A direct biochemical connection between the pluripotency regulator, NANOG and RNA Polymerase II

The aim of the proposed work is to study how gene regulators known as transcription factors (TFs) work in a specific type of cell termed a pluripotent cell. These cells arise early in mammalian development and can differentiate into all adult cell types, defining them as pluripotent. Pluripotent cells can also be cultured in the lab in specific culture conditions as embryonic stem cells (ESCs). During culture, ESCs divide extensively to produce identical daughter cells, in a process termed self-renewal. At the same time, ESCs retain their multilineage differentiation capacity but this is only unmasked if the culture environment is altered from that supporting self-renewal. Due to these combined properties, ESCs hold great promise in regenerative medicine. However, to effectively realise that potential we need to understand how ESC growth and identity is controlled. ESCs are best characterised in the mouse and for that reason, our study focusses on mouse ESCs.
ESC identity is controlled by a cohort of TFs including a master regulator called NANOG. These TFs bind to sites on chromosomes and some of these DNA sites can influence the extent to which a nearby gene is switched ON. The process of switching a gene on initiates a process known as transcription in which DNA is decoded into mRNA by an enzyme called RNA polymerase II (RNAP2).
In recent exciting work we have identified a direct physical contact between NANOG and RNAP2. This is the first example of a direct contact between a sequence specific DNA binding TF and the central enzyme that transcribes DNA into mRNA. We will investigate this interaction to ascertain how it occurs and determine its function. We will do this by mutating specific residues on each protein separately. This will allow us to identify the parts of the molecules that interact. Our initial analysis of has given us a broad overview of the interaction but to maximise what we learn from these studies we will perform more comprehensive mutagenesis to deliver a high resolution view of the interaction.
Recently, it has been proposed that when TFs and other proteins interact at regulatory sites such on chromosomes, a physical transition occurs called 'phase change', similar to the distinction between oil and water. Phase change occurs when the interacting molecules reach a very high local concentration. We have shown that NANOG undergoes phase change and we will further investigate this to determine whether phase change plays a role in the function of NANOG.
We will also investigate how the interaction between NANOG and RNAP2 is regulated. Transcription is controlled by several phosphorylation enzymes and one of these, called CDK9, acts on both NANOG and RNAP2. Using a technique called mass spectrometry we will identify sites of phosphorylation on NANOG. We will then investigate the effect of phosphorylation on NANOG function by mutagenesis. We will also use mass spectrometry to analyse NANOG-RNAP2 complexes purified from ESCs. Other proteins that bind specifically to the NANOG-RNAP2 complex will be identified by this technique and this will give insights into how the complex functions.
The three-dimensional organisation of chromosomes has a profound effect on the regulation of gene transcription. We will study how the spatial organization changes when the NANOG-RNAP2 complex forms on chromosomal DNA and determine the effect of any changes on the regulation of transcription.
Our study will lead to a more complete understanding of the processes regulating transcription and will have implications for understanding processes fundamental to maintenance of all cell types, how they are regulated and how they may be subverted in pathological states.

Self-renewal of pluripotent mouse ESCs is directly related to the concentration of the transcription factor (TF) NANOG. However, the mechanisms through which NANOG mediates function are unclear. We have identified a direct interaction between NANOG and RNA polymerase II (RNAP2), the central enzyme of the transcriptional machinery. Here we will characterise the NANOG-RNAP2 interaction in molecular and functional terms.
The aims of this work are:
1. Characterisation and functional analysis of NANOG sequences affecting RNAP2 CTD interaction
2. Biochemical and functional analysis of sequence requirements within the RNAP2 CTD
3. Determining the influence of the NANOG-RNAP2 complex on chromatin
In Aim 1, we will define the sequences within a low complexity domain (LCD) in NANOG that mediates interaction with RNAP2. We have found that the LCD enables NANOG to undergo phase transition, consistent with reports of phase change mediated by other, distinct LCDs. We will analyse our NANOG mutant panel to determine if NANOG activity is a function of phase change.
Nanog and RNAP2 are targets of CDK9, a key regulator of transcription. We will identify the Nanog residues phosphorylated by CDK9 and determine the effects of phosphorylation on NANOG function by mutagenesis.
In Aim 2 we will characterize the residues of RNAP2 that interact with NANOG. We will also identify proteins that bind specifically to the NANOG-RNAP2 complex and identify downstream functional mediators.
In Aim 3 we will investigate the role of the NANOG-RNAP2 interaction in chromatin function. We will determine changes in transcription of enhancers upon formation of the NANOG-RNAP2 complex and will ask whether formation of the NANOG-RNAP2 complex at specific chromosomal sites alters the chromatin architecture.
Together, the proposed studies will characterise the NANOG-RNAP2 interaction and advance understanding of enhancer and TF function.

The social beneficiaries of the proposed research are to be found within the wider public.
The general public will benefit from the results of the proposed work mainly in three ways:
1) The proposed research has potential medical implications in the broad field of regenerative medicine. The proposed work will study a direct connection between a key pluripotency transcription factor and RNA polymerase II. As this is the first example of an interaction between a mammalian sequence specific DNA binding transcription factor and RNA polymerase II, this work has the potential to improve understanding of fundamental transcriptional mechanisms. Also, as the work examines a key transcription factor required for pluripotent cell identity, it has the potential to enhance the current understanding of the principles of gene regulation governing the specification of cell identity and the induction of pluripotency during reprogramming. Since the ability to promote reprogramming to pluripotency of differentiated cells from patients is a key step in most cellular replacement therapies, the direct investigation of the principles and factors governing this process is of immediate relevance to the field.
2) The biological research carried out during the proposed project will contribute towards maintaining the high standard of academic excellence currently enjoyed by the Centre for Regenerative Medicine. This will be reflected in the ability of the Centre for Regenerative Medicine and the University of Edinburgh being able to offer educational opportunities for undergraduate and post-graduate students training in our group.
3) The conceptual advances and tangible material, such as pictures, diagrams and illustrations generated to present the results of the proposed experiments will add to the resources used by our science communication staff during outreach activities aimed at disseminating knowledge and raising awareness of the latest advances in the field of stem cell biology and regenerative medicine.

The immediate academic beneficiaries of the proposed research are researchers funded by the proposed research and additional researchers in the Centre for Regenerative Medicine that will benefit from scientific and methodological advances made during the research and disseminated outwith the immediate research group. This will help ensure that young Centre for Regenerative Medicine researchers derive the maximum benefit from the proposed research to apply to their own work.