Wei-Li Liu

Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo

BACKGROUND The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the proteins stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised Mdm2-binding peptide into the active-site loop of thioredoxin. RESULTS When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity. CONCLUSIONS These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.

Structural Changes in TAF4b-TFIID Correlate with Promoter Selectivity

Proper ovarian development requires the cell type-specific transcription factor TAF4b, a subunit of the core promoter recognition complex TFIID. We present the 35 A structure of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. As a first step toward correlating potential structure/function relationships of the prototypic TFIID versus 4b/4-IID, we have compared their 3D structures by electron microscopy and single-particle reconstruction. These studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this open conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. By combining functional and structural analysis, we find that distinct localized structural changes in a megadalton macromolecular assembly can significantly alter its activity and lead to a TAF4b-induced reprogramming of promoter specificity.

Structures of three distinct activator-TFIID complexes.

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, we determined the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) and compared their structures as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, our results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation.

p53 dynamically directs TFIID assembly on target gene promoters

ABSTRACT p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of 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 issue, we have undertaken an integrated approach involving single-molecule fluorescence microscopy, single-particle cryo-electron microscopy, and biochemistry. Our real-time single-molecule imaging data demonstrate that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIIDs ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53s response element. 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 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, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters.

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

ATP-dependent chromatin-remodeling complexes such as PBAF mediate changes in chromatin structure, leading to regulation of transcriptional bursting. PBAF is targeted to genomic loci by histone acetylation. Despite extensive in vitro studies, how these chromatin remodelers rapidly bind and discriminate genomic targets in vivo remains unclear. Therefore, we sought to understand how the PBAF complex interacts with different chromatin states using live-cell single molecule fluorescence microscopy. Dual color tracking revealed that PBAF binds H3.3 marked chromatin within actively transcribed regions for faster time periods relative to binding to HP1α containing heterochromatin. Notably, elevation of histone acetylation levels increased the frequency of PBAF revisiting to genomic foci as defined by clustered binding. Furthermore, deletion of six bromodomains within the BAF180 subunit of PBAF reduced chromatin target search efficiency, clustered binding activity, and anchoring to the genome. These findings suggest that acetyl-lysine dependent clustered binding of PBAF to select genomic loci may facilitate rapid chromatin remodeling in actively transcribed regions. Our work also indicates that the dynamics of PBAF mediated chromatin state alterations proceed at fast timescales that may fine-tune transcription regulation.Rapid changes in chromatin structure via the action of ATP-dependent chromatin-remodeling complexes are thought to dynamically regulate transcriptional bursting. Chromatin-remodeling complexes are targeted to genomic loci by histone post-translational modifications (PTMs) including acetylation. Despite extensive in vitro studies, much is still unknown about how chromatin-remodeling complexes rapidly bind genomic targets and function in vivo. We sought to understand how the PBAF chromatin-remodeling complex interacts with different chromatin states using live-cell single particle tracking of the BAF180 subunit. Dual color tracking of PBAF with either H3.3 or HP1α revealed that PBAF binds chromatin within actively transcribed regions for shorter time periods relative to heterochromatin. We also found that deletion of BAF180s six bromodomains reduced both the association and dissociation of PBAF with chromatin. Finally, elevation of histone acetylation levels increased the frequency of PBAF revisiting to genomic foci. Together, these results suggest that acetyl-lysine dependent clustered binding of PBAF to select genomic loci may facilitate rapid chromatin-remodeling in actively transcribed regions. Overall our work also indicates that the dynamics of chromatin state alterations proceed at fast timescales to potentially regulate transcriptional bursting.Transcriptionally active genes contain acetyl-rich chromatin and are organized in distinct nuclear compartments that are spatially separated from transcriptionally inactive genes. It is not known how this compartmentalized acetylated chromatin is targeted and regulated by chromatin remodelers such as PBAF. Thus, we sought to understand how PBAF targets chromatin and modulates compartmentalization of transcriptionally active genes using live-cell single molecule fluorescence microscopy. Our work reveals chromatin hubs throughout the nucleus where PBAF cycles on and off the genome. Deletion of PBAF’s bromodomains impairs recognition of hubs and cycling on chromatin. Interestingly, markers for transcriptionally active and inactive genes can be found in compartments harboring acetylated chromatin at the periphery that is selectively recognized by PBAF via bromodomains. Defects in PBAF’s peripheral targeting lead to a select reduction in the size and number of compartments containing transcriptionally active genes. Our data, combined with previous work in Yeast and Drosophila, suggest that PBAF activity serves as a barrier to heterochromatin spreading. Overall, our findings suggest that chromatin compartments are highly structured with unique peripherally associated acetylation marks. PBAF utilizes these marks to help shape nuclear compartments containing transcriptionally active genes, thereby aiding genomic organization.

A new era of studying p53-mediated transcription activation

ABSTRACT To prevent tumorigenesis, p53 stimulates transcription by facilitating the recruitment of the transcription machinery on target gene promoters. Cryo-Electron Microscopy studies on p53-bound RNA Polymerase II (Pol II) reveal that p53 structurally regulates Pol II to affect its DNA binding and elongation, providing new insights into p53-mediated transcriptional regulation.

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.