With the completion of the human genome-sequencing project, the next major step in understanding genome function is to elucidate the precise molecular steps involved in the regulation of gene expression including long-term gene silencing within a chromatin context. This is crucial to understand the mechanisms that underpin genome instability, and to understand a number of disease states such as cancer and other syndromes involving chromosomal instability. Very little is known however, about the mechanisms by which chromatin structures are remodelled and transmit epigenetic information from one cell generation to the next, to establish stable epigenetic states which are the cell’s memory bank that restricts or permits gene activation. Our research activity over the last few years suggests that silencing histone markers such as H3 K9-3xMe and H2A.Z are enriched at a specific retrotransposon class, LINE L1 (The human genome is composed of 44% retrotransposons, 52% noncoding sequences and < 4% protein-coding sequences.). Our small scale sequencing of ~150 randomly picked ChIP DNA indicated that compared to histone H2A, the majority of H2A.Z clones (~30%) are LINE retrotransposons but not LTR elements. Strikingly, loss of this histone variant by RNAi produced a significant increase in the transcription of retrotransposable L1 elements. Therefore, our hypothesis is that the cell has utilised the ability of silencing histone marks to assemble condensed heterochromatic structures to prevent LINE transcription thus keeping them at bay. Clearly, the loss of LINE silencing would have a disastrous affect on genomic stability. The current research activity will test this novel hypothesis and provide new mechanistic information into the functions and expression of endogenous retrotransposable elements in human diseases.
Specific aims being investigated include
- Genome-wide organization of retrotransposons by ChIP-on-ChIP using tiling arrays and deep sequencing in elucidating the role of chromatin architecture in silencing retrotransposon activity.
- Understand the role of repeat-associated endo-siRNAs and RNAi machinery such as Dicer and AGO2 complexes that can act to silence retrotransposons.
- Investigate the role of epigenetics factors in activation and silencing of retrotransposition.
This study provides new insight into the role of histone components in silencing retrotransposons within the context of chromatin structure and further corroborates the emerging link between dysfunction of L1 activity and genomic instability in mammalian cells.