Cell-Specific PRC1 Accessory Proteins and the Regulation of Mammalian Neurodevelopment

Mammalian embryonic development is the process whereby a single naïve cell is expanded, and serially specialised, to produce all of the functional cell types of the adult body. This intricate process is governed by proteins, which bind to regulatory DNA sequences and establish the appropriate temporal and spatial patterns of gene expression. One such family of protein effectors form multi-subunit-assemblies termed polycomb repressive complexes (PRCs), which function to block gene expression by chemically and physically modifying chromatin - the packaged form of DNA.

In the developing brain, changes in cell state occur as progenitor cells proliferate, migrate and specialise, and PRCs have been shown to be important for these steps. Consequently, loss of PRC function in this context leads to cellular and structural abnormalities. Weaver syndrome, a disorder characterised by cognitive impairment and often brain overgrowth, is caused by the loss of PRC function. Moreover, mutations that block PRC activity occur in the majority of cases of diffuse intrinsic pontine glioma (DIPG) in which they promote tumour formation. PRCs are also required for the regulation of cell proliferation, specialisation and protection against neurodegeneration in the adult brain. Mechanistic interpretation of these cellular and structural abnormalities, however, is hindered by the lack of understanding of how PRCs regulate neurodevelopmental gene expression patterns. Filling this knowledge gap is key to understanding not only the molecular basis of brain development, but also in determining the underlying deficits in developmental brain disorders.

To this end, it is important to determine the molecular mechanics of PRC function and targeting during neurodevelopment. My key aims are: 1). Determine the extent and functional importance of alterations in PRC complex composition during neurodevelopment. 2). Identify and manipulate proteins which act either directly, or in collaboration with chromatin modifications, to recruit PRCs to their target genes during neurodevelopment.

To address these aims I will utilise artificially differentiated and primary cultured neural progenitor cells (NPCs) in conjunction with high-throughput analysis of chromatin state, quantitative analysis of protein composition of PRC bound chromatin, genome-engineering and synthetic protein targeting approaches.

A complete understanding of the molecular basis of disorders of the brain which arise from PRC dysfunction will be critical as a step towards establishing effective and safe therapeutic approaches, prognostic and diagnostic tests and to inform clinical support.

The polycomb system is an important epigenetic regulator involved in directing differentiation, and cell identity. In the neural lineage, perturbations to this system are causal in both congenital and acquired brain pathologies, yet how polycomb proteins regulate transcriptional programmes during normal neurodevelopment remains poorly understood. I aim to elucidate how polycomb regulates neurodevelopment by determining how cell-type-specific protein factors alter the targeting and function of polycomb repressive complex 1 (PRC1). To address this I will combine state-of-the-art molecular approaches and advanced computational analysis to: 1). Determine the protein composition of PRC1 complexes in in vitro differentiated and ex vivo cultured neural progenitor cells (NPCs). 2) Apply auxin-induced degradation (AID) to reversibly deplete PRC1 components in mESCs and determine the impact of depletion on gene repression and the capacity to differentiate ESCs into NPCs. 3). Determine if neurodevelopment is disrupted, both in vitro and in vivo, in separation of function mutants (Ring1b I53A) in which the E3 ubiquitin ligase activity but not the structural function of RING1B is abolished. 4). Identify and validate NPC-specific PRC1 targeting factors using a combination of quantitative proteomic analysis of PRC bound chromatin and AID. 5). Determine the logic of PRC1 recruitment in NPCs by targeting prospective chromatin-recruitment factors with synthetic tools (TALs) in isolation or in combination with chromatin modifiers. Though these objectives I will determine not only the molecular basis of neuropathologies associated with PRC dysfunction but also provide insight into how cell-type-specific proteins adapt or target epigenetic systems to regulate gene expression.

Neurodevelopmental disorders result from a variety of insults that lead to impaired growth and development of the brain. In some instances such as Fragile X-syndrome, the underlying genetic cause and mechanistic impairments are clearly demonstrated. For autism spectrum disorders (ASDs) however, several hundred genes, with seemingly diverse functions, are associated with disordered brain development. This obscures our ability to identify clear unifying models of disease causation and progression. In this proposal, I focus on the role played by polycomb proteins in directing normal neurodevelopment, so as to better understand the mechanistic perturbations which arise from polycomb dysfunction (e.g. Weaver syndrome, DIPG).

Who will benefit from the research and how?

1. The RA recruited on to this project will benefit from opportunities to develop professional and technical skills throughout the course of the project. Recruited principally for their skills in molecular biology and biochemitsry, The RA will develop additional skills (e.g. computational analysis, stem cell biology) through exposure to local expertise and designated University training opportunities.

2. The immediate scientific community represents the primary beneficiaries of discoveries arising from this project. A detailed understanding of one biological system will likely identify generalities that can be explored in other cell types, tissues and disease settings. In combination with the data and technical resources that will be generated during the course of this project, this will provide new research avenues to explore.

3. At the stage of peer-reviewed publication (years 3-5), comprehensive mechanistic insights into neurodevelopmental regulation will directly inform Biomedical Research Science by:
a). Providing unifying mechanisms of disease progression (e.g. ASD).
b). The capacity to ascribe functional relevance to disease associated genetic lesions (genome-wide association studies - GWAS).
c). Informing the use of appropriate epigenetic small molecule inhibitors to impact on the molecular basis of the disease (e.g. EZH2 inhibition in DIPG - PMID: 28263309)

4. A significant impact beyond the timescale of this proposal will be to inform clinical decision-making. The development of genetic screening assays based on a clear understanding of the molecular basis of the disease, could be used to predict both the progression and severity of a disorder. In many cases, neurodevelopmental disorders have no viable cure, however informed early intervention provides the most opportunity for developing a full range of skills, and in so doing has the potential to enhance the quality of life of the patient.

5. How polycomb regulates neurodevelopment could be leveraged to great effect in optimising culture based model systems. Models which more closely recapitulate some aspects of tissue structure and composition (e.g. brain organoids), is central to the reduction of animal use in scientific research (NC3Rs framework). By modulating polycomb function or targeting through either small molecule inhibition or synthetic targeting approaches there is significant potential to skew the production of more functionally desired model systems of benefit to much of the scientific community.

6. This project involved the development of state-of-the-art technology, both at a molecular and computational level. Having been involved in intellectual property protection in the past, I will look to capitalise on novel technologies that arise during the course of this work where commercialisation opportunities exist.