The goal of this proposal is to understand the dynamics of chromatin in various cellular differentiation states and how alteration of this dynamic may contribute to cancer development and progression. Our major findings are outlined as follows and further elaborated below.
1) Among the various acetylation sites of histones, H3K18ac has a unique distribution in hESCs and is specifically affected during oncogenic transformation. As part of a screen to discover upstream regulators of this modification site (described in previous reports), we identified a non-coding RNA that is required for maintenance of H3K18ac, expression of SOX2 and its target genes, and growth of hESCs.
2) We have discovered a highly novel and unanticipated role for histone acetylation. We have found that global histone acetylation and deacetylation coupled with flux of acetic acid in and out of the cells acts as a buffering system for regulation of intracellular pH. This phenomenon is a fundamental biological process and occurs in hESCs, cancer cells as well as normal differentiated human cells. (A paper reporting this finding is currently being reviewed at Nature.)
3) We are continuing our efforts on the role of linker histone H1.5 in transcriptional regulation of terminally differentiated cells vs hESCs. This is a continuation project from a CIRM SEED grant. A manuscript on this project was submitted to Cell but was not accepted. We have performed additional experiments and preparing a new manuscript.
I. A non-coding RNA is required for hESC growth.
This aim was designed to understand how the global levels of histone modifications are regulated. As reported in previous progress reports, we carried out a kinase screen in which ~800 kinases were knocked down individually using siRNAs and the levels of two histone modifications were examined. We validated the top hits which were reported last year. The most significant effect on histone modifications, especially H3K18ac, was observed in knockdown of TPRXL (tetra-peptide repeat homeobox-like). We found that knockdown of TPRXL causes ~50-70% reductions in the global levels of H3K18ac specifically, suggesting that TPRXL is required for maintenance of a portion of H3K18ac throughout the genome. It turned out that the identification of TPRXL was a fortuitous finding. TPRXL is not a kinase but has been mis-annotated as a kinase in certain databases, hence its inclusion in the kinase siRNA library. TPRXL is a member of the TPRX homeobox gene family and is designated as a non-functional retrotransposed pseudogene (Booth and Holland, 2007). It is suggested that TPRXL was generated by reverse transcription of TPRX1 mRNA which was then integrated near an enhancer active in placenta. Consistently, TPRXL has a very high expression in placenta compared to other tissues. Subsequent to integration, TPRXL sequence has diverged from that of TPRX1 in an unusual way. In certain regions, such as over the homebox domain, TPRXL has retained 81% nucleotide identity but only 66% amino acid identity compared to TPRX1 (Booth and Holland, 2007). Despite its designation, TPRXL could possibly be a functional retrogene as it is transcribed and contains two potential open reading frames (ORFs). One ORF can code for a short protein (139 a.a.) that would contain the homeodomain and a polyglutamine stretch. Another ORF codes for a longer protein that would consist mostly of a long polyserine/proline stretch.