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:We measured genome-wide gene expression of embryonic stem cells derived from two different inbred mouse genetic backgrounds. For each genetic background we also conducted an allele swap at SNP rs50454566 upstream of the Lifr gene. We profiled cells from each of the four strains in triplicate (technical replicates). We cultured cells in media with LIF + GSK3-beta inhibitor CHIR99021. Cells remained unfed until harvest, six days later.
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:CRISPR/Cas9 system was used to generate mediator complex subunit 1 (MED1) knockout human pre-B ALL cell line 697. RNA-seq was performed to observe the effects of MED1 deletion on gene expression in 697.
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:Lysine lactylation (Kla), a newly identified epigenetic mark triggered by lactate during glycolysis, including the Warburg effect, marks a pivotal juncture between metabolic pathways and gene regulation. The discovery of enzymes such as p300 and HDAC1/3 has been pivotal in deciphering the regulatory dynamics of Kla, though questions about additional regulatory enzymes, their specific Kla substrates, and the underlying functional mechanisms persist. Our investigation bridges these knowledge gaps by identifying SIRT1 and SIRT3 as key "erasers" of Kla, providing insights into their selective regulatory impact on both histone and non-histone proteins. Through a quantitative proteomic analysis in wildtype, SIRT1 knockout, and SIRT3 knockout HepG2 cells, we delineated a comprehensive landscape of Kla and lysine acetylation (Kac) sites. The results demonstrate a distinct specificity in the substrates modified by SIRT1 and SIRT3, underscoring their differentiated roles in cellular signaling pathways. Particularly, we highlight the role of specific Kla modifications, such as those on the M2 splice isoform of pyruvate kinase (PKM2), in modulating metabolic pathways and cell proliferation, thereby expanding the recognized implications of Kla beyond its epigenetic roles. Therefore, this study paves the way for deeper understanding of the functional phenotypes and mechanisms of Kla, offering new insights into its broader biological significance.