About my research
”Silencing” is a type of epigenetic gene regulation mechanism. In silencing, a gene would be involved in heterochromatin and loses its activity. Depending on situation, some genes are activated by being released from heterochromatin. However, it is unclear that how the genes are released from heterochromatin. I am trying to clarify this mechanism by using budding yeast as a model organism.
I focused on DDI2 and DDI3 gene because these expressions were regulated epigenetically under DNA damage. To analyze epigenetic gene expression of DDI3, I constructed yeast strain that have EYFP, the yellow fluorescent protein coding gene, instead of DDI3 ORF. Promoter and terminator of DDI3 are left because they are important for gene regulation. I am conducting single cell analysis using this strain and I use MMS ( Methyl Methanesulfonate ) for DNA damage induction and chase the change of the expression. I use the culture plate designated for single cell analysis (CellASIC ONIX plate for haploid yeast cells), and acquire fluorescence image of cells.
From the experiment described above, I confirmed that the expression state become ON from OFF when I added MMS and the switch speed of the expression state differs among individual cells. This suggested the heterochromatin expansion and contraction speed are different in individual cells, and the possibility that the fluctuation of heterochromatin controls the epigenetic gene expression state.
To find the factors which participate in the DDI3 regulation, I am doing knockout gene screening. I compare WT with gene deletion strain by real time PCR. Furthermore, I compare them at single cell level using the system described above. So far, I think stress response factors such as YAP1 may involve in DDI3 regulation. There should be the regulation factor which interacts with DDI3 directly, so I want to find out it.
Furthermore, I observed the synchronized gene expression of DDI2 and DDI3 from the single cell analysis of yeast strain which DDI2 and DDI3 were replaced ECFP and EYFP respectively. When I add MMS, the fluorescence of ECFP and EYFP appeared at the same time. This suggests the mechanism that simultaneously regulates genes which are located in different chromosomes.
I focused on DDI2 and DDI3 gene because these expressions were regulated epigenetically under DNA damage. To analyze epigenetic gene expression of DDI3, I constructed yeast strain that have EYFP, the yellow fluorescent protein coding gene, instead of DDI3 ORF. Promoter and terminator of DDI3 are left because they are important for gene regulation. I am conducting single cell analysis using this strain and I use MMS ( Methyl Methanesulfonate ) for DNA damage induction and chase the change of the expression. I use the culture plate designated for single cell analysis (CellASIC ONIX plate for haploid yeast cells), and acquire fluorescence image of cells.
From the experiment described above, I confirmed that the expression state become ON from OFF when I added MMS and the switch speed of the expression state differs among individual cells. This suggested the heterochromatin expansion and contraction speed are different in individual cells, and the possibility that the fluctuation of heterochromatin controls the epigenetic gene expression state.
To find the factors which participate in the DDI3 regulation, I am doing knockout gene screening. I compare WT with gene deletion strain by real time PCR. Furthermore, I compare them at single cell level using the system described above. So far, I think stress response factors such as YAP1 may involve in DDI3 regulation. There should be the regulation factor which interacts with DDI3 directly, so I want to find out it.
Furthermore, I observed the synchronized gene expression of DDI2 and DDI3 from the single cell analysis of yeast strain which DDI2 and DDI3 were replaced ECFP and EYFP respectively. When I add MMS, the fluorescence of ECFP and EYFP appeared at the same time. This suggests the mechanism that simultaneously regulates genes which are located in different chromosomes.