Principal Supervisor: Professor Daniel Panne
Second Supervisor: Dr Andrey Revyakin
PhD project title: Structural mechanism of signal-dependent gene transcription
University of Registration: University of Leicester
The control of gene transcription is the main regulatory step for cellular gene regulation. Transcriptional activation is triggered by signal-dependent assembly of transcription factor (TF) and co-activator complexes on enhancers to initiate epigenetic modification, local remodeling of chromatin, and, ultimately, recruitment of the RNA polymerase II preinitiation complex (RNAPII, PIC) to promoters. Over the last 50 years, great progress has been made, using X-ray crystallography, cryo-electron microscopy (cryoEM), biochemical and single-molecule methods, in our understanding of structures and functions of individual TFs, coactivators, components of the PIC, and RNA polymerase itself. However, it is not understood how these components work together in space and time, and how cellular signaling controls transcriptional activation,
One of the best-understood model systems for metazoan gene regulation is found in the innate immune system, which is crucial to limit pathogen infections. In the innate immune system, several groups of pattern-recognition receptors induce different signalling pathways leading to production of a variety of antiviral molecules, including type I interferons (IFNs) and proinflammatory cytokines. Previous work from the Panne laboratory has revealed mechanistic and structural insights into how the interplay between cellular signalling and TF activation, controls the function of the co-activator (Ortega et al., Nature 2018).
It is now important to ask how assembly of such complexes controls gene expression. The IFN enhancer system is particularly suitable for structural studies due to its naturally compact organization (about 100 base-pairs) and the proximity of the enhancer DNA elements to the +1 transcription start site. The IFN enhancer is recognized by four activators which synergistically recruit coactivator CBP/p300, a modular protein containing several well-defined domains, and CBP/p300, in turn, is believed to recruit components of the PIC and RNAPII. CBP/p300 is a central player in transcription activation, believed to interact with more than 400 TFs including important tumour suppressors (such as Fos/Jun, MYC, NFkB, p53 and BRACA1) as well as oncogenic fusion proteins (such as BRD4-NUT). Interaction of p300 with BRD4-NUT results in an aggressive subtype of lung and head and neck cancer, called NUT midline carcinoma. BRD4-NUT binds to and activates p300 resulting in chromatin hyperaceylation and dysregulation of genome expression. However, direct pharmacological targeting of such TFs remains an elusive challenge.
Recent developments in cryo EM and advances in the structural understanding of RNAPII and PIC now make it realistic to study the entire process of transcription activation, including recruitment of TFs to the enhancer, recognition of the TF surfaces by the coactivator CBP/p300, and, ultimately, recruitment of the PIC components (more than 55 polypeptides) and RNAPII.
The overall focus of the project is to use, the well-defined, natural, compact IFN enhancer to gain mechanistic insights into the assembly of the multicomponent molecular machinery that controls transcriptional activation. Understanding the detailed mode of how enhancers control transcriptional activation will aid in the development of new pharmacological approaches in cancer and other critical human diseases. This is particular important for developing new anti-cancer drugs, because classical tumour suppressors are difficult to target with small-molecule drugs
The first major aim of this project is to use cryoEM to determine the structure of the full 4-MDa enhanceosome poised for PIC recruitment, comprised of the IFN DNA template, DNA-bound activated TFs, the transcriptional coactivator CBP/p300, and +1 nucleosome.
The second major aim is to use cryoEM to determine the structure of the IFN enhanceosome in complex with the recruited PIC and RNAPII. To that end, you will produce the components of the PIC (six general transcription factors and RNAPII), and carryout stepwise assembly of the PIC by sequential incubation on a DNA template with the desired GTFs and enhancer components. The resulting complexes will be purified and imaged by cryoEM and single particle reconstruction. The expertise for purification of components of the PIC will be provided by the co-supervisor lab (Dr Revyakin) whose group has recently visualized the process of assembly of the PIC and RNAP II on promoters in real-time, at single-molecule resolution. Having access to a biochemically well-defined model system will allow visualization, in real-time, the dynamics of enhancer complex assembly and control of the PIC machinery during transcription initiation.
BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology
Techniques that will be undertaken during the project:
Structural biochemistry, protein expression in various expression systems including E. coli, Insect cells and Mammalian cell culture, affinity and ion-exchange protein purification, radioactivity-based activity assays, single-molecule imaging, super-resolution microscopy, optics, surface chemistry, engineering, coding (C++, Python, Matlab) and digital signal processing. Complexes will be reconstituted and imaged in the Midlands Regional cryoEM facility or at the eBIC facility at the Diamond Light Source. The material developed will also be used for single-molecule imaging with the goal to reveal insights into the dynamic nature of the transcriptional machinery.
Contact: Professor Daniel Panne, University of Leicester