Project description:We addressed the lack of experimentally supported transcript annotations in the Rhesus macaque genome by ab initio identification of the transcription start sites (TSSs). We took advantage of histone H3 lysine 4 trimethylation (H3K4me3)'s ability to mark TSSs and the recently developed ChIP-Seq and RNA-Seq technology to survey the transcript structures in the macaque brain. We then integrated the two types of our newly generated data with genomic sequence features and extended a TSS prediction algorithm to ab initio predict and verify 16,833 of previously electronically annotated transcription start sites at 500 bp resolution and predicted ~10,000 new TSSs.
Project description:We addressed the lack of experimentally supported transcript annotations in the Rhesus macaque genome by ab initio identification of the transcription start sites (TSSs). We took advantage of histone H3 lysine 4 trimethylation (H3K4me3)'s ability to mark TSSs and the recently developed ChIP-Seq and RNA-Seq technology to survey the transcript structures in the macaque brain. We then integrated the two types of our newly generated data with genomic sequence features and extended a TSS prediction algorithm to ab initio predict and verify 16,833 of previously electronically annotated transcription start sites at 500 bp resolution and predicted ~10,000 new TSSs. We took advantage of histone H3 lysine 4 trimethylation (H3K4me3)M-bM-^@M-^Ys ability to mark transcription start sites (TSSs) and the recently developed ChIP-Seq and RNA-Seq technology to survey the transcript structures. By integrating the ChIP-seq, RNA-seq and small RNA-seq data (previously uploaded to GEO as GSM450615 by our collaborator) with genomic sequence features and extending and improving a state-of-the-art TSS prediction algorithm, we ab initio predicted and verified previously electronically annotated TSSs at a high resolution, and predicted some novel TSSs.
Project description:The alphaproteobacteria have metabolic activities and lifestyles of societal and industrial importance that differ from those in many other bacteria. Here we report the genome-wide identification of transcription start sites (TSSs) from two alphaproteobacteria grown under conditions that result in significant changes in gene expression. TSSs that were identified as present in one condition or both will be an important resource for future studies of these, and possibly other, alphaproetobacteria.
Project description:BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5′-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3′–5′ degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells.
Project description:miRNAs are key post-transcriptional regulators of gene expression. However, it is still poorly understood how miRNAs themselves are regulated, mainly due to the sparse annotation of miRNA transcription start sites (TSSs). Here, we developed a novel method for identifying active miRNA TSSs from nascent transcriptomes generated by nuclear run-on sequencing. With the least data requirement, our method demonstrated better performance than existing methods. Moreover, it provided ways not only to recognize miRNA TSSs but also to quantify primary miRNA expression in one experiment, which is very useful for revealing miRNAs directly regulated by the regulator(s) of interest.
Project description:BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5â²-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3â²â5â² degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells. Two cell lines were used. K562 cells were mock-irradiated (control) or UVC-irradiated at two different doses (25 and 100 J/m^2). HF1 cells were UVC-irradiated (20 J/m^2) in three independent experiments (nfUV4,nfUV3a, and nfUV3b). In one experiment, HF1 cells were also treated with TNF (10 ng/mL) 1 h prior to UV irradiation (tnfpreUV2, paired with nfUV4).
Project description:More than half of human protein-coding genes have an alternative transcription start site (TSS). We aimed to investigate the contribution of alternative TSSs to the acute-stress–induced transcriptome response in human tissue (skeletal muscle) using the cap analysis of gene expression approach. TSSs were examined at baseline and during recovery after acute stress (a cycling exercise). We identified 44,680 CAGE TSS clusters (including 3,764 first defined) belonging to 12,268 genes and annotated for the first time 290 TSSs belonging to 163 genes. The transcriptome dynamically changes during the first hours after acute stress; the change in the expression of 10% of genes was associated with the activation of alternative TSSs, indicating differential TSSs usage. The majority of the alternative TSSs do not increase proteome complexity suggesting that the function of thousands of alternative TSSs is associated with the fine regulation of mRNA isoform expression from a gene due to the transcription factor-specific activation of various alternative TSSs. We identified individual muscle promoter regions for each TSS using muscle open chromatin data (ATAC-seq and DNase-seq). Then, using the positional weight matrix approach we predicted time course activation of “classic” transcription factors involved in response of skeletal muscle to contractile activity, as well as diversity of less/un-investigated factors. Transcriptome response induced by acute stress related to activation of the alternative TSSs indicates that differential TSSs usage is an essential mechanism of fine regulation of gene response to stress stimulus. A comprehensive resource of accurate TSSs and individual promoter regions for each TSS in muscle was created. This resource together with the positional weight matrix approach can be used to accurate prediction of TFs in any gene(s) of interest involved in the response to various stimuli, interventions or pathological conditions in human skeletal muscle.
Project description:The 5’ LongSAGE (5’LS) approach provides a powerful genomic tool for identifying Transcription start sites (TSSs) in sequenced genome. The main purpose of this study is to identify the actual TSSs of expressed genes as well as the usage of different TSSs in Apis mellifera. We also wish to provide expression evidence for the in silico predicted genes and reveal some previously undiscovered genes.
Project description:Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. Here, we report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. Here, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNA, then dual priming oligonucleotide system to specifically enrich 5’-end tags for genomic analysis.
Project description:Both, acetylation of histones and of histone variant H2A.Z are conserved features of eukaryotic transcription start sites (TSSs) and both features appear to be critical for correct transcription initiation. However, complex patterns of transcriptional regulation have complicated the establishment of functional links between histone acetylation, H2A.Z deposition and their importance in transcription regulation. To elucidate these links, we took advantage of the unusual genome organization in Trypanosoma brucei, a highly divergent eukaryote. In T. brucei genes are organized in long polycistronic transcription units, drastically reducing the sites of transcription initiation. Employing a highly sensitive and quantitative mass-spectrometry-based approach, we quantified the genome-wide histone acetylation and methylation pattern and identified various acetyl and methyl marks exclusively enriched at TSSs In addition, we show that deletion of histone acetyltransferase 2 results in a loss of H4 acetylation at TSSs, a loss of H2A.Z deposition at TSSs and a shift in the sites of transcription initiation. Combined, our findings demonstrate an evolutionary conserved link between histone H4 acetylation, H2A.Z deposition and RNA transcription initiation.