Principal Supervisor: Dr Andrey Revyakin, Department of Molecular and Cell Biology
Co-supervisor: Dr Olga Makarova
PhD project title: Single-molecule nanoscopy in vivo: assembly of the human transcription initiation machinery at endogenous promoters.
University of Registration: University of Leicester
Regulation of gene expression in mammalian cells is the driving force behind all cell-state decisions, including growth, differentiation, de-differentiation, and malignant transformation. At the molecular level, the commitment of a cell to express, or to stop expressing, a particular gene is made at the core promoter locus -- a specific sequence of genomic DNA a few hundred nanometres (~1 kb) in length. After ~50 years of research, it is now understood that transcription initiation requires six General Transcription Factors (TFIIA, IIB, IIE, IIF, IIE, and IIH), promoter-specific activators, and a cadre of co-activator, chromatin-remodelling and histone-deacetylase complexes. These players interact with each other, the promoter DNA, and chromatin, to recruit RNA polymerase II (pol 2) -- the molecular nanomachine which initiates transcription and produces pre-mRNA. However, a real-time picture of the transcription initiation process is entirely missing. How long does each molecular player spend at the promoter? How many attempts does an activator molecule make to remodel chromatin and to recruit pol 2? Do some players bind pre-assembled? Do interactions of activators continue after escape, as to maintain the promoter in a permissive state? Do pol 2 molecules act as pre-assembled ‘transcription factories’? Single-molecule fluorescence microscopy, at present, is the only practical approach to probe molecular interactions in live cells and, therefore, is the only method to tackle these questions.
Recently, we developed a single-molecule technology to image the assembly of molecular complexes on DNA, and used our method to visualize, in real-time, the assembly of human transcription machineries in vitro. The next logical step is to apply our technology in vivo. Thus, the focus of this Ph. D. proposal is dissect the mechanism of assembly of the transcription machinery on endogenous promoters in live cells, at the single-molecule level.
As the first step, most recently, we have harnessed CRISPR technology to generate human cell lines that express key components of the human transcription machinery, tagged with the Halo domain for live-cell labelling -- TFIID (the main promoter-recognition factor), TFIIH (the main promoter-melting factor), and Pol 2 itself. The specific aim of this Ph D proposal is to dissect the single-molecule dynamics of human TFIID, TFIIH, and RNA pol 2 during transcription initiation and promoter escape, in live cell nuclei.
The timeline of the project is as follows:
Months 1-6: Purify, label, and biochemically characterize human endogenous Halo-TFIID, Halo-TFIIH, and Halo-RNA pol 2, natively expressed in Halo-IID, Halo-IIH, and Halo-Pol2 cell lines (available at the lab).
Months 7-12: Use single-molecule imaging to dissect the single-molecule promoter assembly and promoter escape pathways for the Halo-TFIID, Halo-TFIIH, and Halo-Pol 2, in a reconstituted in vitro system.
Months 13-18: Use single-molecule imaging to dissect the single-molecule promoter assembly and promoter escape pathways for the Halo-tagged factors, in nuclear extracts prepared from the Halo-IID, Halo-IIH, and Hao-Pol2 cell lines.
Months 19-24: Use CRISPR technology and the existing Halo-IID, Halo-IIH, and Halo-Pol2 cell lines to insert a model inducible promoter into the AAVS “safe harbour” locus in each of the cell lines, fused to a tandem array of MS2-RNA hairpins for single-molecule detection of transcription.
Months 25-30: Use an inducible degradation-domain technology to optimize the level of expression of Halo-TFIID, Halo-TFIIH, and Halo-RNA in the respective cell lines (or isolated nuclei) to achieve signal-to-noise compatible with the detection of molecular events at the model AAVS promoter locus.
Months 31-36: Carry out single-molecule imaging experiments in Halo-IID, Halo-IIH, and Halo-Pol2 cell lines (or isolated nuclei), to probe single-molecule interactions during initiation and escape at the model AAVS promoter.
BBSRC Strategic Research Priority: Molecules, cells and systems
- Real-time single molecule fluorescence imaging
- Surface chemistry and microfluidics
- Super-resolution microscopy (Total-internal-reflection, epiillumination, oblique angle illumination)
- Laser optics
- Bio-conjugation (NHS, maleimide, Halo-tag, click-chemistry)
- Biochemical purification of proteins from bacteria and mammalian cells (FPLC, ultracentrifugation, pull-down, affinity purification)
- Protein and DNA electrophoresis
- Biochemical activity assays (gel-shifts, transcription assays)
- Molecular cloning (PCR, Gibson assembly, “cut and paste” DNA cloning, transformation)
- CRISPR pipeline (HDR construct design, gRNA design, transfection, cell culture, screening by genomic PCR and Western)
- Digital image and signal processing and algorithm optimization (FFT, filters, edge detectors, correlation, wavelet analysis, GPU CUDA programming)
- Basic electronics
- Basic engineering (metal endmilling, drilling, grinding, 3D printing)