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Investigation of chromatin remodelling proteins in human trophoblast stem cell identity, differentiation and function

Primary Supervisor: Dr Andrew Nelson, School of Life Sciences

Secondary supervisor: Dr Sascha Ott, Warwick Medical School

PhD project title:Investigation of chromatin remodelling proteins in human trophoblast stem cell identity, differentiation and function

University of Registration: University of Warwick

Project outline:

The placenta, a complex organ composed of both maternal and foetal tissue, is essential for foetal growth and survival in the uterus. Defects in placenta formation and function can have profound and lifelong consequences to both mother and child. The foetal component of the placenta consists largely of highly specialized cells known as “trophoblasts”, of which there are multiple subtypes of distinct morphology and function. Vital aspects of placental function such as hormone production, nutrient exchange, evasion of the maternal immune response and expansion of the maternal blood supply to the foetus are controlled by different trophoblast subtypes [1]. For example, invasive Spiral Artery-Associated Trophoblast Giant Cells (SpA-TGC) derived from the spongiotrophoblast layer of the rodent placenta invade the maternal decidua to replace the lining of maternal blood vessels, thus enhancing blood supply to the developing foetus, while maternal-foetal exchange is regulated by layers of multinucleate syncytiotrophoblasts, which line the interface between maternal and foetal blood spaces along with sinusoidal TGCs within the placental labyrinth. We and others have identified various chromatin-level mechanisms controlling trophoblast fate and behaviour, however, the extent to which this represents human biology is unclear.

All trophoblast subtypes arise from a common trophoblast stem cell (TSC) pool [2,3]. Both the function of individual cell types and their emergence during development are controlled by elaborate gene expression programmes, in part dictated by sequence-specific transcription factors which may either promote or repress gene expression in a context-specific manner. Transcription factor access to gene regulatory DNA elements (known as cis-regulatory modules), however, is controlled by the chromatin landscape wherein nucleosomes act as an accessibility barrier [4]. Thus, changes in the chromatin landscape are critical to the emergence of distinct cell types during development. In this project we will use human TSCs to study chromatin-level gene regulatory programmes controlling the correct formation of different trophoblast cell types from TSCs, and compare with mouse TSCs to explore human mechanisms and the applicability of rodent models. This will be achieved through use of small molecule inhibitors targeting chromatin remodelling proteins and epigenetic modifying enzymes. We will then assess the effects on TSC differentiation using a range of classical approaches, as well as functional genomics and transcriptomics including assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA-seq [5]. This will allow us to predict which regions of accessible chromatin are likely to be functional cis-regulatory modules that control formation of different trophoblast cell populations. We will then use CRISPR/Cas9 epigenome editing to explore the function of putative cis-regulatory modules in target gene regulation and cell fate specification [6]. The result will be a compendium and functionally characterised subset of cis-regulatory modules controlling the formation of distinct trophoblast cell types.

References:

  1. Watson and Cross (2005). Development of structures and transport functions in the mouse placenta. Physiology (Bethesda) 20:180-93
  2. Okae, et al., (2018). Derivation of Human Trophoblast Stem Cells. Cell Stem Cell 22:50-63 e6
  3. Tanaka, et al., (1998). Promotion of trophoblast stem cell proliferation by FGF4. Science 282:2072-5
  4. Shlyueva, et al. (2014). Transcriptional enhancers: from properties to genome-wide predictions. Nature Reviews Genetics15:272–86
  5. Buenrostro, et al. (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 10:1213-8
  6. Thakore, et al. (2015). Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods. 12:1143-9

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

      Techniques that will be undertaken during the project:

      • Functional genomics including ATAC-seq
      • Transcriptomics including RNA-seq
      • Next Generation Sequence data analysis and bioinformatics
      • Stem cell maintenance, differentiation and manipulation
      • CRISPR/Cas9 genome editing

      Contact: Dr Andrew Nelson, University of Warwick