Gene Amplification, DNA Rearrangements and Chromosome Instability in Eukaryotes
The primary focus of this laboratory is on molecular processes that affect genome stability, including chromosome breakages, DNA deletions and gene amplification. We have been using the ciliated protozoan Tetrahymena thermophila as a model eukaryote for this analysis. Tetrahymena cells carry out extensive DNA rearrangements during somatic nuclear differentiation, including chromosome breakage at hundred of specific sites, deletion of thousands of specific DNA segments ( IES deletion, Internal Eliminated Sequence deletion) and amplification of the ribosomal RNA gene. We have identified critical cis-acting sequences that regulate these processes, including a 15 bp sequence for chromosome breakage sites, two sets of sequences for DNA deletion boundaries, and a pair of inverted repeats for large DNA palindrome formation during gene amplification. We have also begun to identify proteins involved in their regulations.
Based on studies from this and others labs, DNA deletion had been shown to be an atypical RNAi process. It is only silenced the transcription of specific DNA fragments but further removed these DNA from the genome. Before DNA elimination, double strand RNA are transcripted from corresponding sequences and processed into sRNA. These sRNA targeted the DNA, leading to modifications of chromatin, including H3K9 and H3K27 trimethylation, during nuclear differentiation. The marked chromatin formed heterochromatin similar to that in the fission yeast and fly, but are further deleted in Tetrahymena. In this unique process, we found that a chromodomain protein, pdd1p (a HP1 homolog), was expressed specifically during nuclear differentiation and closely associated with the deleted DNA. Reverse genetics studies showed that pdd1p played an essential role in nuclear differentiation and DNA deletion. In addition, we characterized Tbp2p, a domesticated transposase, and found it was used by Tetrahymena to cut out the deleted DNA. We are also investigating the possible roles of other chromosomal proteins, and have established genetic and cytological approaches to screen for new genes involved in these processes.
The mechanism of gene amplification described in Tetrahymena also occurs in other organisms. We found that large DNA palindromes can be produced in budding yeast, just like in Tetrahymena, when a double stranded DNA break is made at a site adjacent to a pair of short inverted repeats. This finding suggests a general mechanism for gene amplification in eukaryotes. We have now successfully tested this idea in Chinese hamster ovary cells, and developed a genome-wide defection method to examine its role in human cancer progression, which is often accompanied by gene amplification.