Less than 2% of the human genome encodes protein coding genes. But many trait-specific and disease specific mutations seem to map away from such coding sequences. This paradox is partially resolved by observation that some of the noncoding sequences are involved in regulation of when and where in the developing organism genes are to be turned on and off. One class of such regulatory sequences is called enhancers, since they have a property to greatly enhance gene expression. Genomic DNA in the cells is physically organized in the form of chromatin, which consists of DNA wrapped around histone proteins. Specific combinations of chemical modifications of histones form a basis of epigenetic marking system, which helps to organize the genome into functional domains, some of which are active, while others are silenced.
In human embryonic stem cells two different epigenetic signatures are associated with, and specifically distinguish, two classes of enhancer elements. One signature marks enhancers that are actively turned on in embryonic stem cells, and another marks class of enhancers that we dubbed "poised enhancers", which are not active, but are kept in a state of anticipation that allows them to become rapidly activated when stem cells undergo a decision to differentiate. Here we propose a series of experiments aimed at elucidating why and how the poised enhancer signature is formed, and how it transitions to an active signature during differentiation and does so in a cell-type specific manner. Results of such experiments will greatly extend our understanding of how the genomic information is interpreted to form the multitude of human tissues during development.
Why is enhancer regulation important for stem cell biology and its biomedical applications? Basic research on enhancer regulation in embryonic cell types proposed here is important for uncovering fundamentals of early human development and understanding of nature of pluripotency and commitment. In addition to novel insights into developmental gene regulation in humans this work may have unexpected, immediate and broad applications for regenerative medicine. For example, discovery of poised enhancer signature in embryonic stem cells identified a set of over 2,000 putative early developmental enhancers in a single study, thereby creating an invaluable resource for generation of reporters for lineage tracking and isolation of transient cell populations representing early steps of human development.
Our research will uncover fundamentals of gene expression regulation during early human development and further our understanding of pluripotency and cell commitment, and will create a solid foundation of knowledge as well as novel tools for translational research aimed at development of new stem cell therapies based on directed differentiation of stem cell populations. Our research will also uncover and characterize novel human regulatory sequences that can be utilized in personalized medicine.
Other tangible and immediate benefits for the community include:
- contribution to the training of new workforce in a set of unique skills in human stem cell technology
- creation of new intellectual property that would benefit local institution and by extension local community.
- boosting local economy since we buy our supplies from local vendors whenever possible.
The overall goal of this proposal is to dissect the molecular mechanisms by which specific classes of gene regulatory elements, called enhancers, act to control cell fate and orchestrate the early stages of development. The applicant describes two classes of enhancers in human embryonic stem cells (hESC), one associated with active genes, and the other associated with genes that are poised to become active upon differentiation. As each class can be distinguished by an epigenetic signature, the applicant hopes to elucidate how the poised state becomes converted to an active one as hESC differentiate into neural cells. Three specific aims are proposed to assess these phenomena including 1) exploring the role of specific demethylases; 2) probing the dynamics and nature of long-range chromosomal interactions; and 3) investigating the coordinated regulation of specific histone modifications.
Significance and Innovation:
- The experiments proposed are cutting edge and technologically innovative.
- If successful, the project will yield novel insights about fundamental mechanisms of pluripotency, differentiation and epigenetic regulation in stem cells. The identification of novel enhancer regions has the potential to inform all aspects of stem cell biology and may be relevant to many other developmental transitions.
Feasibility and Experimental Design:
- The proposal is very well written and based on a comprehensive and compelling body of preliminary data, much of which has been published by the applicant.
- The experimental plan is logical and sophisticated. Potential pitfalls and alternative strategies are identified and described.
- The proposed approaches are well within the capabilities of the applicant and collaborators.
Principal Investigator (PI) and Research Team:
- The team is well qualified to succeed. The PI has emerged as a leader in the field of chromatin biology with an outstanding track record and impressive list of publications.
- Trainees and co-investigators with established expertise are in place and poised to move this project forward rapidly. An ongoing collaboration with an inventor of the cutting edge methodologies employed in Aim 2 ensures a high likelihood of success.
- The research environment and facilities are excellent.
Responsiveness to the RFA:
- The proposed studies utilize human cells and directly address molecular mechanisms that are relevant to stem cell biology.
- Ali Brivanlou