The role of Nuclear phosphoinositides in epigenetic signalling

Lead Research Organisation: University of Southampton
Department Name: Sch of Biological Sciences

Abstract

If genetics is represented by a book containing the sequence of DNA encoding humans then epigenetics can be considered as the notes written in the margins by a previous reader. They do not alter the code itself but remind and instruct the next reader how they might interpret the written code. Epigenetics provides the means for environmental signals, such as how much food has been eaten or how warm it is, to be written on top of the genetic code to help instruct how cells and organisms might respond. Like the DNA code the notes can also be inherited. In essence epigenetics provides a rational model for the nature versus nurture hypothesis.

DNA is complexed with proteins to form a structure called chromatin that enables the large amount of DNA to be packed into the very small volume of the cell. Problematically this packing prevents the DNA from being used easily. The epigenetic notes which are deposited on to the components of chromatin help to pack and un-pack the chromatin. The notes are deposited, removed and interpreted by a series of enzyme complexes. Exactly how these complexes are regulated in response to environmental signals is not very well understood.

We have found that one of these enzyme complexes is regulated by an enzyme called PIP5K1A. In response to environmental signals PIP5K1A produces a small molecule called PIP2. Our studies suggest that PIP2 sticks to the enzyme complex and controls how well it works. This leads to changes in the epigenetic notes that are deposited and eventually to how the cell behaves. In this way we think that PIP2 acts a messenger informing and controlling the enzyme complex in response to changes in the environment.

In this proposal we intend to understand how PIP2 sticks to and changes how well the enzyme complex works. The enzyme complex is made up of many different protein components and we have found that one of these proteins can stick to PIP2. By using different molecular technologies we want to find out exactly how PIP2 sticks to this protein and if this alters how well the enzyme complex deposits the epigenetic notes. Understanding the molecular details of this process is important as it increases our basic understanding of how cells respond to their environment. Furthermore the enzyme complexes that deposit epigenetic notes often do not work properly which can change how organisms age. It can also lead to the development of different human diseases such as cancer and diabetes. Understanding how the enzyme complexes are controlled will help us to design medicines that alter how well these complexes work which could be useful in modulating how well we age and how we combat diseases.

Technical Summary

DNA and histones are packaged into chromatin. Epigenetic signaling enzymes that modify chromatin alter the packaging and consequently transcriptional output. For example histoneH3 lysine 4 (H3K4) tri-methylation is associated with enhanced transcription. H3K4 methylation is controlled by six different methylation complexes (MLL-SET) and impacts on differentiation, development, aging and transgenerational epigenetic memory. While much is known about H3K4 signalling and its relationship to transcription much less is known about how MLL-SET complexes are regulated.

Nuclear Phosphoinositides (PI), such as Phosphatidylinositol(4,5)bisphosphate (PIP2), are lipid messengers that change during cell cycle progression, differentiation and adaptive stress responses. Nuclear PI operate by interacting with and regulating proteins often involved in chromatin regulation.

We have identified a direct functional interaction between PIP5K1A, which synthesises PIP2, and an essential core component of all MLL-SET complexes. We suggest that nuclear PIP2 regulates H3K4 methylation by interacting with this core component. We will use an array of molecular and structural technologies to identify: A. components that constitute the nuclear PIP2/H3K4 methylation pathway; B. how PIP5K1A and PIP2 interact with the MLL-SET core component and C. how these interactions might regulate MLL-SET activity in vitro and in cells. Next generation sequencing will investigate how nuclear PIP2 influences global transcriptional output and H3K4 methylation. Finally, we will develop novel technologies to investigate how different sub-nuclear pools of PIP2 impact on H3K4 methylation and transcription.

Epigenetics will provide novel targets to impact on healthy ageing and diseases. Defining how small molecule such as PIP2 might allosterically regulate MLL-SET activity will likely provide highly selective, less toxic approaches to intervene in H3K4 methylation signalling.

Planned Impact

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 these 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 molecule, PIP2, interacts with and controls an epigenetic signalling complex providing novel target sites for commercially exploitation. Industrial partnerships could be instigated within two years. These partnerships will likely be aimed at developing proof of principle cellular and animal models. Within the time frame of the grant we would hope to strengthen and generate productive industrial partnership interactions.

Economy. If target molecules can be realised then the 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.
 
Description we have discovered where nuclear PIP2 is localised and that this pool of nuclear PIP2 is controlled by a nuclear PIP5K1A. surprisingly we have also shown nuclear PIP2 appears to be essential for cells to survive. In the context of a knockout of PIP5k1A , the nuclear Pool of PIP2 strongly decreases but within a number of days, the pool of PIP2 is reconstituted. How this occurs is not clear but suggests a highly dynamic mechanism employed in the nucleus to maintain its effects on transcriptional output. we have also developed a number of novel cell lines in which we have targeted lipid phosphatase domains to the nucleus in order to monitor if they can be used ot modulate the levels of nuclear PIP2 and thereby generate a technology to control PIP2 at will. Furthermore using CRISPR cas9 we have developed PIP5K1A knock out lines and have targeted luciferase to the last exon of a PIP2 dependent gene. this latter cell lines enables us to easily monitor effects of changing PIP2 on transcriptional output both in vivo and in vitro.

we have now discovered how PI interact with ASH2l and have novel data showing that the interaction of PIP2 with ASh2L is important for regulating output signalling by ASh2L and the h3k4methylase complex. our studies also suggest that targeting the interaction domain could be useful as a target to prevent cancer cell growth
Exploitation Route our studies suggest that a number of components that constitute this nuclear transcriptional signalling unit might be useful as targets for a number of different signaling inputs as well as being a target for cancer therapeutic development. furthermore we believe our cell lines will be useful to the research community and that there could be possibilities to develop the luciferase cell line as a bioassay for TNF signalling.

we are interested in targeting the pI interaction site to develop novel allosteric regulators of H3K4me 3 signalling
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title ASHL wild type and PI interaction mutant reconstitution cell lines 
Description we have determined how ASH2l interacts with phosphoinositides and therefore to assess how PI interaction modulates ASH2L outcomes we have developed a cell line that expresses wild type or mutant ASH2l. the endogenous protein can be removed using allele specific RNAI constructs. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2019 
Provided To Others? No  
Impact we have used this to demonstrate the importance of the PI interaction site on ASH2l in controlling various epigenetic signalling outputs as well as tumour cell growth 
 
Title C2C12 expressing TIR1 (for inducible degradation of degron tagged alleles) 
Description in order to degrade AID tagged proteins, a cell lines needs to express the TIR1 protein. TIR1 is auxin sensitive and targets the AID tagged proteins for degradation. we have generated a C2C12 cell line that expresses a doxycyclin inducible TIR1 construct. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact this has enabled us to control the expression of PIP4k2B rapidly and reversibly in order to study downstream effects such as differentiation, cell cycle progression and myoblast fusion. in addition we expect that there will be interest in this cell line from the scientific community. has been used to tag PIP4k2B PIp4k2A in human cells 
 
Title CRISPR_knockout of PIP5K1A in U2OS cells 
Description using CRISPR-CAS9 we have generated a number of PIP5K1A knock out cell lines. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact these are being used to assess how PIP5K1A modulates gene expression and epigenetic signalling. they will also be used to enhance our collaboration with Babraham institute to assess nuclear PIP2 levels 
 
Title U2OS cell line with ASH2 tagged with GFP 
Description CRISPR cas9 used to generate a GFP tagged ASh2l which will be used for chip-seq, analysis of interaction partners and for live cell imaging 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? No  
Impact used chip interaction partner analysis 
 
Title U2OS cells line with knockin flag tag into the endogenous ASH2 loci 
Description we have used CRISPR CAS9 to generate a cell line in which ASH2L has been tagged with the FLAG epitope. 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? Yes  
Impact the antibodies to ASH2l are not sufficiently good enough to be used for CHIP-seq studies. we have developed this line in order to establish a tool for this technique. this should be useful to the epigenetic community 
 
Title manipulating nuclear phosphoinositides 
Description We have generated a panel of U2OS cells lines that express various nuclear targeted PIP2 phosphatases. these are all Ha tagged and will be used to assess how they might manipulate nuclear PIP2 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact these are being used by two other projects within the lab. we will also advertise these on our web site. 
 
Title monitoring PIP2 dependent modulation of the transcription of the BIRC3 gene 
Description in order to easily monitor in vivo and in vitro the effect of PIP2 on transcription we have used CRISP -CAS9 to knockin a luciferase gene into the last exon of BIRC3 . The luciferase is expressed downstream of BIRC3, under the control of P2A self cleavage site, and is a direct measure of BIRC3 transcription. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact we have shown that the transcription of this gene is strongly regulated by TNFa in both a PIP2 and histone methylation dependent manner. The stimulation by TNF suggests that is is a highly sensitive assay that could also be used to generate a bioassay for TNFa have used this line to assess how PIP5K and ash2l depletion modulates luciferase activity. is being used as a screen to test for PI and H3K4me3 modulators in the control of Birc3 
 
Title selection plasmids in order to generate knockin alleles tagged with the auxin inducible degron (AID) domain 
Description In order to carry out rapid and reversible degradation of a protein we have utilised the AID tagging system. In order to use this in the most rigourous manner tagging the endogenous allele in cells is a requisite. the simplest way to do this is to utilise CRISPR CAS9. however the efficiency of tagging is still low especially as both alleles need to be tagged. we have generated a set of plasmids that allow selection of tagging using seven different types of selection markers. Some of these are based on antibiotic selection and others are based on their use with fluorescence cell sorting (FACS) in addition we have incorporated 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? Yes  
Impact this tool has now been used to generate AID Knockin alleles for two separate projects within the department (update 2018) and has been used by others in our department to knockin AID into various endogenous loci 
 
Title tools to deplete nuclear phosphoinostides 
Description we have generated genetically encoded lipid phosphatases that are targeted to the nucleus. the phosphatase domains from 10 different lipid phosphatases known to regulate PIP2 have been cloned in to inducible vectors that target the protein domain to the nucleus. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact these tools are being utilised to assess the role of nuclear PIP2 in nuclear functions. 
 
Title genes regulated by changes in PIP5K1A 
Description gene expression array data set after knocking down PIP5K1A 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact we have been able to define a set of genes that are regulated by changes in PIP5K1A 
 
Title impact of PIP5K1A modulation on H3K4me3 
Description data set to understand how PIP5K1A modulates histone methylation and its relationship to ASH2L 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact defining mechanistically how nuclear PIP2 impacts on gene expression. data has been curated and used to show that PIP5K modulates H3K4me3 signalling similarly to ASH2L 
 
Description measuring nuclear PIP2 in response to depletion of kinases, phosphatases and phospholipases 
Organisation Babraham Institute
Department Signalling
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution we are knocking down various enzymes that are known to regulate PIP2 levels and isolating nuclei to bu used to measure the levels of nuclear PIP2
Collaborator Contribution They are measuring the levels of nuclear PIP2 using mass spectroscopy
Impact multi disciplinary : encompassing genetic modulation of enzyme components with the use of mass spectroscopy.
Start Year 2018
 
Description measuring the impact of PIP2 depletion on histone modifications 
Organisation University of Southampton
Department Centre for Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution we are generating PIP5K1A knockdown and CRISP cas 9 knockout cells and are measuring the extent of PIP2 depletion. we have already shown that this leads to a decrease in nuclear PIP2 levels and in decrease in the extent of H3K3Me3 at specific loci.
Collaborator Contribution They are using Mass spectrometry to determine the extent of other histone modifications that might also act directly downstream of changes in nuclear PIP2
Impact this is a multidisciplinary collaboration encompassing genetic modulation of components of a nuclear phosphoinositide pathway with quantitative mass spectrometry to determine changes in histone modification.
Start Year 2018
 
Description the role of PIP2 in regulating histone methylation complexes 
Organisation State University of New York
Country United States 
Sector Academic/University 
PI Contribution We are identifying how PIP2 interacts with components of the histone methylation complex in order to identify mutants that may no longer interact.
Collaborator Contribution they are measuring how PIP2 might impact on the in vitro activity of the methylation complex. they have purified and reconstituted a core methylation complex composed of ASH2l, wdr5, wd-40 and a methylation subunit such as the set domain from MLL1.
Impact none yet
Start Year 2018
 
Description developement of CRISPR CAS9 techonology for education 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact we developed a CRISPR CAS9 project in order to illustrate the potential of the ues of this technology to students . in essence 18 students were associated with six different research groups and carried out CRISPR to develop novel models of knockouts and knockins for primary research activity. these models have been taken further by researchers within the department and likely will form a basis for novel grant proposals.

the project was developed primarily to enable undergraduate students to experience the excitement of primary research
Year(s) Of Engagement Activity 2018,2019
 
Description hosting summer students within the laboratory 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact six week placement for two summer students to experience primary research activity.
of the two participants (Steven John and Lakshay Nayyar) steven has now taken up a Ph.D studentship within the department.
Year(s) Of Engagement Activity 2018
 
Description university open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact to increase awareness of school students and parents of the educational and research objectives of the centre for biological sciences
Year(s) Of Engagement Activity 2017,2018