Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: This proposal addresses the functions and activities of non-coding RNAs emanating from homeotic genes (hom-ncRNA). The Principal Investigator (PI) has identified such RNAs in Drosophila where they appear to facilitate recruitment of epigenetic activators to host genes and oppose repression of homeotic genes by polycomb components. The PI has identified 32 such hom-ncRNAs in humans. These are not expressed in human embryonic stem cells (hESCs). In the first aim the effects of expression of hom-ncRNAs on homeotic gene expression in hESCs will be examined. Subsequently the recruitment of epigenetic regulators (eg MLL) to homeotic genes will be assessed by chromatin immunoprecipitation (IP). In the third aim, markers of differentiation will be assayed in hESCs expressing the hom-ncRNAs. The hope is that these studies will provide novel insights into control of differentiation in hES cells. INNOVATION & SIGNIFICANCE: The proposal is novel in its focus on hom-ncRNAs. The significance of studying control of hESC differentiation is high. How study of hom-ncRNAs will impact this area is less clear. This proposal seeks to determine whether the conserved pathway of homologous non-coding RNA-regulated HOX genes can be harnessed to control differentiation of hESCs. This is a highly innovative approach that could be employed to drive differentiation into defined lineages using small RNA transgenes to induce a more differentiated chromatin state at selected HOX genes. The model system chosen to prove the approach is hematopoietic differentiation, which is already well described, but is important for determining if the approach extends beyond existing approaches for protein overexpression. STRENGTHS: This proposal has outstanding merit as it is based on a solid hypothesis and the research team is highly qualified to carry out the proposed experiments. In particular, Dr Sauer’s lab has recently shown homologous non-coding RNAs regulate epigenetic control of HOX genes in the Drosophila model system. They have extended these observations in mammals to show that nearly all the murine HOX cluster genes have non-coding RNAs that recruit the activating histone methyltransferase MLL concomitant with gene activation. Aims 1 and 2 extend these observations by asking whether human non-coding RNA can be transiently introduced into hESCs to induce HOX gene transcription dependent upon MLL binding. Aim 3 gets to the heart of the biology of the system, and addresses whether this regulatory pathway can be harnessed to selectively induce lineage specification. In summary, the strengths of this proposal include (1) the PI's experience with hom-ncRNAs and preliminary identification in humans; (2) a collaborator within the PI's department for hESC research; and (3) the potential of developing new insights into how differentiation genes are repressed in hESCs. WEAKNESSES: The main weakness in this proposal is that the first two aims are not at all risky, because they only modestly extend the high profile recent observations from the PI's lab. The heart of the interesting science is in Aim 3. Other weaknesses include: (1) lack of a priori reasons to study hom-ncRNAs; (2) since hom-ncRNAs are not expressed in hESCs, it is uncertain what may be learned from their expression; and (3) the proposal may be construed only to meet the requirements of the RFA. DISCUSSION: This is an interesting topic where the PI is trying to move from work on the fruit fly Drosophila melanogaster into hESC research. One reviewer felt that perhaps there was no reason a priori to study homeotic gene ncRNA since they are not expressed in hESCs. Another reviewer was more enthusiastic, stating that the PI has the expertise to perform this work and understands the important questions. This reviewer felt that even though there is not much preliminary data, the first part of the proposal is almost certain to work. The HOX5A model for hematopoiesis is good, and the expectations are clear.