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Single cell resolution analysis of transcriptional regulation in development by in vivo 4D imaging

Primary Supervisor: Professor Ferenc Mueller, MDS

Secondary supervisor: Dr John Fossey, School of Chemistry

PhD project title: Single cell resolution analysis of transcriptional regulation in development by in vivo 4D imaging

University of Registration: University of Birmingham

Project outline:

Transcriptional regulation is the central mode of regulation of embryonic development. Its disruption, often due to mutations in enhancers and promoters, leads to multifactorial and congenital disorders. Due to the lack of understanding of the underlying transcriptional codes and their roles in developmental gene regulation, however, we still are unable to predict the developmental consequences of changes in transcriptional regulatory elements at various stages of development, from the onset of transcription to completion of morphogenesis. The primary aim of this studentship is to understand the mechanisms, by which transcription regulation coordinates the first steps towards differentiation of pluripotent stem cells of the early embryo. We will study the control of promoter and enhancer activity dynamics at the first developmental transition in the early embryo and will aim to understand how dynamic transcription regulation of individual genes in single cells contribute to morphogenesis. This project will build on our previous work, which combined computational genome-wide analysis (e.g. Haberle et al., 2014) with the recording of transcriptional initiation events at nascent transcripts by high-resolution in vivo imaging and genome editing at key individual genes in an early model vertebrate embryo. We shall exploit the transparency of zebrafish embryos and utilize a novel 4D visualization technology we recently developed (Hadzhiev et al., 2019) to detect transcription initiation on individual genes. We shall ask the question: how is transcription initiation dynamics regulated at the level of a single gene in cells of the differentiating embryo? We will predict promoter sequence determinants of transcription dynamics by genomics data analysis and subsequently we will monitor transcription of individual genes by using antisense oligomeric probes and various aptamers (Autour et al., 2018), which we will generate in collaboration with with co-supervisor Fossey (School of Chemistry).We will exploit genome editing of candidate regulatory elements in embryos (Hadzhiev et al., 2021) to validate predicted sequence determinants. We shall integrate the single-cell based transcription data visualized with global analysis of transcription regulation by computational genomic approaches (e.g. CAGE- sequencing) to model transcription regulation during early embryo development.

References:

  1. Hadzhiev, L. Wheatley, L. Cooper, F. Ansaloni, C.Whalley, Z. Chen, S. Gustincich, R. Sanges, S.M. Burgess, A. D Beggs, F. Mueller. (2021)The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation. bioRxiv 2021.08.09.455629;
  2. Hadzhiev, Y., Qureshi, H., Wheatley, L., Cooper, L. Jasiulewicz, A., Wragg, J., Nguyen, H., Poovathumkadavil, D., Conic, S., Bajan, S., Sik, A., Hutvagner, G., Tora, L., Gambus, A., Fossey J., and Müller F. (2019) A cell cycle-coordinated nuclear compartment for Polymerase II transcription encompasses the earliest gene expression before global genome activation Commun. 11;10(1):691.
  3. Haberle, , Li, N., Hadzhiev, Y., et al., Carninci, P., Müller, F.*, Lenhard B. Two independent transcription initiation codes overlap on vertebrate core promoters. Nature, 507(7492):381-5.*co-corresponding author
  4. Autour A, C Y Jeng S, D Cawte A, Abdolahzadeh A, Galli A, Panchapakesan SSS, Rueda D, Ryckelynck M, Unrau PJ. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nat Commun. 2018 Feb 13;9(1):656.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Stem Cells & Systems Biology

Techniques that will be undertaken during the project:

  1. 4D Imaging with light sheet microscopy and image analysis of hundreds of gigabytes of light sheet image data (Mueller)
  2. Image processing and large data analysis (Mueller
  3. CrisprR targeting to generate lesions in genomes (Mueller)
  4. Synthetic chemistry of Morpolino oligonucleotides and aptamers (Fossey)

Contact: Professor Ferenc Mueller, University of Birmingham