Project description:Enhancer loci identified by ChIP-Seq or other experimental methods occupy hundreds of base pairs on the genome. It is the paradox comparing with the motif analysis, which usually contains only a few or tens nucleotides, achieved by bioinformatics analysis. To address this issue, we develop an experimental method, termed as massive active enhancer sequencing (MAE-Seq), to designate active enhancer sequences from arbitrary sources of 25bp random DNA libraries. These sequences are constructed in a mini-promoter vector with fluorescent reporter. After transfection, positive cells are sorted out and for sequencing. With the results, we successfully identify hundreds known accurate active enhancer sequences as expected. Besides that, large amounts of unmarked regulatory elements (UREs) that might control gene expression without epigenetic features are also been spotted. In conclusion, MAE-Seq would be useful to refine enhancer sequences and precisely annotate the genome in eukaryotes.
Project description:The budding yeast genome is marked by 250-350 origins of DNA replication. These origins are bound by the origin recognition complex (ORC) throughout the cell cycle. ORC has known DNA binding sequence preferences which, though necessary for binding, are not sufficient to fully specify a genomic locus as being bound by ORC, indicating that the cell must use additional chromosomal cues to specify ORC binding sites and origins of replication. Using high-throughput sequencing to precisely locate both ORC binding sites and nucleosome locations genome-wide, we find that a nucleosome depleted region (NDR) and precisely positioned nucleosomes are a ubiquitous feature of yeast replication origins. The ARS consensus sequence (ACS) and adjacent sequences are sufficient to maintain the nucleosome-free properties of the NDR. We use a temperature sensitive ORC1 mutant to demonstrate that ORC is required to maintain precisely positioned nucleosomes at origins of replication. These findings demonstrate the importance of local nucleosome positioning at replication origins, and that chromatin organization is an important determinant of origin selection. Examination of nucleosome positioning in wild-type and orc1-161ts mutant S. cerevisiae at room temperature and heatshock temperatures. Examination of ORC binding locations by ChIP-seq. All reported coordinates are based on the SGD genome build released 12/16/2005.
Project description:Background: The expression of microRNAs (miRNAs) is primarily regulated during their transcription. However, the transcriptional regulation of miRNA genes has not been studied extensively owing to the lack of sufficient information about the promoters and transcription start sites of most miRNAs. Results: In this study, we identified the transcription start sites of human primary miRNAs (pri-miRNAs) using DROSHA knockout cells. DROSHA knockout resulted in increased accumulation of pri-miRNAs and facilitated the precise mapping of their 5′ end nucleotides using the rapid amplification of cDNA ends (RACE) technique. By analyzing the promoter region encompassing the transcription start sites of miRNAs, we found that the unrelated miRNAs in their sequences have many common elements in their promoters for binding the same transcription factors. Moreover, by analyzing intronic miRNAs, we also obtained comprehensive evidence that miRNA-harboring introns are spliced more slowly than other introns. Conclusions: The precisely mapped transcription start sites of pri-miRNAs, and the list of transcription factors for pri-miRNAs regulation, will be valuable resources for future studies to understand the regulatory network of miRNAs.
Project description:The budding yeast genome is marked by 250-350 origins of DNA replication. These origins are bound by the origin recognition complex (ORC) throughout the cell cycle. ORC has known DNA binding sequence preferences which, though necessary for binding, are not sufficient to fully specify a genomic locus as being bound by ORC, indicating that the cell must use additional chromosomal cues to specify ORC binding sites and origins of replication. Using high-throughput sequencing to precisely locate both ORC binding sites and nucleosome locations genome-wide, we find that a nucleosome depleted region (NDR) and precisely positioned nucleosomes are a ubiquitous feature of yeast replication origins. The ARS consensus sequence (ACS) and adjacent sequences are sufficient to maintain the nucleosome-free properties of the NDR. We use a temperature sensitive ORC1 mutant to demonstrate that ORC is required to maintain precisely positioned nucleosomes at origins of replication. These findings demonstrate the importance of local nucleosome positioning at replication origins, and that chromatin organization is an important determinant of origin selection.