Investigating the role of PIP4K2B, nuclear phosphoinositides and TAF3 in transcription and genome organisation during myogenic differentiation

Cells are constantly being instructed (programmed) to modulate their behaviour because of changes to environment of the body. In particular, continual repair of muscle tissue is essential to our daily lives but unfortunately is a process which deteriorates as we age. Deterioration in muscle function not only decreases our movement and function but also makes us more susceptible to metabolic type diseases such as diabetes and cardiovascular disease.
Satellite cells are specialised cells that are present in small numbers within muscle tissue. When muscles are damaged either by exercise or during disease, the satellite cells become active and produce more muscle cells, a process called differentiation that helps to repair muscle tissue. The activation of satellite cells is complex and depends on signals generated within the environment of the damaged muscle. As organisms age the ability of satellite cells to respond to these signals and generate more muscle cells decreases and in part is responsible for age-induced deterioration in muscle function.

Using a model of muscle cell generation we have found that by controlling an enzyme called PIP4K2B we can increase the ability of a cell to differentiate to produce muscle cells. PIP4K2B controls the levels of naturally occurring molecules called phosphoinositides that are present in the cells. It is well established knowledge that phosphoinositides are present in the plasma membrane of cells where they control many different cellular functions. However, over the years there has been growing evidence that phosphoinositides are also present in the nucleus, the cells' control centre, where their levels change in response to different environments. In fact in response to signals generated during muscle cell differentiation the levels of phosphoinositides in the nucleus go up. We now know that nuclear phosphoinositides interact with and change the function of special proteins in the nucleus that are involved in generating instructions that control the behaviour of the cells. One such protein is TAF3, which is part of different protein complexes that control the programming of cells. Interaction of TAF3 with phosphoinositides reprograms the cell to increase muscle cell differentiation.

In this proposal we will use a novel state of the art technology called CRISPR CAS to change the DNA of cells so that we can investigate how PIP4K2B and nuclear phosphoinositides control TAF3 and its various complexes to increase muscle differentiation. We believe that specialised TAF3 complexes direct specific instructions to the cell in response to the interaction of TAF3 with phosphoinositides and we can investigate this using a technique called CHiP-Seq. We also think that nuclear phosphoinositides together with TAF3 act as a platform that helps to organise which instructions are given and how these are coordinated to increase muscle differentiation. We will investigate this using a novel technique called promoter capture HiC which will allow us to understand the three dimensional aspect of DNA in the nucleus that is used to generate these instructions.

PIP4K2B is a very druggable protein as is the site of interaction between phosphoinositides and TAF3 and we hope that eventually we might be able to use our knowledge to develop drugs to harness this control process within a cell's nucleus and help satellite cells differentiate more effectively, thus aiding the process of muscle tissue repair.

We have uncovered a novel and evolutionarily conserved pathway linking PIP4K2B, which phosphorylates and controls nuclear PtdIns5P, with TAF3, a transcriptional regulator, that together impacts on myogenic differentiation. Inhibiting PIP4K2B leads to increased nuclear PtdIns5P which interacts with the PHD finger of TAF3 to increase the transcription of a selective group of myogenic genes. The TAF3 PHD finger is a multi-ligand binding domain that also acts as an epigenetic reader to interpret histone methylation.

Recent exciting studies have shown that although the rate of transcription is controlled at many levels including by promoter specific transcription factors and RNA polymerase regulation, by juxtaposing distal regulatory elements with promoters chromatin conformation is the level at which many of these control mechanisms are integrated. How dynamic signalling reactions shape these events is poorly understood.
We hypothesise that nuclear phosphoinositide domains function as recruitment platforms where PIP4K2B and TAF3 signalling is integrated to effect different levels of transcriptional control including chromatin conformation to regulate selective gene expression.

We will use CRISPR CAS9 to introduce mutations that dissociate TAF3-PHD finger multi-ligand binding into the endogenous TAF3 gene loci to generate a unique set of isogenic cell lines. These cells lines will also carry a genetic regulatory element that enables the rapid, and reversible control of PIP4K2B protein expression. We will use promoter capture HiC, allowing unbiased analysis of the 3D interaction space of specific loci, coupled with RNA-Seq and CHiP-seq as a powerful approach to interrogate the impact of PIP4K2B and TAF3 on different levels of transcriptional control during myogenic differentiation.

These studies will for the first time reveal how nuclear phosphoinositide signalling can initiate and coordinate dynamic reorganisation of chromatin to impact gene expression output.

People. This project has the potential to impact directly on the general public through impacting on health. PI and epigenetic signalling are implicated in cancer, muscular degeneration and in mental health and inflammatory diseases. Our studies bring these two pathways together and in doing so reveal novel target sites for the potential development of therapies that could be used to combat human diseases. Furthermore recent studies in model organisms show that PI and epigenetic signalling impact on the ageing process and on health span, potentially placing our studies of value in this area. The western world has an increasing aged population above 65 and both medicinal and non-medicinal interventions will be essential to maximise the health span of the population. Therefore the outputs from this research and their further commercial developments have potentially enormous impact on public health, although the realistic timescales to such therapeutic use are in the order of tens of years.The increased knowledge gained from this proposal and its impact on human health and ageing are topics frequently highlighted in the news and are of direct interest to the public. Engaging the public in discussions of the wider issues encompassing our research increases knowledge and fosters an information based society which indirectly impacts on the ability of research councils and universities to lobby government on research funding policies.

Industrial beneficiaries. The research outputs outlined in this proposal could impact on industrial beneficiaries such as small and larger scale pharmaceutical companies. Development of therapies aimed at components of PI and epigenetic signalling pathways are already being developed illustrating their known potential in human health. Our studies will define exactly how a small regulatory molecules in the nucleus interacts with and controls epigenetic signalling and how they impact on the 3D topology of the genome to impact on specific transcriptional output that modulate myogenic differentiation. The PI binding site of TAF3 provides a potential novel target site for commercial exploitation. Small molecules that impact on this site might show allosteric specificity that can be used to harness beneficial effects of regulating TAF3 to restimulate exhausted satellite stem cells for regeneration purposes. Within the time frame of the grant we would hope to generate and strengthen productive industrial partnership interactions which will likely be aimed at developing proof of principle cellular and animal models.

Economy. If target molecules that can impact on TAF3 function and on muscle regeneration can be realised then the eventual potential to the overall economy is large. Revenue from the technology and its exploitation together with increased societal health will impact on the economy of the nation. The second potential for impact into the economy is through training and teaching. The research participants within this project will be trained in sophisticated biological and non-biological areas thereby enabling their contribution to both the academic and professional science base as well as non-academic environments. This research will also provide research placements and lecture material for undergraduate students, thereby contributing to the development of an educated workforce.