The physiological template of our genome, called chromatin, is composed of DNA wrapped around histone proteins. In the process of development the genome is interpreted in a way that is dynamic, and yet, often heritable, to produce different specialized tissues and organs. A substantial portion of information that is required for proper interpretation of the genome is transmitted in a form of methylation of histones and associated DNA. Methylation marks are written by specific enzymatic activities, called methyltransferases. Different methyltransferases can activate or repress genes, and the right balance between the two is critical for proper execution of the developmental programs. Thus, not surprisingly, deregulation of methyltransferases leads to human disease, most notably cancer.
Here we propose to address how the interplay between “activating” and “silencing” methylation signals regulates gene expression patterns in embryonic stem cells and during their differentiation along the neural lineage. These studies will advance our knowledge of the unique properties of chromatin in embryonic stem cells and will address the mechanisms of gene expression during neural commitment. This basic science foundation will be necessary for development of efficient protocols to direct the differentiation of stem cells into therapeutically useful neural tissues. In addition to advancing basic knowledge, our studies will lead to development of novel technology that takes advantage of the latest developments in bioengineering and proteomics. This technology will be broadly applicable in studies of stem and progenitor cells, human and from different model organisms and will push stem cell research forward.
We believe the proposed research will benefit people of California in the following ways:
It will result in development of novel technology that will be broadly applicable to study different stem and progenitor cells and will help position us and other Californian scientists at the forefront of stem cell research.
It will help uncover unique biological properties of embryonic stem cells.
It will generate wealth of information on novel molecules involved in embryonic stem cell pluripotency and differentiation. This information will provide a foundation for development of stem cell-based therapies.
It will increase experience and knowledge of embryonic stem cells among residents of California. This project involves cooperation between three laboratories with complementary expertise. The interaction will facilitate skill exchange and staff training in cutting edge multidisciplinary approaches.
SYNOPSIS: This application is aimed at better understanding the mechanism by which stem cells direct pathways of either self-renewal or cell differentiation to specific lineages. The Principal Investigator Dr. Wysocka, an Assistant Professor of Chemistry and Systems Biology at Stanford, proposes to examine the chromatin modifications that may be associated with pluripotency in embryonic stem (ES) cells. At a basic transcriptional level, this involves methylation of histone lysine residues, and recent studies have identified two H3K methylation sites that if doubly modified represent bivalent domains that are highly enriched in ES cells. It is hypothesized that these bivalent domains represent chromatin that is poised to become either activated or repressed. The first aim seeks to identify proteins that may recognize the bivalent mark (H3K4 and H3K27). The second aim addresses the mechanism of bivalent domain formation. Is the bivalent structure linked to stalled PolII? Finally, the third aim focuses on the role of Polycomb and Trithorax methyltransferase complexes in undifferentiated ES cells and during differentiation toward a neural fate. In this latter aim the PI will seek new protein partners of these complexes using state of the art proteomics.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The major benefit of this research is that it will significantly expand our basic knowledge of the fundamental mechanisms governing gene regulation in mammalian development and specifically fate determination in hESCs. Understanding how chromatin regulation influences the differentiation state and pluripotency is a major topic in the stem cell field, and the impact of an improved understanding may be great in terms of somatic cell reprogramming. This application rests on the strong background of the investigator. The PI has extensive biochemical experience and is linked up appropriately with collaborators for proteomics and for ES cell studies. The application is clearly laid out and the likelihood is high that this productive young investigator will obtain meaningful results.
The proposal is divided into three hypothesis-driven aims. Aim 1 seeks to use an unbiased biochemical pulldown approach to identify factors that are bound to methylated histone tails. This experiment addresses the important question of how bivalent domains may be translated into gene expression changes and proposes to test the hypothesis that there may be a factor that specifically recognizes K4/K27 marks. The PI has successfully used this technology in her previous lab and the aim should be feasible; however, one criticism of Aim 1 is that it is very risky and will not be informative if no proteins are pulled down.
The second aim will characterize the state of PolII activity at bivalent domains and its dependence on the Polycomb member Eed. Little is known about the mechanism by which genes are silenced at bivalent domains and how this inhibition is released upon differentiation. The PI will examine the state of PolII (stalled or elongating form) in the absence or presence of the Polycomb member Eed to determine (i) if silencing is due to stalled PolII and (ii) if this is dependent on Polycomb binding in ES cells and neural progenitors. Provided that permanganate footprinting is an established technology in the lab (there is no description of the technique in the application), this seems like a feasible experiment in the PI’s lab that will shed light on the role of PolII stalling and Polycomb in the silencing of bivalent gene expression. Because of the expertise available, this project will focus on murine ES cells. In preliminary studies the investigator has demonstrated feasibility including the ability to differentiate mouse ES cells to Nestin positive enriched cultures and to use a peptide pulldown assay for identifying proteins associated with methylated histones thus demonstrating proof of principle for accomplishing Aims 1 and 2. Overall, the experiments appear quite feasible and are likely to yield new discoveries.
Aim 3 will identify interacting proteins of several Polycomb and Trithorax proteins in ES cells and neural progenitors to identify potential non-histone targets. This is an interesting question that will be addressed by using novel microfluidic affinity purification approaches combined with mass-spec, which will be facilitated through collaborations with Drs. Ma, Burlingame and Quake who are world leaders in microfluidics and mass spectrometry technologies. In addition, the investigators have established mouse ES cell lines that express flag epitope tagged Eed and Wdr5, which are complements of the polycomb and trithorax complexes that will be used in Aim 3 experiments. There is some concern that constitutive overexpression of PRC2 and MLL members may prevent differentiation into neural lineages or may cause toxicity. The use of an inducible lentiviral system seems more intuitive. Also, the microfluidic chip and lab on a chip technology is still in the development phase. If the technology is not fully developed or unavailable it may limit the ability to carry out experiments proposed in Aim 3.
Finally, reviewers have little doubt that this investigator will be productive and successful; however, the proposal has two general weaknesses. The main criticism relates to the weight placed in the application on the "bivalent" domain model put forward by Bernstein, Lander and colleagues. Although it was first portrayed as a mark specific for embryonic cells, increasingly it is apparent that this is not the case and its relationship to the unique properties of ES cells and pluripotency is not so clear. One reviewer believes that Aim 3 dealing with the Trithorax and Polycomb complexes may be more informative as to mechanisms specific for stem cells. Also, there is a general lack of explanation for certain techniques (e.g., permanganate footprinting, lentiviral transduction in ES/NSC cells), and the level of sensitivity of the mass spectrometry for detecting protein subunits of low abundance could be a concern.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Wysocka has been an Assistant Professor in the Department of Chemical and Systems Biology and Developmental Biology at Stanford University School of Medicine for the past year. The PI is exceptionally well trained. Her PhD focused on globin gene regulation and chromatin biochemistry with Emery Bresnick at Wisconsin, and her postdoctoral studies at Rockefeller with Dr. Allis represented superb training in the biochemistry of histone modifications. She is the recipient of a CIRM SEED grant entitled “Role of Chromatin Modifiers in Regulating Human Embryonic Stem Cell Pluripotency” which has some parallels to the current study but is focused on hESCs whereas the current proposal involves murine ES cell studies. Dr. Wysocka has an exceptionally strong publication record with many publications in high impact journals including several publications in Cell, Science, and Nature as well as Molecular Cell. Her letters demonstrate that she is an “extremely gifted young scientist” and a “top notch candidate", and she is highly competitive externally as shown by the Searle Scholar Award. Her career development plan is clear and appropriate, and she has a high chance of becoming a leader in the field.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: In addition to her very strong pedigree, Dr. Wysocka is in a fruitful and supportive environment at Stanford. The institutional commitment is outstanding with an 11 bench laboratory and minimal teaching and administrative responsibilities. This PI was highly sought after by Stanford and the institution is clear in its support of her career. The strong institutional commitment and excellent track record predicts a high likelihood of success in completing the aims of the proposal.
DISCUSSION: Two reviewers called this the best application they reviewed. Significant questions are being asked, and the PI has unique expertise, a proven track record, and strong collaborators. One reviewer noted that the only reason the score was not 100 is based on the focus on the bivalent domain as a specific marker for pluripotency, which it may not be. The PI will take what has been learned in mouse and give it a human focus. This is a rational approach with hypothesis driven aims, and the only other minor weaknesses are the lack of detail in the description of certain experiments, and the micro fluidic chip technology proposed with collaborators seems to be in development and not yet validated.