Project description:Anti-sense non-coding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops, but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo results in RNAPII stopping as well, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII poly-ubiquitylation, the half-life of collided polymerases increases, so that these can be detected between convergent genes by ChIP-Seq. These results provide new insight into fundamental mechanisms of gene traffic control, and point to an unexplored effect of anti-sense transcription on gene regulation via polymerase collision. Total RNA was extracted from WT or Elongin C deletion mutant (elc1M-bM-^HM-^F) cells and strand-specific RNA-Seq was performed. Three biological replicates were performed for WT and elc1M-bM-^HM-^F.
Project description:Anti-sense non-coding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops, but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo results in RNAPII stopping as well, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII poly-ubiquitylation, the half-life of collided polymerases increases, so that these can be detected between convergent genes by ChIP-Seq. These results provide new insight into fundamental mechanisms of gene traffic control, and point to an unexplored effect of anti-sense transcription on gene regulation via polymerase collision. ChIP-Seq of RNA polymerase II was performed with WT and Elongain C deletion mutant (elc1M-bM-^HM-^F) cells. 4H8 antibody against the Rpb1 C-terminal domain was used for RNA polymerase II immunoprecipitation, whilst mouse IgG antibody was used for control immunoprecipitations. Two biological replicates were performed for both WT and elc1M-bM-^HM-^F.
Project description:RNA sequencing was performed to characterize transcriptomic changes in the human hepatocellular carcinoma cell line HepG2 following treatment with doxorubicin (DOX), either alone or in combination with the histone deacetylase inhibitors valproic acid (VPA) or suberoylanilide hydroxamic acid (SAHA), relative to untreated controls. HepG2 cells were exposed to the indicated treatments for 48 hours. Total RNA was extracted, and poly(A)-enriched stranded libraries were prepared. Paired-end sequencing was conducted on an Illumina platform. Reads were aligned to the human reference genome (GRCh38), followed by gene-level quantification and differential gene expression analysis. This dataset enables analysis of transcriptional programs associated with HDAC inhibition and modulation of DOX response, including pathways related to DNA repair, stress response, and p53 signaling.
Project description:The purpose of this experiment was to generate an RNAseq dataset of human cardiomyocytes with lower levels of FOXO3 exposed to doxorubicin for 24 hours. Stable knock-down of FOXO3 was achieved using lentiviral shRNA construct. As negative control cells bearing an shRNA targeting GFP was used
Project description:Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesinâs Scc1, Smc1, and Smc3 subunits is generated during S and eventually destroyed at anaphase through cleavage of Scc1 by separase. Throughout the cell cycle, cohesinâs association with chromosomes is controlled by opposing activities: loading by the Scc2/4 complex and release by a separase independent releasing activity. Co-entrapment of sister DNAs during replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain stably connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We now show that all mutations capable of bypassing Eco1, be they in cohesinâs Smc1, Smc3, Scc1,Wapl, Pds5, or Scc3 subunits, greatly reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. We show that this process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation Effect of mutations QQ and EQ in Smc3 on cohesin loading onto chromosomes
Project description:Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesinâs Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover an unexpected functional asymmetry within the heart of cohesinâs highly conserved ABC-like ATPase machinery and show that an activity associated with one of cohesinâs two ATPase sites drives DNA release from cohesin rings. This key mechanism is conserved from yeast to humans. We propose that Eco1 locks cohesin rings around the sister chromatids by counteracting an asymmetric cohesin-associated ATPase activity. Effect of mutations in Smc1 and Smc3 on cohesin loading onto chromosomes
Project description:Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein’s distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C.glabrata cells (the calibration genome) with S.cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this “internal standard” calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using PCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics. Develop a method for quantitative ChIP-seq
Project description:The study was designed to compare whether and how muscle fibroblasts (FIB) and muscle stem cells (MYO) interact with motor neurons. Moreover, we aimed to investigate whether muscle cells, isolated from lifelong exercisers, exerted a protective and supportive effect on motor neurons. These objectives were investigated in vitro using primary muscle cells from human donors and primary motor neurons from rat embryos and were analyzed by immunocytochemistry and species-specific RNA sequencing. Rat neuron cells were treated for 24 hours with conditioned media from human skeletal muscle fibroblasts and myoblasts.
Project description:Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with its Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing at the Mediator level. In sum, we provide insight into how NPC-associated adaptor complexes can access the core transcription machinery. RNAseq was performed from WT, sac3∆, cdk8∆ and Sac3 R288D mutant cells. For each strain triplicates were analyzed. WT strain was sac3∆ transformed with pRS315 SAC3 WT