p300/CBP-dependent enhancer’s dynamic activation
Figure 1 – Hypothetical model for p300/CBP activity in enhancer dynamic activation, focusing on PIC assembly. RNAPII recruitment and RNAPII pause-and-release mechanism.
Abstract
A recent study published on Molecular Cell, 2021 reports new discoveries about p300/CBP acetylation activity on dynamic regulation of enhancers. Using both EU-seq and ChIP-seq analysis, Narita and colleagues showed how the catalyzed acetylation of H3K27 is fundamental for dynamic enhancer activation, unravelling a novel “recruit-and-release” mechanism used by p300/CBP to concurrently promote RNAPII recruitment, PIC assembly and pause release.
Review
Introduction
p300/CBP are two paralogs lysine acetyltransferases that have a function in the regulation of gene expression. These acetyltransferases have a role in several cellular processes, such as cell proliferation or differentiation (Dancy et al., 2015). They are also involved in several diseases, including neurodegenerative disorders. (Kazantsev and Thompson, 2008). Recent studies reported how p300/CBP activity regards hundreds of acetylation sites on enhancers as well as their involvement in mediating the interaction with general transcription factors (GTFs) and RNAPII (Weinert et al.,2018, Kim et al., 1998, Wang et al., 2013).
However, the precise molecular mechanisms underpinning dynamic enhancer activation are still poorly understood. Starting from here, Narita et al. presented clarifications on p300/CBP activity, unravelling new mechanisms of enhancer control.
Discussion
p300/CBP is crucial for enhancer-dependent trascription
To investigate the activity of p300/CBP, the authors performed in vivo nascent RNA labeling by 5-ethynyl uridine (5-EU) followed by sequencing (EU-seq) analysis to profile nascent transcription in mouse embryonic stem cells (ESCs) treated with A-485, a selective inhibitor of p300/CBP acetyltransferase activity (Lasko et al., 2017). The treatment resulted in a broad transcriptional inhibition affecting mostly cell type-specific (CTS) genes, showing the crucial role of p300/CBP in enhancer activation. In particular, based on their kinetic expression pattern, three gene families were identified: Downregulated, Slightly downregulated and Not Changed.
p300/CBP and KDAC synergistic activity
Next, the authors investigated the interplay between p300/CBP and lysine deacetylases (KDACs). First, using Trichostatine-A (TSA) as a KDAC inhibitor, they observed that the treatment of ESC with TSA alone has a modest effect on transcription, not increasing the expression of p300/CBP regulated genes (identified by A-485 treatment). However, after a co-treatment of ESC using both A-485 and TSA, they observed that the inhibition of KDAC prevented A-485-induced transcription downregulation. These results show how the two enzymes work in a synergistic way on enhancer regulation, in which KDAC have a direct role in their inactivation.
p300/CBP inhibition on TF and p300 binding
It is well known that the bromodomain-containing protein 4 (BRD4), by recognising p300/CBP-catalyzed acetylation and recruiting P-TEFb, plays a key role in the RNA polymerase II (RNAPII) pause release mechanism.
For this reason, the authors investigated whether the downregulation of transcription following p300/CBP inhibition might be a consequence of impaired BRD4 recruitment and RNAPII pause release. Upon A-485 treatment, they observed reduced BRD4 binding at enhancers and enhancer-regulated gene promoters. Next, using JQ1 as BRD4 inhibitor and comparing the induced transcription changes with those resulting from A-485 treatment, the authors observed that the transcriptional downregulation induced by BRD4 inhibition was less severe, not fully explaining the effect of p300/CBP inhibition. They next turned to the investigation of RNAPII recruitment, analysing both unphosphorilated RNAPII and the phosphorylated forms RNAPII Ser5P (which marks promoter-proximal paused RNAPII) and RNAPII Ser5P (which associates with productive elongation). Interestingly, by ChIP-seq analysis, the authors observed decreased binding of all RNAPII forms upon A-485 treatment, including a strong binding impairment of the unphosphorilated form, thus suggesting a novel role of p300/CBP in promoting RNAPII recruitment independent from pause release. Moreover, using degradation tag (dTAG) to rapidly deplete BRD4 together with A-485 treatment, they showed that the decreased binding of all RNAPII forms is not related to the presence of BRD4, revealing that p300/CBP-catalyzed acetylation is fundamental in RNAPII recruitment independently of BRD4.
p300/CBP promotes PIC assembly
Finally, the authors investigated the relation between p300/CBP activity and pre-initiation complex (PIC) assembly, focusing both on TAF1 (the subunit of TFIID that contains two bromodomains) and TBP (TATA-Box binding protein TFIID subunit) general transcription factors. TFIID is involved in the early steps of PIC assembly, and TAF1 is crucial to promote the binding of TBP, required to initiate transcription. ChIP-seq analysis evaluating the binding of these two elements upon A-485 treatment showed again a decrease of their binding, demonstrating the direct involvement of p300/CBP-mediated acetylation in the assembly of PIC.
Conclusions
In conclusions, this study presents a new working model for the role of p300/CBP in enhancer dynamic activation. Especially, it is unrevealed a new role of p300/CBP in PIC assembly, RNAPII recruitment and pause release mechanisms. It is also highlighted the crucial role of KDAC in enhancer reversible decommissioning. This study can have an important impact on the research of new therapies for several diseases that are related to compromised p300/CBP activity, such as Rubinstein–Taybi syndrome or poly-glutamine expansion repeat-associated neurodegenerative disorders (Kazantsev and Thompson, 2008), as well as several types of tumours (Chen et al., 2022).
References
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