Transcription: molecular mechanisms & dynamics

Project Outline

Background/Questions

Transcription is central to all life, but many things remain unclear. In particular in eukaryotes, many contributing factors and mechanistic details have been identified for transcription of mRNAs by RNA polymerase II (Pol2), but a number of phenomena remain enigmatic, including:

• action-at-a-distance type of regulation of gene expression by enhancers
• the observation of localized hotspots of transcribing polymerase in the nucleus, known as ‘transcription factories’
• the natures of the local genic and nuclear environment in terms of topological constraints, dynamic rearrangements of these, and factor composition & turnover
• origins of stochastic fluctuations in the process of transcription, known as ‘transcriptional bursting’
• the ‘pausing’ of polymerase at certain positions in a gene
• the interplay of transcription and DNA with its attached proteins and the varying post-translational modifications on these (‘chromatin landscape’)

A major challenge is that many of these questions are believed to be connected and difficult to study in a reductionist and/or isolated way.

Aims

The overall aim of our research is to provide insights into the mechanisms underlying the above phenomena and the relationships among them in human cells.

We are pursuing an interdisciplinary approach based on genome-wide data accumulation (mostly via next generation sequencing methods) and quantitative analysis and results interpretation (bioinformatics, modelling), accompanied by targeted system perturbations (mostly via dCas9 methodology) and imaging strategies.

This overall strategy splits into several complementary research undertakings and associated (sub)topics that are well suited as PhD projects (see below).

Strategies / Thesis Topics

We rely on human cell lines for our research, such as HEK293, U2OS, or HeLa, which are easy to maintain and serve as excellent models for the basic transcriptional mechanisms we want to investigate. No complex animal or plant models are necessary, thus greatly simplifying the experimental side and minimizing ‘model system housekeeping’.

The interdisciplinary nature of our research means that projects can be designed to any desired mix of wet lab and dry lab work, around the following themes:

Liquid-liquid phase transitions (LLPT)

Pol2 and many transcription factors have intrinsically disordered domains among their protein structures, which have recently been shown to lead to large scale, dynamic aggregations that can mechanically rearrange the genome topology. LLPT thus appear to form a critical link between 3D genome structure, dynamic Pol2 localization and transcriptional mechanisms.

We will induce LLPTs at specific spots in the genome and/or express factors with altered domains to perturb their aggregation properties, followed by NGS-based characterization of affected genes’ transcriptional activities.

Recent research grant from group with more information: https://gtr.ukri.org/projects?ref=EP%2FT002794%2F1

Polymerase pausing

This refers to the transient arrest of Pol2 at promoter proximal regions after transcription initiation. This is a key step in transcription that strongly affects the dynamics of mRNA production and is little understood; it probably relates to the transition of Pol2 from initiation to its release into productive elongation and the tethering this involves.

We will perturb pausing with small molecules, gene editing, and LLPT induction, followed by NGS run-on assays to study the relation between pausing, gene architecture, and other factors, to gain mechanistic insights.

Example paper from our group: bioRxiv 461442

Pol2 positional dynamics

A key to understand the transcriptional dynamics at individual genes is the diffusion and exchange of Pol2 molecules among local niches and how this is constrained by nuclear topology.

We will track individual Pol2 molecules with imaging-based techniques and explore how this relates to DNA topology by conducting ChIA-PET assays, along with gene editing and potential modelling strategies.

Example paper from our group: bioRxiv 514174

New methods

We are constantly developing novel techniques, mostly NGS- and/or software-based, that facilitate the types of experimental studies and analysis approaches described above.

We will test novel experimental and mathematical ideas to extend and expand the currently available methodology and to apply newly developed assays to our research questions.

Example papers from our group: Bioinformatics. 2018, 34(17): i647–i655; Cell Systems. 2016, 3(5):467-479.e12

PhD projects within and across these topics, with different weightings of wet lab and dry lab components, can be discussed and planned.

Techniques

Depending on the precise formulation of the project, it will be a selection from these techniques routinely used in the group:

• NGS; (sc)RNA-seq, ChIP-seq, SLAM-seq, PRO-seq, NET-seq
• Cas9-mediated genome engineering
• Cas-Drop
• Standard molecular biology; (q)PCR, DNA cloning, western blotting, etc
• Mammalian tissue culture; cell line maintenance, transfection etc
• Flow Cytometry / FACS
• Imaging; smRNA-FISH, spinning disc confocal, FRAP, FCS
• Bioinformatics; command line scripting, software tools, R
• Mathematical modelling; Matlab, R, Mathematica, ODEs, stochastic models
• Deep learning