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Final Report Summary - NUC-INOSITIDE (Investigating the topography of nuclear phosphoinositide signalling in relation to chromatin and the genome)

Final publishable report
The two main objectives of the grant were:
Objective I. To understand the topographical environment of nuclear PI and the enzymes that modulate them in relationship to different histone marks.

Objective 2: To determine if and how specific modulation of nuclear PI in different chromatin landscapes functionally regulates transcription.
In order achieve these objectives we have concentrated on one aspect of phosphoinositide modulation within the nucleus of C2C12 cells. These cells are a model for muscle differentiation and enabled us to investigate in detail how modulation of nuclear phosphoinositides impacts on this process. Control of muscle differentiation and function is of considerable interest to the ERA and has strong indications for ERA competitiveness.
PIP4K2B is a lipid kinase that phosphorylates PtdIns5P to generate PtdIns(4,5)P2 and in doing so controls the levels of both of these lipids in various subcellular compartments. This lipid kinase family has been implicated how organism respond to stress, control their immune responses and control tumour development. We have found that by decreasing the expression of PIP4K2B and therefore its activity there is a strong increase in C2C12 muscle cell differentiation. This was observed at the level of transcription. As observed in other cell types, PIP4K2B is present in the nuclei of C2C12 cells and we therefore hypothesised that PIP4K2B controls the topographical environment of nuclear PtdIns5P and nuclear PtdIns(4,5)P2 which in some way impacts directly on differentiation.
Major results.
Objective 1: Our experiments detailed how PIP4K2B impacts on muscle cell differentiation. We found that PIP4K2B is present in the nucleus and upon differentiation relocalises to the cytoplasm. Specifically designed assays revealed an increase in the levels of PtdIns5P associated with nuclear chromatin upon muscle cell differentiation, which was enhanced by knockdown of PIP4K2B. These data suggest that control of PIP4K2B localisation impacts on nuclear PtdIns5P. TAF3 is a component of the basal transcription complex that couples transcriptional output with environmental signalling by sensing the histone mark H3K4me3. We found that TAF3 interacts with PtdIns5P and acts as a nuclear sensor that couples changes in nuclear PtdIns5P to transcriptional output. The development of international collaborations (Dr. S. Lauberth (University of California), Dr. Haramis (Institute of Biology (IBL), Leiden University), Dr. Hart (University of California, San Francisco,), Prof Fischle (Laboratory of Chromatin Biochemistry, Max Planck Institute for Biophysical Chemistry, Germany) ) allowed us to demonstrate that this PIP4K2B/TAF3 pathway was evolutionarily conserved (in zebra fish) and that nuclear PtdIns5P impacted on the ability of TAF3 to sense and interpret the levels of the histone mark H3K4me3. This histone mark is strongly associated with modulating transcriptional output. Our studies reveal a unique relationship between nuclear PI levels and transcriptional output through direct control of a reader protein that interprets changes in the histone mark H3K4me3. The study was published in the high impact journal Molecular Cell and a graphical abstract of the data is presented in figure 1.
The results outlined above enabled the development of a novel technique termed PI-DAMid which in combination with Next Generations Sequencing (NGS) has been used to establish the relationship between nuclear PI rich regions and specific genomic DNA regions. Initial analysis of this NGS data suggests that nuclear PI are not randomly distributed through the nucleus but are associated with specific regions of the genomic DNA. Furthermore associations with genomic regions change upon differentiation suggesting that PI may impact on transcriptional by controlling the topography of genomic DNA regions.
Objective 2 was based around the development of tools to manipulate nuclear PI levels. We hypothesised that we could use a synthetic lipid phosphatase (pseudo-Jannin) to target the removal of nuclear PtdIns4P and PtdIns(4,5)P2. We obtained pseudo-Jannin (Dr. J. Hammond) and re-engineered it to generate an inducible nuclear targeted construct. We initially concentrated on a version of the enzyme able to remove PtdIns(4,5)P2 as we could directly measure the levels of this lipid in the nucleus using antibody staining. Both stable inducible cell lines and transiently transfected cells were used however, extensive experiments led to the conclusion that they were unable to reduce the amount of PtdIns(4,5)P2 within the nucleus. We hypothesised that that this might be due to the specific phosphatase domain used to generate pseudo-jannin and therefore we cloned 10 different lipid phosphatase domains that can hydrolyse PtdIns(4,5)P2 and targeted them to the nucleus. None appeared to be able to decrease PtdIns(4,5)P2 , although they were expressed and targeted to the nucleus. We next manipulated other enzymes that might regulate nuclear PtdIns(4,5)P2 and have recently identified one which apparently controls the levels of nuclear PtdIns(4,5)P2 (see figure 2, manipulated refers to targeting a specific gene to decrease nuclear PtdIns(4,5)P2 levels). In addition we have also targeted a bacterial PtdIns(4,5)P2 phosphatase, IPGD, to histone state specific regions using the TAF3 domain, and showed that this leads to changes in transcriptional output of muscle cell differentiation specific genes. This information is being used to further develop highly controllable tools to manipulate nuclear PI levels in order to investigate their impact on transcription and nuclear function.
Training aspects of the Marie Curie fellowship:
As a consequence of the enabled mobility to the INGM I have fully developed an understanding and a working methodology for carrying out CHiP experiments and their subsequent analysis. These skills have been transferred and implemented in my new laboratory based at the University of Southampton. I have also developed new skills to carry out bioinformatic analysis which includes the curation and analysis of high through put gene expression array analysis and the basics of NGS data analysis. I have also developed basic R scripting skills and have developed intermediate skills in programming with Python. I will further implement these studies by undertaking courses on advanced NGS bioinformatics (Hinxton bioinformatics course) and through their use in the analysis of NGS data generate as a consequence of the development of our new PI-DAMid technology.
In addition through a unique opportunity enabled by the Marie Curie fellowship and the newly established collaboration with Prof. M. Pagani (INGM), I have developed novel expertise in T cell signalling. By transferring my expertise and knowledge in phosphoinositide signalling to members at the INGM, we have developed a new project investigating the role of PIP4Ks in Treg cell proliferation and function. This has led to a proposal to request funding from the AIRC (Italian Cancer research council) to maintain this international collaboration between the University of Southampton and the INGM and extend it to the newly established cancer Immunotherapy centre based in Southampton.

Potential Impacts: As PIP4K2B is a highly druggable enzyme our data suggest that pharmacological modulation of its activity could be used to aid muscle differentiation which could impact on muscle recovery after injury or after disease states that effect muscle function such as cancer. In this respect our studies might have socio-economic impact on patients that suffer from mobility issues as a consequence of muscle dysfunction. The study also outlines the potential for the development of drugs which can target PIP4K2B that could be used to aid muscle cell differentiation, thus opening novel routes to economic impact by initiating pharmaceutical interaction. Furthermore our studies open up new avenues of research based on the ability of nuclear PI to modulate epigenetic signalling. Epigenetic signalling essentially couples environmental changes to genome control of cell fate. This subtle aspect of genomic regulation is important in the different responses observed between patients in their response to pathogenic stimuli and to drugs such as anticancer therapeutics. Understanding how these pathways function should impact on the development of personalised medicine applications.

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