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In Specific Aim 2 we plan to engineer light-inducible systems for the precise control of looping dynamics. Using light activated dimerization domains that can be used in conjunction with designer DNA binding proteins we attempt to engineer factors used to rapidly promote or disrupt chromatin looping at various scales. This technology should enable studies not only in populations but also at the single cell level. In Specific Aim 3: we will develp reagents to study the transcriptional dynamics in relation to looping at the single cell level. We will combine RNA FISH with super-resolution imaging to develop a methodology for exploring the spatial and temporal structure of nascent transcription at high resolution. Combined with high-throughput image acquisition, we will discriminate the temporal dynamics of transcription by measuring the relative intensities arising from the different parts of the transcript. We will employ super-resolution imaging (STORM) to measure the spatial structure of transcription sites. 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Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. 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(2018)", "title": "Transcriptional Burst Initiation and Polymerase Pause Release Are Key Control Points of Transcriptional Regulation.", "authors": ["Bartman CR", "Hamagami N", "Keller CA", "Giardine B", "Hardison RC", "Blobel GA", "Raj A"], "@type": ["Publication", "Item"], "@id": "/publications/c88a1838-2e2c-4f6b-81a4-9d12fd53189e/", "date_published": "2018-12-06", "journal": "Molecular cell", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "pubs_using": [], "publications_of_set": [{"abstract": "Transcriptional regulation occurs via changes to rates of different biochemical steps of transcription, but it remains unclear which rates are subject to change  upon biological perturbation. Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release.", "uuid": "c88a1838-2e2c-4f6b-81a4-9d12fd53189e", "date_published": "2018-12-06", "authors": ["Bartman CR", "Hamagami N", "Keller CA", "Giardine B", "Hardison RC", "Blobel GA", "Raj A"], "title": "Transcriptional Burst Initiation and Polymerase Pause Release Are Key Control Points of Transcriptional Regulation.", "@type": ["Publication", "Item"], "status": "current", "journal": "Molecular cell", "display_title": "Bartman CR et al. (2018) PMID:30554946", "ID": "PMID:30554946", "@id": "/publications/c88a1838-2e2c-4f6b-81a4-9d12fd53189e/", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "number_of_experiments": 3, "@context": "/terms/", "aggregated-items": {"badges": [{"parent": "/biosamples/4DNBSTJXE5FI/", "embedded_path": "experiments_in_set.biosample.badges", "item": {"messages": ["Biosample missing passage_number", "Biosample missing culture_duration", "Biosample missing morphology_image", "Biosample is a stem cell line with unknown passage number missing karyotype"], "badge": {"commendation": null, "warning": "Biosample Metadata Incomplete", "uuid": "2b2cc7ff-b7a8-4138-9a6c-22884fc71690", "@id": "/badges/biosample-metadata-incomplete/", "badge_icon": "/static/img/badges/biosample-icon.svg", "description": "Biosample is missing metadata information required as part of the standards implemented by the 4DN Samples working group."}}}, {"parent": "/biosamples/4DNBSEP2EHKU/", "embedded_path": "experiments_in_set.biosample.badges", "item": {"messages": ["Biosample missing passage_number", "Biosample missing culture_duration", "Biosample missing morphology_image", "Biosample is a stem cell line with unknown passage number missing karyotype"], "badge": {"commendation": null, "warning": "Biosample Metadata Incomplete", "uuid": "2b2cc7ff-b7a8-4138-9a6c-22884fc71690", "@id": "/badges/biosample-metadata-incomplete/", "badge_icon": "/static/img/badges/biosample-icon.svg", "description": "Biosample is missing metadata information required as part of the standards implemented by the 4DN Samples working group."}}}, {"parent": "/biosamples/4DNBS579WEBZ/", "embedded_path": "experiments_in_set.biosample.badges", "item": {"messages": ["Biosample missing passage_number", "Biosample missing culture_duration", "Biosample missing morphology_image", "Biosample is a stem cell line with unknown passage number missing karyotype"], "badge": {"commendation": null, "warning": "Biosample Metadata Incomplete", "uuid": "2b2cc7ff-b7a8-4138-9a6c-22884fc71690", "@id": "/badges/biosample-metadata-incomplete/", "badge_icon": "/static/img/badges/biosample-icon.svg", "description": "Biosample is missing metadata information required as part of the standards implemented by the 4DN Samples working group."}}}]}, "validation-errors": []}