Statement of Benefit to California:
Year 1Epigenetic mechanisms play pivotal roles in cell fate determination during development. Long non-coding RNAs (ncRNAs) are in involved in epigenetic gene expression by recruiting epigenetic regulators to target genes. We are dissecting the role of ncRNAs in the stemcellness and differentiation of human embryonic stem cells (hESC). We have used biochemical and molecular approaches to identify ncRNAs, which associate with epigenetic regulators in hESC and are involved in the regulation of the expression of homeotic (HOX) genes. HOX genes encode for key regulators of cell differentiation in Arthropods and Chordates. We have identified 32 ncRNAs, which are transcribed during hESC differentiation. The detailed dissection of the role of two of the identified ncRNAs in HOX gene expression has resulted in novel insights into the role of ncRNAs in hESC differentiation. The ncRNA Mistral plays an important role in ectoderm development, one of the three germ layers that give rise to all cells, tissues and organs. Inactivation of Mistral ncRNA during hESC development prevents ectoderm development, indicating that Mistral is a key regulator of ectoderm development. The second ncRNA Scirocco is involved in epigenetic activation of HOX genes, controlling mesoderm development.
The obtained results support a model in which ncRNAs regulate important steps during hESC differentiation. Our results lay a foundation for the application of ncRNAs in hESC differentiation and the development of diagnostic and therapeutic assays to detect and manipulate the differentiation of hESC. The obtained results open novel areas in the filed of stem cell research by providing tools and assays that actively control the differentiation of hESC. These assays represent valuable additions to the efforts aimed at the active control hESC differentiation in order to obtain desired cell types.
Year 2The establishment and maintenance of mitotically and meitotically stable -epigenetic- gene expression patterns is paramount for cell proliferation and differentiation. Long non-coding RNAs (ncRNAs) have emerged as important regulators of epigenetic gene expression, in particular epigenetic gene silencing. NcRNAs have been associated with imprinting, gene dosage compensation, gene silencing and metastasis. NcRNAs silence gene expression by recruiting epigenetic repressors of the Polycomb group family to target genes. However, the functional importance of ncRNAs in stem cell biology remains unknown.
This project is aimed at the dissection of the role and function of ncRNAs in stem cell differentiation. During the funding period, we have focused on the functional characterization of one ncRNA. The ncRNA is transcribed in differentiating embryonic stem cells (ESCs) and facilitates activation of Hox genes. Hox genes are key regulators of cell differentiation and establish the developmental fate of cells. Destruction of Mistral through RNAi attenuates the differentiation of ESCs. Collectively, our results uncover a role for the ncRNAin epigenetic activation of gene expression and stem cell differentiation and identify ncRNAs as important regulators of stem cell differentiation. The obtained results establish a foundation for the development of novel tools and assays that actively control the differentiation of stem cells and direct the differentiation of stem cells into a desired cell type.