Project description:ATF6 is a key regulator of the unfolded protein response. Through use of zebrafish and cultured cells we demonstrate that ATF6 drives fatty liver disease by interaction with fatty acid synthase (FASN). Total small RNA from livers of 5 dpf larval zebrafish were collected: 2 batches of Tg(fabp10:nls-mCherry) control larvae, 2 batches of ethanol-treated Tg(fabp10:nls-mCherry) larvae, and 1 batch of Tg(fabp10:nAtf6-cherry; cmlc2:GFP). Each batch was purified for preparation of high-throughput sequencing libraries.
Project description:Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1, also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution, with at any given time a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage likely under pressure of host restriction factors, which act notably by silencing L1 expression during early embryogenesis. Here, we demonstrate that in human embryonic stem cells (hESC) KAP1, the master co-factor of KRAB-containing zinc finger proteins (KRAB-ZFP) previously implicated in the restriction of endogenous retroviruses, represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 and 7.6 million years ago. In the mouse, we documented a similar chronologically conditioned pattern, albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP, suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hESC induced the expression of KAP1-bound L1 elements, but their younger, human-specific counterparts (L1Hs) were unaffected. Instead, they were stimulated by depleting DNA methyltransferases, consistent with recent evidence demonstrating that the PIWI-piRNA pathway regulates L1Hs in hESC. Altogether, these data indicate that the early embryonic control of L1 is an evolutionary dynamic process, and support a model whereby newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms, before KAP1-recruiting protein repressors are selected. HA-tagged Gm6871 ChIP-seq in mES cells, RNA-seq in control and Gm6871 KD mES cells, KAP1 ChIP-seq in WT mES cells, RNA-seq in control and DNMTs KD hES cells.
Project description:Transcriptomic analysis was performed to gain further insights into the molecular and cellular mechanisms underlying cardiac pathology.
Project description:Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2 (IKK2-CA). In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b+ cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. To gain insight into proximal events after activation of IKK2/NF-κB, we isolated cardiomyocytes and activated the transgene in vitro, thus avoiding interference of other cell types. Gene expression analysis revealed up-regulation of transcripts of chemotactic cytokines, adhesion molecules, and a striking number of IFN-regulated genes in cardiomyocytes expressing IKK2-CA. In addition, apoptotic and anti-apoptotic genes were up-regulated.
Project description:Reverse transcription-derived sequences account for at least half of the human genome. Although these retroelements are formidable motors of evolution, they can occasionally cause disease, and accordingly are inactivated during early embryogenesis through epigenetic mechanisms. In the mouse, at least for endogenous retroviruses, important mediators of this process are the tetrapod-specific KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor TRIM28. The present study demonstrates that KRAB/TRIM28-mediated regulation is responsible for controlling a very broad range of human-specific endogenous retroelements (EREs) in human embryonic stem (ES) cells and that it exerts, as a consequence, a marked effect on the transcriptional dynamics of these cells. It further reveals reciprocal dependence between TRIM28 recruitment at specific families of EREs and DNA methylation. It finally points to the importance of persistent TRIM28-mediated control of ERE transcriptional impact beyond their presumed inactivation by DNA methylation. Analyses of epigentic effectors and marks in KAP1 WT and KD human embryonic stem cells
Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.
Project description:Here, we characterize the transcriptome of the mouse embryonic stem cell line CM7-1 during differentiation into beating cardiomyocytes and compared the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium.
Project description:Morphogenesis requires the dynamic regulation of gene expression, including transcription, mRNA maturation and translation. Dysfunction of general components of the splicing machinery can cause surprisingly specific phenotypes, but the basis for these cell-type specific effects is not clear. Here we show that the faint sausage (fas) locus, implicated in epithelial morphogenesis and previously reported to encode a secreted immunoglobulin domain protein, in fact encodes a subunit of the Prp19 complex that is essential for efficient pre-mRNA splicing. Loss of zygotic fas function impairs the efficiency of splicing, associated with widespread retention of introns in mature mRNAs and dramatic changes in gene expression. Surprisingly, despite these general effects, zygotic fas mutants show specific defects in tracheal cell migration. Zygotic fas function becomes essential during late embryogenesis when maternally supplied splicing factors decline. We propose that tracheal branching, which relies on dynamic changes in gene expression, is particularly sensitive for efficient spliceosome function. Our results provide an entry point to study functions of splicing during organogenesis and provide a better understanding of specific disease phenotypes associated with mutations in general splicing factors.