Project description:Mice lacking the stem/progenitor cell transcription factor, Sex determining region Y-box 2 (SOX2), in neurons of the suprachiasmatic nuclei display imprecise, poorly consolidated behavioral rhythms that do not entrain efficiently to environmental light cycles. RNA sequencing revealed that Sox2 deficiency alters the circadian transcriptome of the SCN, reducing the expression of many core clock genes and neuropeptide-receptor systems.
Project description:Maternal inheritance of mitochondrial DNA (mtDNA) is highly conserved in metazoans. While many species eliminate paternal mtDNA during late sperm development to foster maternal inheritance, the regulatory mechanisms governing this process remain elusive. Through a large-scale genetic screen in Drosophila, we identified 47 mutant lines exhibiting substantial retention of mtDNA in mature sperm. We mapped one line to Poldip2, a gene predominantly expressed in the testis. Disruption of Poldip2 led to pronounced mtDNA retention in mature sperm and subsequent paternal transmission to progeny. Further investigation via imaging, biochemical analyses and ChIP assays revealed that POLDIP2 is a mitochondrial matrix protein capable of binding to mtDNA. Moreover, we uncovered that CLPX, a key component of the major mitochondrial protease, binds to POLDIP2 to co-regulate mtDNA elimination in Drosophila spermatids. This study shed light on the mechanisms underlying mtDNA removal during spermatogenesis, underscoring the pivotal role of this process in safeguarding maternal inheritance.
Project description:Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi-piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, while those in bovine ovary just express PIWIL1. In human, macaque and bovine ovaries we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis we show that these maturing oocytes strongly and specifically express the thus-far uncharacterized PIWIL3 protein, alongside other known piRNA-pathway components. In bovine early embryos these piRNAs are still abundant, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal unexpected, highly dynamic piRNA pathways in mammalian oocytes and early embryos. Analyses of multiple small RNA libraries obtained from fetal/adult oocytes, cumulus cells, ovary, testis and 2-4 cell stage ivf embryos of multiple mammalian species.
Project description:This study aims to identify combination treatments capable of inducing improved IO responses in lung tumours and thus, help guide decisions on the next combination arms for the HUDSON trial (post-IO). For that purpose, a lung tumour GEMM model was treated with either vehicle, PD-L1, ATR, ATR/PD-L1; Cisplatin/PD-L1/Ctla4 or VEGFR/PD-L1 and tumours collected for transcriptional profiling.
Project description:To identify the role of BLIMP1 in Waldenström's macroglobulinemia, the PRDM1 transcript was targeted using an artificial miRNA. RNAseq was used to compare it to a non-targeting control in the RPCI-WM1 cell line. To determine the role of EZH2 in Waldenström's macroglobulinemia, the RPCI-WM1 cell line was treated with 0.3µM of the EZH2 inhibitor Tazemetostat, compared to DMSO vehicle control by RNAseq. ChIPseq was performed for the factors BLIMP1 and H3K27me3 in the RPCI-WM1, OPM-2 and NCI-H929 cell lines, along with ChIPseq for EZH2 in the NCI-H929 cell line.
Project description:Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. We previously identified an activator-targeted ~85 amino acid three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11 Mediator subunits in fungi. The Gal11 KIX domain is engaged by pleiotropic drug resistance transcription factor (Pdr1) orthologues, key regulators of the multidrug resistance (MDR) pathway in S. cerevisiae and in the clinically important human pathogen Candida glabrata. Drug-resistant clinical isolates of C. glabrata most commonly harbour point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway may represent a lynchpin in C. glabrata MDR. We have now carried out sequential biochemical and in vivo high-throughput screens to identify small molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation in both S. cerevisiae and C. glabrata and re-sensitizes drug-resistant C. glabrata to effective azole antifungal concentrations in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Samples are generated in triplicate for four conditions (DMSO/vehicle-treated, iKIX1-treated, DMSO/vehicle and ketoconazole-treated. and iKIX1-ketoconazole treated) in both Saccharomyces cerevisiae and Candida glabrata
Project description:We performed RNA sequencing on grafts after heart transplantation under three conditions: syngeneic, allogeneic, and allogeneic with anti-CD80/86 antibody treatment.
Project description:The activating signal co-integrator 1 complex subunit 3 (ASCC3), a multifunctional protein, has been implicated as a prognostic marker in several types of cancer. However, mechanisms underlying its prognostic value are not fully understood. Here, we report that ASCC3 promotes sensitivity to chemotherapeutic drugs, such as 5-fluorouracil, cisplatin, and hydroxyurea, in colorectal cancer (CRC) cells, likely in a cancer type dependent manner. Increased chemoresistance resulting from ASCC3 loss is not due to reduced genomic instability as evidenced by enhanced accumulation of DNA damage and micronuclei following exposure to these drugs. RNA-seq analysis reveals that ASCC3 stimulates the expression of gene sets associated with mTORC1 signaling, glycolysis, and protein folding pathways in CRC cells. While promoting the serine biosynthesis pathway, we demonstrate, through extracellular flux assays and stable isotopes tracer analysis, that ASCC3 reprograms energy metabolism, favoring glycolysis over oxidative phosphorylation. Furthermore, we find that ASCC3 is required for PERK production upon ER stress. Impaired PERK production is associated with reduced levels of CHOP and caspase 3 following treatment with 5-fluorouracil, indicating that ASCC3 promotes PERK production to enhance cell death upon chemotherapy. Collectively, our work underscores molecular complexities underlying chemoresistance in the wake of ASCC3 loss in CRC cells.
Project description:Long non-coding (lnc)RNAs play key roles in many biological processes. Elucidating the function of lncRNAs in cell type specification during organ development requires knowledge about their expression in individual progenitor types rather than in whole tissues. To achieve this during cortical development, we used a dual-reporter mouse line to isolate coexisting proliferating neural stem cells, differentiating neurogenic progenitors and newborn neurons and assessed the expression of lncRNAs by paired-end, high-throughput sequencing. We identified 379 genomic loci encoding novel lncRNAs and performed a comprehensive assessment of cell-specific expression patterns for all, annotated and novel, lncRNAs described to date. Our study provides a powerful new resource for studying these elusive transcripts during stem cell commitment and neurogenesis. mRNA profiles of Proliferating Progenitors, Differentiating Progenitors and Neurons from lateral cortex of E14.5 mouse embryos. Each cell type in three biological replicates.
Project description:Mammalian cells contain copious amounts of RNA including both coding and non-coding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) binds directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155 in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, we propose that overexpression of specific miRNAs in human cancer may lead to aberrant DNA methylation and altered gene-expression. Examine of the DNA methylation and mRNA profile of HCT 116 cells transfected by random 23-mer or miR-155 RNA