Histone arginine methylation and the control of neural stem cell proliferation and differentiation.

The central nervous system (brain and spinal cord, CNS) is formed from a founding population of committed stem cells, the neural stem cells (NSCs). These cells give rise to the three major cell types of the CNS: neurons (cells that process the information we receive from the outside world and control our behaviour), astrocytes (cells that provide support to the neurons) and oligodendrocytes (cells that form the insulating fatty myelin sheath around the neurons that ensures rapid electrical transmission of electrical impulses). Amazingly, all NSCs are endowed with identical genetic material (DNA), yet they generate many different types of neurons, distinct from each other in their physiological properties and wiring diagram, as well as an unknown number of astrocyte and oligodendrocyte subtypes. How is such cellular diversity achieved from a common set of precursors? The key mechanisms that control NSC differentiation (the generation of specialized cells from immature NSCs) are referred to as epigenetic mechanisms. Such mechanisms do not change the genetic material within the cells, but rather ensure that the right sets of genes are turned "on" or kept "off" within the differentiated cells. These different gene sets comprise the genetic programs that make the cell a neuron, oligodendrocyte or astrocyte. Understanding the epigenetic mechanisms that govern the process of differentiation will provide us with the possibility of manipulating NSCs to form specific cell types that may be required for future stem cell-based
therapies for neurological diseases - e.g. multiple sclerosis, injury, stroke and other neuro-degenerative conditions. We have recently discovered a novel protein, called Schwann cell factor 1 (SC1 or PRDM4) which is an epigenetic regulator of gene expression in developing NSCs. The levels of SC1 must be lowered in order for differentiation to begin. Therefore, SC1 might provide a novel target to direct differentiation of NSCs. However, we first need to understand how the activity of SC1 contributes to development in vivo. We found that SC1 binds to and acts in partnership with another protein, PRMT5, which is an enzyme that modifies chromatin (the complex of DNA and its protein wrapper) in an unusual way. This gives us a handle to start unravelling the mechanism of action of the SC1:PRMT5 complex and its biological consequences. Ultimately, we hope to gain information on how SC1:PRMT5 might be used in re-programming NSCs to adopt specific cell fates for cell replacement therapies.

Recently, we provided evidence that a protein from the PRDM family of transcription factors, SC1/PRDM4, recruits a chromatin modifier, the histone arginine methyltransferase PRMT5, to maintain the undifferentiated cellular state of neural stem cells (NSCs). SC1 and PRMT5 protein expression is high in NSCs and proliferating glial (oligodendrocyte) precursors (OLPs). SC1 is down-regulated at the onset of neuronal or glial differentiation suggesting that control of the "stem-like" state of NSCs might be regulated by activity of the SC1:PRMT5 complex. This further suggests the possibility that the specific histone modification mediated by SC1:PRMT5 (H4R3me2s) forms part of the histone code "signature" of multipotent NSCs and that this modification might be inherited asymmetrically during neurogenic divisions of the NSCs.
The aim of this investigation is to test this possibility in an in vivo animal model in which SC1 is conditionally ablated (SC1flox/flox) in the developing CNS. This conditional knockout will be used in combination with region- or cell type-specific Cre lines to ablate SC1 in specific populations of neural precursors in vivo, in order to investigate the downstream genetic and phenotypic consequences. Our main aims are:
1) to ablate SC1 in all cortical progenitors, by crossing SC1flox/flox mice with Emx1-Cre mice
2) to ablate SC1 in developing OLPs, by crossing SC1flox/flox mice with Sox10-Cre mice
3) to uncover cell type-specific gene networks defined by SC1:PRMT5-mediated deposition of H4R3me2s on a genome-wide scale. We will achieve this by Chromatin immunoprecipitation and DNA Sequencing (ChIP-Seq) with anti-H4R3me2s antibodies, and RNA-Seq from SC1 null and wild type animals. The combined information from these screens will be used to test direct regulation of candidate genes by performing ChIP with anti-PRMT5 and anti-H4R3me2s antibodies.

Regulation of gene expression underpins all biological processes in health and disease. Gene expression is controlled by 1) DNA sequence-specific binding proteins (transcription factors) and 2) chromatin folding and accessibility of DNA (epigenetics). Transcription factors modulate the activity of the basal transcriptional machinery upstream of genes. By binding to and recruiting chromatin modifier, they can also target epigenetic modifications to specific genes. Epigenetic mechanisms that influence the global structure of chromatin can affect many genes simultaneously. Both things go on in parallel with substantial cross-talk between these two levels of control. How a given programme of gene expression is established is therefore a complex genome-level problem and systems-level approaches are required to make progress in understanding the problem.
Our research will cast light on how a novel histone modification - arginine methylation - is targeted to specific genes during NSC development, what genes are targeted and how the different patterns of gene activity are inherited (or modified) from one cell generation to the next. This impinges on the BBSRC priority area "Systems approaches to biological research" and will add substantially to our understanding of how patterns of gene expression are established and controlled, with far-reaching implications across biology and biomedicine.
A major goal of stem cell research is to learn how to direct production of specific cell populations for regenerative medicine, e.g. to replace dying dopaminergic neurons in Parkinson's, or myelin-forming oligodendrocytes in demyelinating diseases like multiple sclerosis (MS). A thorough understanding of how programmes of gene expression are established is essential if we are to make strong progress towards this goal. Much effort is going into generating induced pluripotent stem cells (iPSCs) from somatic cells for this purpose, but it is becoming clear that iPSCs do not regain the epigenetic state of genuine embryonic stem cells (ESCs). This is likely to limit the therapeutic value of current protocols for iPSC production. By throwing light on how a potentially crucial form of epigenetic modification is controlled and by clarifying its role during NSC division and differentiation, our research will make a substantial and lasting contribution towards the common goal of stem cell-mediated regenerative medicine. Our proposed project therefore fits into the BBSRC priority area "Ageing Research: Lifelong health and well-being", with special emphasis on "Developmental factors and health during ageing".
The research is at the "basic science" end of the translational spectrum. However, it is directed to the heart of a crucially important biological problem that is bound to have a major influence on clinical practice. Immediate beneficiaries of our research will include the wider biological and biomedical research communities and charitable organizations dedicated to finding treatments for particular neurological disorders e.g., the MS Society, Autism speaks, Rethink, the Alzheimer's Society. These charities will communicate this to their stakeholders (patients, carers and medical professionals) who are eager to hear of advances in stem cell research and its potential applications. By opening up avenues for future drug discovery our work will also be of interest and benefit to the pharmaceutical industry. Moreover, a new Research Assistant will be trained in the course of the project.
Stem cell research is a topical area of great interest to the public. Therefore, Dr Chittka runs a series of popular public seminars at a local secondary school to explain our research and help them appreciate the medical potential of stem cell research generally.