Mechanisms of chromatin dynamics at enhancers during ES cell differentiation

Mechanisms of chromatin dynamics at enhancers during ES cell differentiation

Funding Type: 
New Faculty II
Grant Number: 
RN2-00905
Award Value: 
$1,726,564
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
The human ES cells are euploid cells that can proliferate without limit and maintain the potential to differentiate into all cell types. Differentiation of human ES cells involves selective activation or silencing of genes, a process that involves not only combinatorial interactions between the cis-regulatory sequences and DNA binding transcription factors, but also post-translational histone modifications and other epigenetic mechanisms such as DNA methylation and non-coding RNAs. A number of transcription factors have been found to be essential for the ES cells to maintain their identity or differentiate along specific lineages. These regulators exert their effects through interacting with the promoters, enhancers or silencer elements to modulate the expression of target genes. Currently, the molecular details of how transcription factors modulate target gene expression upon binding to DNA and how they mediate ES cell differentiation are still unclear. The proposed project is aimed to determine the role that histone modifications play in this process. We will conduct experiments to identify the DNA binding proteins, chromatin modifying enzymes and chromatin binding proteins that are responsible for the specific histone modification profiles at regulatory DNA sequences , and investigate how these proteins activate target genes and contribute to the unique properties of human ES cells. Results from the proposed study will improve our understanding of the mechanisms that control pluripotency and lineage specification, and lay a foundation for development of better tools for manipulating and reprogramming human ES cells for regenerative medicine.
Statement of Benefit to California: 
Our research will provide better understanding of the mechanisms by which human ES cells differentiate along specific cell lineages. Such knowledge will facilitate the development of new methods for manipulating the ES cells, and enable a mechanistic understanding of the reprogramming of somatic cells to the pluripotent state. These results will directly support the efforts by us and other California researchers to investigate the mechanisms of stem cell biology, and design new stem cell therapies.
Progress Report: 

Year 1

The human ES cells have the remarkable properties of self-renewal and unlimited potential to differentiate into other cell types in the body. Cell fate determination in ES cells involves selective activation and silencing of specific genes, a process that is accompanied by dynamic modifications of the histone proteins. Among the histone modifications that take place during ES cell differentiation are dynamic methylation of histone H3 at a class of regulatory DNA known as enhancers. Currently, the molecular mechanisms leading to the formation of this characteristic histone modification at enhancers are still unknown. The proposed project is aimed to answer this question. We hypothesize that enhancer function requires binding of sequence specific transcription factors that recruit histone modification enzymes. We also hypothesize that the histone modifications at enhancers in turn facilitate recruitment of chromatin binding proteins and possibly additional transcription factors to mediate transcriptional activation. In the first year of the 5-year project, we have carried out experiments to identify the DNA binding sites for transcription factors Oct4, Sox2, Nanog and Klf4. The results identify a large number of enhancers where these factors act to control gene expression in human ES cells. We have also begun to characterize a chromatin binding protein suspected to recognize the chromatin signature at enhancers.

Year 2

The human ES cells have the remarkable properties of self-renewal and unlimited potential to differentiate into other cell types in the body. Cell fate determination in ES cells involves selective activation and silencing of specific genes, a process that is accompanied by dynamic modifications of the histone proteins. Among the histone modifications that take place during ES cell differentiation are establishment of unique chromatin signatures at a class of regulatory DNA known as enhancers. Currently, the molecular mechanisms leading to the formation of characteristic histone modification signatures at enhancers are unknown. The proposed project is aimed to answer this question. We hypothesize that enhancer function requires binding of sequence specific transcription factors that recruit histone modification enzymes responsible for the unique histone modification pattern at these sequences. We also hypothesize that the histone modifications at enhancers in turn facilitate recruitment of chromatin binding proteins and possibly additional transcription factors to mediate transcriptional activation. During the second year of the five-year project, we have been able to identify one putative chromatin-modifying enzyme recruited to the enhancers and possibly involved in establishing the chromatin modification signature there. We have also identified a potential effector protein that bind to the chromatin modification at enhancers and potentially participate in the transcription activation of target genes. The research lays a foundation for mechanistic study of transcriptional enhancers in the human ES cells.

Year 3

The pluripotency of the embryonic stem (ES) cells, i.e. the properties to proliferate indefinitely in culture and differentiate into virtually every other cell type in the body, is controlled by a handful of transcription factors, which are proteins that bind to DNA and selectively activate expression of specific genes. The main objective of this project is to understand how transcription factors mediate the selective activation of genes involved in pluripotency of the ES cells. In the current funding period, we have demonstrated that ES cell differentiation is accompanied by dynamic modifications of the histone proteins, the basic building blocks of the chromosomes that play critical roles in gene regulation. The dynamic histone modifications occur where these transcription factors bind, and appear to be both dependent on binding of the transcription factors, and capable of facilitating the their binding to the same regions. Thus, histone modifications and transcription factors together regulate the self-renewal and pluripotency of the ES cells.

Year 4

The pluripotency of the embryonic stem (ES) cells, i.e. the properties to proliferate indefinitely in culture and differentiate into virtually every other cell type in the body, is controlled by a handful of transcription factors, which are proteins that bind to DNA and selectively activate expression of specific genes. The main objective of this project is to understand how transcription factors mediate the selective activation of genes involved in pluripotency of the ES cells. In the previous funding periods, we demonstrated that ES cell differentiation is accompanied by dynamic modifications of the histone proteins at genomic regions where the key stem cell regulatory factors bind. In the current funding cycle, we identified a protein involved in regulating the chromatin modification state in the ES cell genome, and showed that loss of this protein results in up-regulation of more than a thousand genes in the ES cell. We also identified a protein that recognizes the chromatin modification mark at transcription factor binding sites, and showed that its genomic distribution is consistent with the role in mediating transcriptional activation in the ES cells. Finally, in order to better understand the mechanisms of transcriptional regulation, we determined the 3-dimensional genome organization in the human and mouse embryonic stem cells. We found that the chromosomes in these cells are partitioned into mega-base sized genomic domains that are largely stable and invariant through cell differentiation, and highly conserved during evolution. Results from the present research have shed new lights into the mechanisms of gene activation in ES cells, and will help us devise better strategies to manipulate the embryonic stem cells for cell-based therapeutics.

Year 5

The pluripotency of the embryonic stem (ES) cells, i.e. the properties to proliferate indefinitely in culture and differentiate into virtually every other cell type in the body, is controlled by a handful of transcription factors, which are proteins that bind to DNA and selectively activate expression of specific genes. The main objective of this project is to understand how transcription factors mediate the selective activation of genes involved in pluripotency of the ES cells. Previous studies have shown that transcription factors act by interacting with specific DNA sequences with regulatory function. How these factors regulate the levels of gene expression has not been fully understood. Previously, we demonstrated that ES cell differentiation is accompanied by dynamic and characteristic modifications of the histone proteins at genomic regions where the key stem cell regulatory factors bind. In the current funding cycle, we determined global dynamic histone modifications in human ES cells as they differentiate into four distinct cell lineages that represent major cell types in early embryos. We identified over 100,000 potential regulatory sequences that could participate in the maintenance of pluripotency or drive the differentiation of ES cells to specific lineages. Using bioinformatic analysis we identified a number of candidate transcription factors that recognize these candidate regulatory sequences and may play a role in ES cell differentiation. We further identified four large protein complexes that selectively bind to the regulatory sequences in ES cells in a way that depend on the presence of the characteristic histone modification. Results from the present research advanced our understanding of the mechanisms of gene activation in ES cells, and will help us devise better strategies to manipulate the embryonic stem cells for cell-based therapeutics.

© 2013 California Institute for Regenerative Medicine