group
Project description:To address the mechanism of action of EFA6B we have knocked-out its gene PSD4 using the CRISPR/Cas9 technology. The MCF10A human mammary cell line was used for the knock-out as it is a well-characterized normal human mammary cell line and thus enabled us to study the effect of deleting PSD4 in a non-transformed genetic background.
Project description:During canonical Wnt signalling the activity of nuclear beta-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones simultaneously carrying loss-of-function alleles of all four TCF/LEF genes. Exploiting unbiased whole transcriptome sequencing studies, we found that a subset of beta-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that beta-catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of beta-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as beta-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of beta-catenin that bypasses the TCF/LEF transcription factors.
Project description:Using a high-end mass spectrometry, we screened phosphoproteins and phosphopeptides in five types of Alzheimer's disease (AD) mouse models (5xFAD, APP-tg, PS1-tg, PS2-tg and APP-KI) and four types of frontotemporal lobar degeneration (FTLD) mouse models(CHMP2B-KI, PGRN-KI, VCP-KI and TDP43-KI) at multiple time points (1, 3 and 6 months).
Project description:The effect of the presence of HMEC-1 cells on HK-2 cell gene expression was investigated. Cells were cultured in mono or co-culture on opposite sides of aluminium oxide filters, 0.2 ?µM pore size. These conditions provide a close proximity but not direct contact. RNA from HK-2 cells was assayed with Affymetrix HGU133 Plus 2 arrays. 3 Biological samples were used per group.