Project description:Implantation is dependent on synchronized interactions between the conceptus and surrounding decidual cells but the involvement of clock genes in this process is not well understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK and BMAL1 and repressors encoded by PER and CRY genes. Here we show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Downregulation was preceded by reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter and occurred between 12 - 24 h after exposure to a deciduogenic stimulus. RNA sequencing revealed that premature inhibition of PER2 by siRNA knockdown leads to a grossly disorganised decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators amongst the 1,121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of mid-luteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure. Thus, PER2 synchronizes mitotic expansion of HESCs with a periodic decidual gene expression; uncoupling of these events may cause persistent pregnancy failure. Endometrial mRNA profiles of paired control (siRNA-NT) and siRNA-PER2 were generated by deep sequencing, in triplicate using Illumina
Project description:To date there are very few tools to reverse the induced dedifferentiation program in CRPC and to improve the response to the androgen deprivation therapy. Here we report that MAT2A is an important oncogenic cofactor of ERG/EZH2 transcriptional reprogramming impacting significantly the androgenic pathway. Using RNA sequencing coupled with ATAC, here we reveal an important link between ERG/MAT2A and EZH2 that impact on AR signaling pathway. This aberrant epigenetic program can be reversed by MAT2A inhibition which establish a near physiologic AR transcriptional program. Targeting MAT2A alone or in combination with EZH2 inhibitors reverse stemness in multiple models including prostatospheres from human PDX and GEM models of aggressive prostate cancer. Targeting MAT2A enhance the sensitivity to the androgenic blockade by Enzalutamide and to EZH2 inhibitors
Project description:Conversion between phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate on endosomal membranes is critical for maturation of early endosomes to late endosomes/lysosomes, and is regulated by the PIKfyve-Vac14-Fig4 complex. In this study, we screened for the cellular interactome of Vac14 and Fig4 using proximity-dependent biotin labelling (BioID) in 293T cells.
Project description:CSF-1R is recruited on EGR1 motifs in monocytes where it colocalizes with EGR1. To address if EGR1 required for CSF-1R recruitment on chromatin, THP-1 monocytic cell line has been deleted for EGR1 by CRISPR-Cas9 approach. 3 clones were generated by single cell cloning and CSF-1R localization on chromatin was compared to two unmodified THP-1 clones by ChIP-sequencing. Since the read number was strongly decreased in the EGR1-deleted clones, the three clones were pooled for the comparison with wild-type clones. ChIPseq of CSF-1R (Nter Antibody) in monocytes of two CMML patients (CMML2130 and CMML2609)
Project description:Lariat is formed by excised intron, in which the 5' splice site joints with the branchpoint (BP) during splicing. Although lariat RNAs are usually degraded by DBR1 (RNA debranching enzyme 1), recent findings in animals showed that many lariat RNAs were widely detected under physiological conditions. In contrast, the features of BPs and to what extent lariat RNAs accumulate naturally are largely unexplored in plants. Here, we analyzed 948 RNA sequencing datasets to document plant BPs and lariat RNAs on a genome-wide scale. In total, we identified 13872, 5199, 29582, and 13478 BPs in Arabidopsis, tomato, rice, and maize, respectively. Features of plant BPs are highly similar to those in yeast and human, in that BPs are adenine-preferred and flanked by uracil-enriched sequences. Intriguingly, ~20% of introns harbor multiple BPs, and BP usage is tissue-specific. Furthermore, 10580 lariat RNAs accumulate in wild type Arabidopsis plants, and most of these lariat RNAs originate from longer or retroelement-depleted introns, and the expression of these lariat RNAs significantly correlated with the incidence of back-splicing of parent exons. Collectively, our results provide the first comprehensive map of intron BPs and lariat RNAs in plants, and uncover a novel link between lariat turnover and splicing.
Project description:Introns in pre-mRNAs must be spliced out prior to their translation. During splicing, introns are removed in the form of a lariat, in which the 5' end is linked to the 2' hydroxyl of an internal adenosine. Lariat degradation is initiated by an 2'-5' phosphodiester-specific RNA endonuclease which debranches these lariat RNAs to linear form. Deletion of the debranching enzyme is yeast results in the accumulation of lariat introns. We used this accumulation to identify spliced lariat introns on a genome-wide scale in S. cerevisiae using tiling microarrays. Keywords: two sample comparison, 3 biological replicates
Project description:We performed a massively parallel reporter assay on 2,396 genomic regions containing single nucleotide polymorphisms that are in high linkage disequilibrium with 97 lead variants from an obesity GWAS (PMID: 25673413). Regions were transfected into human SGBS preadipocytes, SGBS mature adipocytes, 3T3-L1 preadipocytes, HT22 hippocampal cells, and GT1-7 cells and assessed for enhancer activity. The processed file contains the MPRA barcodes.
Project description:We constructed a DBR1 knockout cell line (C22) using CRISPR in HEK293T cells. Through mapping of lariat reads, lariat levels in the DBR1 - samples are shown to increase dramatically (~20x) relative to wild type cells. Over 60% of this increase in lariat levels is abrogated upon rescue of DBR1 - cells with a DBR1 expression vector
Project description:Using wild type HEK293T cells and our DBR1 CRISPR knockout cell line (C22), we investigated the effect of AQR knockdown on cellular lariat levels. Samples were taken after transfection with either one of two AQR-targeting siRNAs or a non-targeting control siRNA. After sequencing the samples, lariat mapping of the reads revealed that AQR knockdown increased the lariat recovery rate by ~50% relative to the level observed in the negative control samples.