Project description:DNA-Diffusion is a novel generative approach leveraging diffusion probabilistic models for the design of cell type-specific DNA regulatory sequences. To evaluate the capacity of DNA-Diffusion sequences to endogenously alter AXIN2 transcription, we employed a novel MPRA-like system that utilizes CRE-recombinase mediated cassette exchange and long-read sequencing to measure the gene’s transcriptional output in response to enhancer sequences several kilobases away. We evaluated a total of 100 sequences using the AXIN2 endogenous replacement experiment using the following sequence groups: GM12878 DNA-Diffusion, GM12878 Positive Controls, GM12878 Negative Controls, K562 DNA-Diffusion, HepG2 DNA-Diffusion, and shuffled GM12878 DNA-Diffusion. Our findings demonstrate the potential of DNA-Diffusion to design sequences with therapeutic potential, showing their effectiveness in an endogenous setting.
Project description:DNA-Diffusion is a novel generative approach leveraging diffusion probabilistic models for the design of cell type-specific DNA regulatory sequences. To evaluate the capacity of DNA-Diffusion sequences to endogenously alter AXIN2 transcription, we employed a novel MPRA-like system that utilizes CRE-recombinase mediated cassette exchange and long-read sequencing to measure the gene’s transcriptional output in response to enhancer sequences several kilobases away. We evaluated a total of 100 sequences using the AXIN2 endogenous replacement experiment using the following sequence groups: GM12878 DNA-Diffusion, GM12878 Positive Controls, GM12878 Negative Controls, K562 DNA-Diffusion, HepG2 DNA-Diffusion, and shuffled GM12878 DNA-Diffusion. Our findings demonstrate the potential of DNA-Diffusion to design sequences with therapeutic potential, showing their effectiveness in an endogenous setting.
Project description:DNA-Diffusion is a novel generative approach leveraging diffusion probabilistic models for the design of cell type-specific DNA regulatory sequences. To evaluate the capacity of DNA-Diffusion sequences to endogenously alter AXIN2 transcription, we employed a novel MPRA-like system that utilizes CRE-recombinase mediated cassette exchange and long-read sequencing to measure the gene’s transcriptional output in response to enhancer sequences several kilobases away. We evaluated a total of 100 sequences using the AXIN2 endogenous replacement experiment using the following sequence groups: GM12878 DNA-Diffusion, GM12878 Positive Controls, GM12878 Negative Controls, K562 DNA-Diffusion, HepG2 DNA-Diffusion, and shuffled GM12878 DNA-Diffusion. Our findings demonstrate the potential of DNA-Diffusion to design sequences with therapeutic potential, showing their effectiveness in an endogenous setting.
Project description:RNA-Sequencing is a transformative method that captures the quantitative dynamics of a transcriptome with exquisite sensitivity and single-base resolution. There are, however, few computational pipelines for RNA-Seq with statistical tests that evince sufficient robustness and power as demanded by the difficult combination of small sample sizes and high variability in sequence read counts. To this end, we developed GENE-counter, a complete software pipeline for analyzing RNA-Seq data for genome-wide expression differences between replicated treatment groups. One important component of GENE-counter is a statistical test based on the NBP parameterization of the negative binomial distribution for identifying differentially expressed genome features. We used GENE-counter to analyze RNA-Seq data derived from Arabidopsis thaliana infected with a strain of defense-eliciting bacteria. We identified 308 genes that were differentially induced. Using alternative methods, we provided support for the induced expression and biological relevance of a substantial proportion of the genes. These results suggest the NBP parameterization of the negative binomial distribution is well suited for explaining RNA-Seq data and the statistical test makes GENE-counter a powerful pipeline for studying genome-wide expression changes. GENE-counter is freely available at http://changlab.cgrb.oregonstate.edu/. Our RNA-seq data is uploaded on the NCBI short read archive (SRA) under the SRA025952.
Project description:Microfluidic devices provide a low-input and efficient platform for single-cell RNA-seq (scRNA-Seq). Here we present microfluidic diffusion-based RNA-seq (MID-RNA-seq) for conducting scRNA-seq with a diffusion-based reagent swapping scheme. This device incorporates cell trapping, lysis, reverse transcription and PCR amplification all in one microfluidic chamber. MID-RNA-Seq provides high data quality that is comparable to existing scRNA-seq methods while implementing a simple device design that permits multiplexing. The robustness and scalability of MID-RNA-Seq device will be important for transcriptomic studies of scarce cell samples.
Project description:Here we reveal a biophysical basis for the spreading behavior of Xist RNA on the inactive X-chromosome (Xi). Xist and HNRNPK together drive a liquid-liquid phase separation (LLPS) that encapsulates the Xi. HNRNPK and Xist exert mutual pulling forces that lead to RNA internalization into the condensate. While HNRNPK is sufficient for condensate formation, Xist induces a further phase transition into a "soft" droplet by altering elasticity, adhesiveness, and wetting properties of the condensate in vitro. Once phase transition occurs, other Xist-interacting factors are internalized and concentrated within the condensate. We attribute LLPS to HNRNPK's RGG and Xist's Repeat B (RepB) motifs. Mutating these motifs causes Xist diffusion, disrupts Polycomb recruitment, and precludes the required mixing of Xi chromatin for establishing the Xi superstructure. Thus, phase transitions driven by RepB and HNRNPK create a membrane-less subnuclear compartment for the localization and limited diffusion of Xist ribonucleoprotein complexes on the Xi.
Project description:Expression of virulence genes in pathogenic E. coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters. How H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA-strand sequence called ops (operon polarity suppressor). RfaH–ops interactions establish long-lived RfaH–EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify 8 RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS–binding features, explaining minor-paralog targeting.
Project description:Purpose: The goal of this study is to compare endothelial small RNA transcriptome to identify the target of OASL under basal or stimulated conditions by utilizing miRNA-seq. Methods: Endothelial miRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. After sequencing, the raw sequence reads are filtered based on quality. The adapter sequences are also trimmed off the raw sequence reads. rRNA removed reads are sequentially aligned to reference genome (GRCh38) and miRNA prediction is performed by miRDeep2. Results: We identified known miRNA in species (miRDeep2) in the HUVECs transfected with siCTL or siOASL. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of endothelial miRNA profiles affected by OASL knockdown with biologic replicates.