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Functionally distinct promoter classes initiate transcription via different mechanisms [PRO-Seq]

ABSTRACT: To identify the functional contribution to transcription of various promoter-bound proteins we have endogenously tagged select proteins with an auxin-inducible degron and measured nascent transcription with PRO-seq 6 hours after their depletion

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE181257 | GEO | 2023/03/02

REPOSITORIES: GEO

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Functionally distinct promoter classes initiate transcription via different mechanisms [PRO-Seq]

Project description:Functionally distinct promoter classes initiate transcription via different mechanisms [PRO-Seq]

| PRJNA751383 | ENA

Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns.

Project description:Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.

| S-EPMC10183819 | biostudies-literature

Nascent DNA-associated proteome of POLE1-depleted cells

Project description:iPOND experiment was performed on control and dox-aux treated U2OS cells in which POLE1 was endogenously tagged with an auxin-inducible degron

2022-05-07 | PXD033757 |

Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns

Project description:Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II Pre-Initiation Complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs. Promoter DNA of developmentally-regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.

| S-SCDT-10_15252-EMBJ_2023113519 | biostudies-other

Members of an array of zinc finger proteins specify distinct Hox chromatin boundaries [ChIP-Seq2]

Project description:In this experiment, we sought to identify how the distribution of CTCF, MAZ, and RAD21 change in CTCF-degron (no auxin), CTCF-degron (auxin), CTCF-degron MAZ KO (no auxin), and CTCF-degron MAZ KO (auxin) backgrounds in mouse embryonic stem cells (ESCs).

2024-08-21 | GSE230481 | GEO

Mechanisms that specify promoter nucleosome location and identity

Project description:The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. Here we show that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, we show that NFR establishment is necessary for H2A.Z deposition. These studies suggest an ordered pathway for the assembly of promoter chromatin architecture. Nucleosome positions were mapped in a variety of strains under a variety of conditions to determine how several proteins are involved in nucleosome positioning. All replicates are biological replicates. Each set of replicates is balanced in terms of Cy3:Cy5 such that there are equal numbers of Cy3:Cy5 and Cy5:Cy3 arrays (format: mononucleosomal DNA:reference DNA). Changes in gene expression in a strain carrying either the Sth1-degron or Abf1-Reb1-degron under degron-inducing conditions were profiled by hybridizing labeled cDNA derived from the degron strain against labeled cDNA derived from a control strain lacking the degron, but grown under the same conditions. Four biological replicates for each experiment were done using a balanced dye-swap design.

2009-04-30 | E-GEOD-13446 | biostudies-arrayexpress

One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging

Project description:Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

2024-01-31 | GSE243637 | GEO

Distinct properties and functions of CTCF revealed by a rapidly inducible degron system [PRO-Seq]

Project description:We coupled an auxin-induced degron (AID) system with precision nuclear run-on (PRO-seq). Oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner ostensibly independent of bound CTCF. CTCF binding sites display highly variable resistance to degradation, with persistent sites displaying architectural related features.

2021-02-23 | GSE150414 | GEO

CRAC of yeast RNA polymerase II in anchor-away and auxin-degron strains.

Project description:We obtained RNA polymerase II occupancy profiles across the genome of S.cerevisiae strains: Abf1 anchor-away cells after addition of rapamycin for different time points (including no addition of rapamycin) and Rap1-AID auxin-degron cells after addition of auxin for different time points (including no addition of auxin) . This allowed us to compare polymerase occupancy profiles when these proteins are depleted from the nucleus or degraded.

2018-12-20 | GSE114589 | GEO

RING1B calibrated ChIP-seq (cChIPseq) datasets for control mESCs and an Scc1-AID auxin-inducible degron.

Project description:We examined 3D chromatin structure in the absence of cohesin (Scc1-AID auxin-inducible degron) and a control line (E14-Tir1) and found that PRC1 core promoter component RING1B was one of the most enriched proteins. Hence, we performed calibrated ChIP-seq experiments on the control (E14-Tir1) and the Scc1-AID mESC lines with a spike in of HEK293 human cells to further study this relationship.

2020-01-23 | E-MTAB-7817 | biostudies-arrayexpress

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