Fast-acting chemical tools to delineate causality in transcriptional control

Jaeger, Martin G.; Ciulli, Alessio

doi:10.1016/j.molcel.2021.02.015

Cited by 60 publications

(33 citation statements)

References 146 publications

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“…To examine the direct effect of PAF1 depletion on transcription, we developed the acute degradation system by integrating FKBP12 F36V tag at the N terminus of the endogenous PAF1 locus in DLD-1 cells to generate PAF1-dTAG cells (Fig. 1A) (52)(53)(54). PAF1-dTAG exhibited a marked decrease in levels of tagged endogenous protein in the presence of degradation-inducing ligands (Fig.…”

Section: Acute Paf1 Depletion Compromises the Stability Of Paf1cmentioning

confidence: 99%

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

Wang

Song

et al. 2022

Sci. Adv.

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Pleiotropic transcription regulator RNA polymerase II (Pol II)–associated factor 1 (PAF1) governs multiple transcriptional steps and the deposition of several epigenetic marks. However, it remains unclear how ultimate transcriptional outcome is determined by PAF1 and whether it relates to PAF1-controlled epigenetic marks. We use rapid degradation systems and reveal direct PAF1 functions in governing pausing partially by recruiting Integrator-PP2A (INTAC), in addition to ensuring elongation. Following acute PAF1 degradation, most destabilized polymerase undergoes effective release, which presumably relies on skewed balance between INTAC and P-TEFb, resulting in hyperphosphorylated substrates including SPT5. Impaired Pol II progression during elongation, along with altered pause release frequency, determines the final transcriptional outputs. Moreover, PAF1 degradation causes a cumulative decline in histone modifications. These epigenetic alterations in chromatin likely further influence the production of transcripts from PAF1 target genes.

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Section: Acute Paf1 Depletion Compromises the Stability Of Paf1cmentioning

confidence: 99%

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

Wang

Song

et al. 2022

Sci. Adv.

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“…The development of acute protein degradation approaches has made it possible to investigate the impact of genome-wide depletions of key architectural proteins on genome organization and gene expression 24 . Using such approaches, it has been shown that depletion of cohesin, its loading factor NIPBL, and CTCF result in large-scale changes in the 3D organization of the genome and a loss of TADs 25 – 31 .…”

Section: Introductionmentioning

confidence: 99%

Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF

Aljahani¹,

Hua

Karpinska³

et al. 2022

Nat Commun

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Enhancers and promoters predominantly interact within large-scale topologically associating domains (TADs), which are formed by loop extrusion mediated by cohesin and CTCF. However, it is unclear whether complex chromatin structures exist at sub-kilobase-scale and to what extent fine-scale regulatory interactions depend on loop extrusion. To address these questions, we present an MNase-based chromosome conformation capture (3C) approach, which has enabled us to generate the most detailed local interaction data to date (20 bp resolution) and precisely investigate the effects of cohesin and CTCF depletion on chromatin architecture. Our data reveal that cis-regulatory elements have distinct internal nano-scale structures, within which local insulation is dependent on CTCF, but which are independent of cohesin. In contrast, we find that depletion of cohesin causes a subtle reduction in longer-range enhancer-promoter interactions and that CTCF depletion can cause rewiring of regulatory contacts. Together, our data show that loop extrusion is not essential for enhancer-promoter interactions, but contributes to their robustness and specificity and to precise regulation of gene expression.

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“…Given that transcriptional changes occur rapidly, traditional genetic approaches have failed to effectively define direct targets of sequence-specific transcription factors, and therefore, have inadequately defined mechanisms of transcriptional control by these proteins 9,10 . Thus, CRISPR-based addition of degron tags to endogenous transcription factor proteins has provided a technological breakthrough that is greatly aiding the study of transcription factor function 7,8,34,35 .…”

Section: Discussionmentioning

confidence: 99%

“…Genetic inactivation coupled with RNA-seq and chromatin immunoprecipitation has suggested that DNA-binding transcription factors can control the expression of thousands of genes, but faster methods of transcription factor inactivation suggest that this is an over estimation due to indirect and compensatory transcriptional changes 7,8 . Small molecule "degraders" coupled with the analysis of nascent transcripts and proteomics offer an opportunity to greatly refine these transcription factor networks and define mechanisms of transcriptional control 9,10 .…”

mentioning

confidence: 99%

PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

Zhang¹,

Wang

Liu

et al. 2021

Preprint

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Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues1. Thus, it is difficult to achieve a mechanistic understanding of transcription factor function using traditional genetic deletion or RNAi methods, because these slow approaches make it challenging to distinguish direct from indirect transcriptional effects. Here, we used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO12-6 to define how the t(2;13)(q35;q14) disrupts normal gene expression programs to trigger cancer. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses, we identified a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Moreover, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. The activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective and often only a single element within a complex super-enhancer was affected. In addition, fusion of the endogenous PAX3-FOXO1 with APEX2 identified proteins in close proximity with PAX3-FOXO1, including ARID1A and MYOD1. We found that continued expression of PAX3-FOXO1 was required to maintain chromatin accessibility and allow neighboring DNA binding proteins and chromatin remodeling complexes to associate with this small number of regulated enhancers. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network.

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Fast-acting chemical tools to delineate causality in transcriptional control

Cited by 60 publications

References 146 publications

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF

PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

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