Mechanisms of regulation of RNA polymerase II phosphorylation in embryonic stem cell pluripotency and neuronal differentiation

Stem cell therapies promise cures for a plethora of complex diseases such as cardiac, infertility and neurodegeneration. Embryonic Stem cells (ESCs) have the potential to differentiate into any of the 200 different cell types that make up a higher organism. Complex regulatory mechanisms are present to maintain genes important for cell commitment silent in ESCs, but in a metastate which is compatible with activation upon differentiation. Towards devising efficient and safe stem cell procedures, we, and others, are interested in understanding the control mechanisms that maintain important developmental regulator genes silent in ESCs, and the dynamic changes that occur during cell differentiation. Our group has recently shown that an important cohort of developmental regulator genes, including genes that orchestrate commitment into neurons, are poised for activation in ESC. The poised state is characterized by association of silent genes with active and repressive chromatin, and with RNA Polymerase II, the enzyme that initiates the cascade of gene expression by transcribing the genetic information contained in the DNA into RNA molecules that serve as template for protein synthesis. We have found that developmental regulator genes, which are silent in ESCs, are actively transcribed but associated with polymerase II in a novel 'poised' conformation which uncouples the process of transcription from downstream events in gene expression. The presence of poised polymerase II complexes at developmental regulator genes maintains these genes silent in ESCs, but primed for induction in response to differentiation signals. With this proposal, we aim to understand the mechanisms of gene poising in ESCs and the dynamic changes in polymerase and chromatin conformation that accompany gene expression reprogramming during neuronal differentiation, from ESCs to neural stem cells, to fully differentiated dopaminergic neurons. At poised developmental regulator genes, we aim to investigate the activity of molecular complexes known to modulate polymerase II conformation, either by hindering its elongation during transcription, by preventing its phosphorylation, or by actively increasing dephosphorylation. Polymerase II is crucial to integrate events in the cascade of gene expression, as it not only transcribes the DNA sequence, but also recruits nuclear machineries which modify chromatin to make it compatible with transcription and RNA processing activities which promote the maturation nascent transcripts and transport out of the nucleus for protein synthesis. This research will provide important insights into the mechanisms of chromatin regulation that maintain the pluripotent program of gene expression in ESCs, to help understand the mechanisms of induced pluripotency and lineage commitment towards the design of cell therapies. We also aim to elucidate the roles of RNA polymerase II modification in integrating transcription with RNA processing and chromatin states, to help understand the basic mechanisms of gene regulation during differentiation.

The differentiation from ES to specialized cells is one of the most important fields of research in modern cell biology. To unveil the mechanisms by which programs of gene expression are established during differentiation represents a key challenge towards the development of stem cell therapies and differentiation protocols into specific cellular lineages. Recent work has shown that important cohorts of developmental regulator genes are silent in ESCs but primed for activation upon differentiation. We and others have shown that the poised state reflects an equilibrium between Polycomb silencing by histone modification and the presence of poised RNAPII complexes, which elongate through coding regions, but lack Ser2 phosphorylation which uncouples transcription from downstream RNA processing events such as RNA splicing and polyadenylation. Poised RNAPII have not yet been identified in lineage-committed cells, but are likely to occurs since Polycomb silencing is essential during development. The molecular mechanisms that regulate the poised state of RNAPII at developmental regulator genes in ESCs are still unknown. The aims of this proposal are to: 1) investigate dynamic changes in RNAPII phosphorylation during neuronal commitment of ESCs in neural stem cells and post-mitotic neurons, and 2) to address the mechanisms of RNAPII poising regulated by the modulators of RNAPII activity in ESC pluripotency, using a combination of ChIP, siRNA, and transcriptome analyses. The Systems Approach proposed to integrate epigenetic and expression data obtained here during neuronal commitment and upon interference with the regulatory networks that control RNAPII activity in ESCs will provide important insights about the chromatin priming mechanisms that are active in ESCs. The accomplishment of these objectives will clarify important transcriptional mechanisms of outstanding interest in the biology of pluripotent cells and in the basic mechanisms of gene regulation.

Neurological diseases represent a growing cause of mortality and morbidity in the western world with important socio-economic and welfare impact. Although hereditable mutations on specific genes are often associated with these diseases, most are multifactorial and complex diseases, where the mechanisms underlying the diversity of pathological states are still unknown. A major challenge in devising efficient clinical strategies is still the lack of understanding of the complex molecular interactions associated with normal cell function. Dysregulation of transcriptional mechanisms is observed in several neurological disorders, including Huntington's, Alzheimer's, Amyotrophic Lateral Sclerosis and Rett Syndrome. Our proposal addresses basic aspects of RNAPII regulation at important developmental regulator genes in ESCs and during neuronal differentiation. Understanding gene regulation in stem cells is particularly important towards the development of efficient and safe therapeutic approaches to treat neurological disorders. We propose a systems approach that takes advantage of large whole genome datasets of gene expression and chromatin occupancy by regulatory factors which combines the generation of novel datasets with the integration of publicly available resources, across the whole genome in ESCs, neural stem cells (NSCs) and dopaminergic neurons. The resulting datasets will be of value to academic and industry partners. The research program will establish a pipeline for integrating a whole range of chromatin changes related with gene expression states that occur at multiple scales during normal neuronal development. We and others have interests in studying changes in gene expression and chromatin states during neurodegeneration and so far there is no available data of RNAPII conformation or about the roles of RNAPII modulators in the normal state, before mouse models of disease or human samples can be addressed. The research program combines experimental and computational approaches, which will provide a rich scientific environment for training the new generation of scientists in the current post-genomic era. The proposal also allows us to establish novel collaborations within the Clinical Sciences Division (ICL), with Drs Jesus Gil and Meng Li that bridge between the themes of stem cells and neuronal specification, and make our basic research skills in chromatin and RNAPII regulation relevant in the fields of neuroscience. The immediate beneficiaries of the proposed research will be mostly research scientists engaged in basic academic research. However, our research program on chromatin state and RNAPII conformation will provide important insights into chromatin regulation in ESCs and during neuronal cell commitment, towards improving the design of induced pluripotency and differentiation protocols for stem cell therapies. Our datasets will provide an important resource for the scientific community, but also have an industrial and economical benefit towards developing stem cell therapies in drug screens. We will disseminate the results and conclusions of the research to broader audiences. We will present the work at meetings supported through the grant, and at invited seminars in various institutions. We will publish our findings in international peer-reviewed journals with the best possible impact factors, and make our datasets immediately available to the community at the time of publication. Moreover, we work with science journalists to submit press releases of our most prominent publications in science databases such as AlphaGalileo, which then reach the wider public. Imperial College London, and locally at the MRC-CSC, have in place a press release office which endeavor the public dissemination of the most relevant discoveries. We will use that communication system to reach the goal of engaging a wide public audience.