Project description:Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. In the popular ATAC-seq method, Tn5 transposase inserts sequencing adapters into accessible DNA (‘tagmentation’). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells. Here we show that by simply modifying the tagmentation conditions for histone H3K4me2/3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce accessible DNA maps that are indistinguishable from the best ATAC-seq maps. Thus, DNA accessibility maps can be produced in parallel with CUT&Tag maps of other epitopes with all steps from nuclei to amplified sequencing-ready libraries performed in single PCR tubes in the laboratory or on a home workbench. As H3K4 methylation is produced by transcription at promoters and enhancers, our method identifies transcription-coupled accessible regulatory sites.
Project description:Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. A popular example is ATAC-seq, whereby Tn5 transposase inserts sequencing adapters into accessible DNA ('tagmentation'). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells. Here we show that by simply modifying the tagmentation conditions for histone H3K4me2 or H3K4me3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce chromatin accessibility maps that are indistinguishable from the best ATAC-seq maps. Thus, chromatin accessibility maps can be produced in parallel with CUT&Tag maps of other epitopes with all steps from nuclei to amplified sequencing-ready libraries performed in single PCR tubes in the laboratory or on a home workbench. As H3K4 methylation is produced by transcription at promoters and enhancers, our method identifies transcription-coupled accessible regulatory sites.
Project description:Here, we report a new strategy that combines in situ nuclear proximity ligation and transposase digestion, called ChIATAC, for highly efficient mapping of chromatin interactions between open chromatin loci simultaneously from lower input cells.
Project description:Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that responds to RNA polymerase (RNAP) stalling and removes DNA lesions from transcribed genes. Activation of TC-NER requires specific factors, such as human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. Mutations in CSB are associated with the severe neurological disorder Cockayne syndrome. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of chromatin organization, is not fully understood. Here we used single-nucleotide resolution UV damage mapping data to investigate the genome-wide function of Rad26 in TC-NER. Our data shows that Rad26 is critical for TC-NER in transcribed regions downstream of the first (+1) nucleosome; however, Rad26 is largely dispensable for TC-NER in the +1 nucleosome. We further show that the Rad26-independent TC-NER in the +1 nucleosome is correlated with high occupancy of the transcription initiation/repair factor TFIIH. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER when Rad26 is dysfunctional. Deletion of SPT4 significantly restores TC-NER in the downstream nucleosomes in a rad26∆ mutant. Collectively, these data indicate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5, and Rad26 mainly functions in the downstream nucleosomes to remove TC-NER suppression by Spt4/Spt5.
Project description:Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Idetification of accessible chromatin in forzen adult mouse liver tissue