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.
Project description:anti-ACAA2 and anti-OGDH ChIP-high throughput sequencing (Seq) was performed on, normal and 7 day post-transverse aortic constriction, mouse heart tissue (a pool of 3 hearts for each condition)
Project description:We report genome wide mapping of the histone variant H2A.Z during G0/G1 and mitosis in T24 bladder cancer cells. The results show that the broad enrichment pattern of H2A.Z near transcription start sites of active genes is maintained during mitosis. Furthermore, using H2A.Z localization to visualize nucleosome positioning near the start site, we see that the +1 nucleosome of active genes shifts upstream to occupy the transcription start sites during mitosis and the nucleosome depleted region is shortened. H2A.Z is also maintained on the -2 nucleosome which also shifts towrds the transcription start site during mitosis, further contributing to the shorteneing of the nucleosome depleted region. Examination of H2A.Z duing G0/G1 and mitosis in bladder cancer cells
Project description:We report genome wide mapping of the histone variant H2A.Z during G0/G1 and mitosis in T24 bladder cancer cells. The results show that the broad enrichment pattern of H2A.Z near transcription start sites of active genes is maintained during mitosis. Furthermore, using H2A.Z localization to visualize nucleosome positioning near the start site, we see that the +1 nucleosome of active genes shifts upstream to occupy the transcription start sites during mitosis and the nucleosome depleted region is shortened. H2A.Z is also maintained on the -2 nucleosome which also shifts towrds the transcription start site during mitosis, further contributing to the shorteneing of the nucleosome depleted region.
Project description:Affinity Purification Mass Spectrometry (AP-MS) of Drosophila ovaries expressing an H2A.Z-FlagHA transgene to identify interacting partners of H2A.Z to elucidate potential maternally supplied histone chaperones that deposit H2A.Z on the transcription start site (TSS).
Project description:We have developed both WTSS-seq (whole transcriptome start site sequencing) and WTTS-seq (whole transcriptome termini site sequencing) methods to capture either 5’- or 3’-ends of transcripts. HATT-seq (head and tail tag sequencing) is still under development, which can be used to capture both 5’- and 3’ ends of each transcript simultaneously. Iso-seq was used to produce full-length transcripts, which can be used to validate both alternative transcription start sites and alternative polyadenylation sites. CAGE-seq was used to confirm alternative transcription start sites only.
Project description:Role of alternative transcription start sites in muscle growth and development remains largely undetermined. Our WTSS-seq (whole transcriptome start site sequencing) approach to capture 5'-end of RNAs clearly revealed alternative transcription start events responsible for increased muscle mass in AMPK α2 knockout mice.
Project description:Here we quantified the transcription start site usage in a WT strain (BY4741) and a ∆set2 strain associated with the appearence of cryptic transcription start sites.