During cell differentiation, transcriptional programs are changed, and then must be maintained in turn. Chromatin-based epigenetic mechanisms are at the core of maintenance and switching of transcriptional programs. The fundamental issues of the nature of epigenetic marking and of the mechanisms that switch this marking during differentiation remain unclear due to lack of relevant experimental approaches. We developed new experimental paradigms that allow investigating the structure of chromatin during DNA replication at a single-cell and at a gene-specific levels. Using our new techniques, we found striking differences in the structure of chromatin during differentiation of the pluripotent human embryonic stem cells (hESC) and the antigen-inexperienced (nave) T cells. During the first several hours after induction of differentiation of hESCs to dopamine neuron lineage, or T cells to different T cell subsets, accumulation of H3K27me3 is significantly delayed on nascent DNA. Since the occurrence of H3K27me3 in the genome coincides with the dense structure of nucleosomes, this suggests the existence of a temporarily de-condensed structure of nucleosomes on nascent DNA shortly after induction of cell differentiation. Our preliminary data indicate that the de-condensed, `open' structure of chromatin may be essential for recruitment to DNA of the lineage-specific transcription factors (TFs) that are essential to induce changes in transcriptional programs during cell differentiation. Thus, our results present a molecular explanation of how the vast areas of the repressed genome can be activated during cell differentiation. The goals of this proposal are to test two unique hypotheses using different models of differentiation to various lineages for pluripotent hESCs and for specialized T cells: 1) To examine whether the period of `open' post-replicative chromatin in early differentiating cells is a result of complex interplay of activities of several histone-modifying proteins; and 2) To examine whether this open post-replicative chromatin creates a `window of opportunity' for high accessibility of lineage- specifying TFs that are required to change the transcriptional program during cell differentiation. Examining these unique hypotheses may provide a universal chromatin-based molecular mechanism for biological plasticity of the cell.

Public Health Relevance

Chromatin structure is at the core of the epigenetic inheritance, which is essential for changes and conservation of gene transcription programs during cell differentiation and cell proliferation. Proposed studies will examine whether a newly discovered de-condensed structure of chromatin just after DNA replication may be responsible for changes in epigenetic and transcriptional programming during the early period of cell differentiation. Studies will be performed using a models of differentiation of the pluripotent human embryonic stem cells and differentiation of specialized T cells, and therefore may have important implications for future development of novel strategies to treatment of different human diseases.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Development - 1 Study Section (DEV1)
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Kelly, Halonna R
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Thomas Jefferson University
Schools of Medicine
United States
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Petruk, Svetlana; Cai, Jingli; Sussman, Robyn et al. (2017) Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of Stem Cell Differentiation. Mol Cell 66:247-257.e5
Petruk, Svetlana; Mariani, Samanta A; De Dominici, Marco et al. (2017) Structure of Nascent Chromatin Is Essential for Hematopoietic Lineage Specification. Cell Rep 19:295-306
Petruk, Svetlana; Fenstermaker, Tyler K; Black, Kathryn L et al. (2016) Detection of RNA-DNA association by a proximity ligation-based method. Sci Rep 6:27313