Biology subjects

Liu, W.-L.

Publications and source records attributed to Liu, W.-L..

3 recordsLinked to original sources

bioRxiv2017

PBAF’s genomic binding dynamics are regulated via bromodomain-acetyl-lysine interactions and select chromatin states

Transcriptional bursting involves genes rapidly switching between active and inactive states. Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA Pol II during transcriptional bursting. It is unknown how acetylated chromatin is dynamically targeted and regulated by chromatin remodelers such as PBAF. Thus, we sought to understand how PBAF targets acetylated chromatin using live-cell single molecule fluorescence microscopy. Our work reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAFs bromodomains impairs targeting, stable engagement and persistent binding on chromatin in hubs. Interestingly, PBAF has a higher probability to stably engage chromatin inside hubs indicating that hubs contain a unique nucleosomal scaffold compared to global chromatin. Dual color imaging of PBAF in hubs near H3.3 or HP1 reveals that PBAF targets both euchromatic and heterochromatic regions with distinct genome binding kinetics that mimic chromatin stability. Removal of PBAFs bromodomains stabilizes H3.3 and HP1 binding within chromatin indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data, suggests that PBAF differentially and dynamically engages a variety of chromatin structures involved in both activation and repression of transcription via bromodomains. Furthermore, PBAFs binding stability on chromatin may reflect the chromatin remodeling potential of different bound chromatin states. Statement of SignificanceTranscriptional bursting involves a gene rapidly switching between transcriptionally active and inactive states. To regulate transcriptional bursting, chromatin must interchange between euchromatin and heterochromatin to permit or restrict access of transcription factors including RNA Polymerase II to enhancer and gene promoters. However, little is known regarding how chromatin remodelers dynamically read a rapidly changing 4D epigenome. We used live-cell single molecule imaging to characterize the spatiotemporal chromatin binding dynamics of PBAF, a chromatin remodeler that accesses both euchromatin and heterochromatin to regulate transcription. PBAF cycles on and off chromatin hubs in select nuclear regions where it distinctly engages euchromatin and heterochromatin via bromodomains in its BAF180 subunit. Our study provides the framework to understand how the 4D epigenome is regulated.

cell biology↗

bioRxiv2017

Identifying a TFIID interactome via integrated biochemical and high-throughput proteomic studies

The core promoter recognition TFIID complex acts as a central regulator for eukaryotic gene expression. To direct transcription initiation, TFIID binds the core promoter DNA and aids recruitment of the transcription machinery (e.g., RNA polymerase II) to the transcription start site. Many transcription factors target TFIID to control vital cellular processes. Current studies on finding TFIID interactors have predominantly focused on transcription factors. Yet, a comprehensive interactome of mammalian TFIID has not been established. Therefore, this study sought to reveal potential TFIID-nucleated networks by identifying likely co-regulatory factors that bind TFIID. By using intact native human TFIID complexes, we have exploited three independent approaches including a high-throughput Next Generation DNA sequencing coupled with proteomic analysis. Among these methods, we found some overlapping and new candidates in which we further assessed three putative interactors (i.e., Sox2, H2A and EMSY) by co-immunoprecipitation assays. Notably, in addition to known TFIID interactors, we identified a number of novel factors that participate either in co-regulatory pathways or non-transcription related functions of TFIID. Overal, these results indicate that, in addition to transcription initiation, mammalian TFIID may be involved in broader regulatory pathways than previous studies suggested.

biochemistry↗

bioRxiv2016

p53 dynamically directs TFIID assembly on target gene promoters

The p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIIDs ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIIDs conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIIDs interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

biochemistry↗