Project description:Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single-cell RNA sequencing has been used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA-seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis during tumor processing. Additionally, frozen tumor samples constitute a vast and valuable material bank for cancer research. Therefore, we developed a high-throughput and high-sensitivity method called HH-seq, which combines random primers and a pre-index strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. We also established a robust pipeline to facilitate the analysis of full-length RNA-seq data. We applied HH-seq to more than 730,000 single nuclei from 32 patients with various tumor types. The pan-cancer study enables us to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns and clone dynamic, etc. We identified new malignant cell subclusters and explored their specific function across cancers. Furthermore, we investigated the malignant status of epithelial cells among different cancer types with respect to mutation and splicing patterns. The ability to detect full-length RNA at the single-nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.
Project description:Purpose: Most Hedgehog responsive gene expression is mediated through GLI de-repression. Additionally GLI -repression is proposed to play roles in limb pre-patterning before HH pathway activation. This study evaluates if GLI repression is established prior to HH pathway activation. Methods: To determine if GLI-repression is established prior to pathway activation, we used genomic approaches to study GLI-mediated repression using the mouse developing limb as a model. We identified pre-HH (E9.25, 21-23S) and post-HH (E10.5, 32-25S) GLI3 binding regions using CUT&RUN for endogenous FLAG-tagged GLI3 proteins. Using a combination of ChIP-seq, CUT&RUN, CUT&Tag, ATAC-seq and RNA-seq, we tested whether loss of Gli3 prior to HH signaling was able to de-repress genes and enhancers, as it does after HH signaling. Results: Prior to HH signaling, GLI3 binds to poised, accessible regions with histone deacetylase (HDAC) proteins, similar to post-HH signaling. Despite GLI3 binding to most regions as it does in the post-HH limb, loss of Gli3 is unable to prematurely active target genes or enhancers. Furthermore, we find that GLI3-dependent chromatin compaction does not occur until roughly 10 hours after HH signaling would have normally been induced. Collectively, these results support that GLI repressor proteins are inert prior to HH pathway activation.
Project description:Paracrine Hedgehog (Hh) signaling regulates growth and patterning in many Drosophila organs. We mapped chromatin binding sites for Cubitus interruptus (Ci), the transcription factor that mediates outputs of Hh signal transduction, and we analyzed transcription profiles of control and mutant embryos to identify genes that are regulated by Hh. Putative targets we identified include several Hh pathway components, most previously identified targets, and many targets that are novel. Analysis of expression patterns of pathway components and target genes gave evidence of autocrine Hh signaling in the optic primordium of the embryo. And, every Hh target we analyzed that is not a pathway component appeared to be regulated by Hh in a tissue-specific manner. We present evidence that Hh-dependent tissue specificity is dependent upon transcription factors that are Hh-independent, suggesting that “pre-patterns” of transcription factors partner with Ci to make Hh-dependent gene expression position-specific. Analysis of the expression profiles of loss of function mutantations in core components of the Hh signaling pathway. A total of 14 samples were analysed consisting of comparisons of hh-, ci-, smo-, ptc-, and Cim1-m4 (Activator) mis-expression embryos compared to wt sibling embryos.
Project description:Paracrine Hedgehog (Hh) signaling regulates growth and patterning in many Drosophila organs. We mapped chromatin binding sites for Cubitus interruptus (Ci), the transcription factor that mediates outputs of Hh signal transduction, and we analyzed transcription profiles of control and mutant embryos to identify genes that are regulated by Hh. Putative targets we identified include several Hh pathway components, most previously identified targets, and many targets that are novel. Analysis of expression patterns of pathway components and target genes gave evidence of autocrine Hh signaling in the optic primordium of the embryo. And, every Hh target we analyzed that is not a pathway component appeared to be regulated by Hh in a tissue-specific manner. We present evidence that Hh-dependent tissue specificity is dependent upon transcription factors that are Hh-independent, suggesting that “pre-patterns” of transcription factors partner with Ci to make Hh-dependent gene expression position-specific.
Project description:Current methods for single-nucleus RNA-sequencing (snRNAseq) are limited to assaying endogenously polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNAs enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based single-nucleus transcriptomics to the total transcriptome. We apply standard snRNAseq and total snRNAseq to C2C12 myoblasts. Our in situ polyadenylation strategy relies on a brief, low-cost add-on to a widely used protocol for single-cell/single-nucleus RNA-sequencing, and thus could be broadly and quickly adopted. Single-nucleus RNA-sequencing of the total transcriptome will enable new insights into gene regulation and biology.