Embryonic stem cells (ESCs) are unique in their ability to self-renew while retaining the potential, when appro- priately stimulated, to form the full repertoire of cell lineages present in the adult body. Long-term self-renewal requires stable gene regulatory network that specifies the undifferentiated state. On the other hand, the ge- nome of ESCs should be in a plastic state in order to enter any one distinct differentiation pathways. Despite significant progress in deciphering ESC regulation the molecular machinery that establishes and maintains chromatin in the pluripotent configuration remains poorly understood. During the previous funding period, we have identified chromatin-associated ESC-specific factors Dppa2 and Dppa4 as critical regulators of cellular reprogramming and have shown that they are required for resetting the epigenome to a pluripotent configura- tion. When introduced together with the Yamanaka factors into somatic cells, Dppa2/4 initiate global chromatin decompaction via the recruitment of chromatin remodeling factor Parp1 and activation of the DNA damage re- sponse pathway, whereas local Dppa2/4 binding contributes to silencing of somatic gene promoters and acti- vation of ESC enhancers. Our unpublished data indicate that in addition to the role in reprogramming, Dppa2/4 play critical role in maintaining the developmental capacity of ESCs. Dppa2/4-deleted ESCs fail to form germ layer derivatives, while Dppa2/4 overexpression leads to a more efficient differentiation. Our ChIP-Seq anal- yses identified over 65,000 Dppa2/4 binding peaks, large fraction of which maps onto poised developmental promoters. Dppa2/4-bound developmental genes are rapidly induced upon differentiation of WT, but not Dppa2/4-KO ESCs indicating that the presence of Dppa2/4 at those promoters may be required for their activa- tion during differentiation. Based on these data, we hypothesize that Dppa2/4 and its associated chromatin remodeling complexes maintain open chromatin, both globally and locally at the regulatory elements of devel- opmental genes, to ensure correct execution of lineage-specific programs during differentiation. This hypothe- sis will be tested via three specific aims.
In aim 1, we will identify developmental lineages, stages and genes affected by Dppa2/4 depletion in ESCs using single cell RNA-sequencing and our novel computational tool PHATE.
In aim 2, we will identify global and local changes in chromatin induced in Dppa2/4-KO ESCs and during neuronal differentiation.
In aim 3, we will dissect the functions of Dppa2/4-associated chromatin remod- eling factors Parp1, Myst4, Smarca5a and Kdm2a in the chromatin maintenance. The work proposed will elu- cidate the role of Dppa2/4-associated chromatin remodeling complexes in the establishment and maintenance of the pluripotent capacity of ESCs. The positive impact will be an improved understanding of developmental potency and its regulation at the chromatin level, information to develop more efficient ESC culture systems and new approaches to somatic cell reprogramming.
The proposed research is relevant to public health because a thorough understanding of embryonic stem cell pluripotency is a crucial requirement for developing regenerative therapies to effectively treat human disease. Patients with neurodegenerative diseases such as Parkinson?s and Alzheimer?s diseases and spinal cord inju- ries could potentially benefit from this research. Thus, it is relevant to the part of NIH?s mission that pertains to developing of fundamental knowledge that will help reduce the burdens of illness and disability.
| Hernandez, Charles; Wang, Zheng; Ramazanov, Bulat et al. (2018) Dppa2/4 Facilitate Epigenetic Remodeling during Reprogramming to Pluripotency. Cell Stem Cell 23:396-411.e8 |
| Wang, Zheng; Gearhart, Micah D; Lee, Yu-Wei et al. (2018) A Non-canonical BCOR-PRC1.1 Complex Represses Differentiation Programs in Human ESCs. Cell Stem Cell 22:235-251.e9 |
| Kumar, Ishan; Ivanova, Natalia (2015) Moving Toward the Ground State. Cell Stem Cell 17:375-6 |
