Chemical biology of small RNA chromatin modifiers

2016 Fiscal Year Research-status Report

• Project/Area Number: 16K18496
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: SCHNEIDER TILMAN 国立研究開発法人理化学研究所, 吉田化学遺伝学研究室, 研究員 (30599478)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Keywords: epigenetics / splicing / chromatin / small RNA

Outline of Annual Research Achievements

Research went largely according to plan. We had to redo part of the high throughput sequencing analysis and have sequenced additional sets of samples to get better read coverage. While our previously obtained data still holds, we need to increase the read number to get statistically more reliable data/
We have detected significant redistribution of histone H3 K9m3 and K27m3 marks and it appears that they accumulate at opposite ends of genes. As data analysis proved challenging we have initiated a collaboration with the Nikaido group at RIKEN.
We have identified several putative target genes whose chromatin status changes dependent on small RNA sequences contained in pre-mRNA. It appears these sequences are not exclusively intronic. Analysis is under way whether these sequences are responsible for increased histone methylation within the gene body or closer to transcription start and transcription end sites.
The data acquisition for small RNAs and K9m3 methylation is complete. K27m3 and K36m3 are under way. We are in the process of adding ChIP-Seq libraries on Ago1 and Ago2 proteins to better understand their function in chromatin editing. Validation experiments to strengthen our argument are under way to confirm locations of increased heterochromatin formation. We are currently looking to identify the most likely small RNA sequences to build a follow up experiment and test heterochromatin formation without splicing inhibition. While we are focusing on K9, K27 and K36 methylation, we are taking steps to look into histone acetylation and phosphorylation as well.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The project is progressing roughly as planned. While re-analysis of the data has cost some time, we are now in a better position to use the acquired data for hypothesis testing.

Strategy for Future Research Activity

We will add ChIP sequencing data on the Ago proteins to confirm that they colocalize to positions matching the RNA sequences they harbor.
We have started validation work on detected H3K9m3 peaks and expect to submit a manuscript withing this fiscal year.

Causes of Carryover

We waited with some sequencing experiments until data validation of previous data. Now money will be used to finish sequencing prepared libraries.

Expenditure Plan for Carryover Budget

We are in the process of sending more libraries out for sequencing.