2014 Fiscal Year Research-status Report
Mechanistic link between DNA methylation and H3K9 trimethylation in mammalian cells mediated by two novel SRA proteins- Np95 and Np97
| Project/Area Number |
26870847
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| Research Institution | The Institute of Physical and Chemical Research |
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Principal Investigator |
SHARIF JAFAR 独立行政法人理化学研究所, 統合生命 医科学研究センター, 研究員 (00577968)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Keywords | NP95/UHRF1 / NP97/UHRF2 / DNA methylation / H3K9 trimethylation / Conditional KO / ES cells / ChIP / IAP |
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| Outline of Annual Research Achievements |
Purpose of research:This study aims to elucidate the molecular interplay between multiple epigenetic pathways mediated by the SRA proteins NP95 and Np97.
Research progress:
1. Analysis of DNA methylation (5mC) levels in Np95 or Np97 conditional knockout (Np95cKO, Np97cKO) ES cells:By constructing embryonic stem (ES) cells in which Np95 can be conditionally ablated upon addition of 4-hydroxytamoxifen (4-OHT), I have reconfirmed that global DNA methylation is rapidly depleted in Np95cKO cells as well, consistent with my previous results obtained in constitutive knockout (Np95-/-) cells. As the impact of Np97 deletion on DNA methylation has not been analyzed, I took advantage of a conditional ES cells line (Np97cKO) that I constructed newly for this project, and measured the level of global and local DNA methylation status in these cells. My analyses revealed that, unlike the ablation of Np95, deletion of Np97 does not affect the DNA methylation level in ES cells.
2. Measurements of H3K9 trimethylation levels in Np95cKO and Np97cKO ES cells: By performing global chromatin immunoprecipitation (ChIP-seq) assays I found that in Np95-ablated cells, the H3K9me3 levels were augmented by nearly 2-fold ay specific genomic loci. Surprisingly, knocking out Np97 also reproduced similar results (2-fold increase in H3K9me3)
Enigmatically, the double mutant (Np95 and Np97 double cKO) ES cells showed no change in global or local levels of H3K9me3, implicating an interplay between Np95 and Np97 for regulation of repressive histone modifications.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Summary of progress status:
I have showed that NP95/UHRF1 but not NP97/UHRF2 plays a role in regulating DNA methylation (5mC). Interestingly, deletion of either Np95 or Np97 leads to accumulation of H3K9me3 marks, revealing a role for SRA proteins in modulating H3K9me3 in vivo. Surprisingly, ablation of Np95 and Np97 together (Np95, Np97 double cKO) did not induce any change in global or local H3K9me3 levels, paradoxically implicating that in the absence of Np95, Np97 facilitates the enhancement of H3K9me3 deposition and vice versa.
Reasons for progress:
Construction of the conditional mutant ES cells lines for Np95 and Np97 were finished smoothly, which substantially helped the downstream analyses. The required experimental procedures (ChIP, ChIP-seq etc.) using the conditional ES cells were already well optimized. This was a crucial reason behind obtaining high-quality and reproducible data. I also collaborated with Dr. Matthew Lorincz (The University of British Columbia, Canada) for the detailed bioinformatics analysis of the H3K9me3 ChIP-seq data, enabling me to come up with a model showing that Np95 and Np97 have key roles for regulating IAP ERV sequences.
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| Strategy for Future Research Activity |
While the single knockouts for Np95 or Np97 show gain of H3K9me3, the double (both Np95 and Np97) mutants remain unchanged for this particular histone mark. These results suggest that Np95 and Np97 may compensate for each other’s loss by elevating H3K9me3 at particular loci (i.e. IAPs) to maintain transcriptional silencing. These observations raise several important questions, which are discussed below along with possible experimental approaches to address these questions.
1) How do NP95 and NP97 sense each other’s loss?
My preliminary analyses indicate that NP95 and NP97 physically interact with each other and may form heterodimeric complexes. Thus the loss of one could trigger the disruption of such heterodimeric complexes and allow the other to mount compensatory responses, which are likely modulated by chromatin-mediated interactions.
2) What is the mechanism for augmentation of H3K9me3 in Np95cKO or Np97cKO?
The loss of Np95 or Np97, may allow the other factor to bind more freely to specific chromatin regions such as the IAP retrotransposons, which have the higher CpG density among all retrotransposons and therefore might intrinsically attract the binding of SRA proteins (Np95 and Np97) that have an affinity for CpG motifs. The ChIP-seq based binding profile analysis for Np95 in Np97-depleted cells and vice versa are now being carried out to elucidate whether the loss of one promotes increased chromatin binding by the other.
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| Causes of Carryover |
1. Article costs (JPY630,735): Some of the experiments including ChIP-seq, RNA-seq etc. was delayed and therefore a part of the article cost was carried over to the next (2nd year) of research.
2. Travel costs (394,880): An international conference (Keystone Meeting, Colorado, USA) where the applicant participated, took place from the end of March, 2015 to the first week of April, 2015, thus moving over from fiscal year 2014 to 2015. Due to this reason, the travel costs from fiscal year 2014 was carried over to 2015.
3. Others (90,000): A large part of this cost was unused in fiscal year 2014 and therefore was carried over to 2015.
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| Expenditure Plan for Carryover Budget |
1. Article costs: The amount carried over to fiscal year 2015 will be used to purchase reagents mainly for molecular biology experiments such as ChIP-seq, RNA-seq etc.
2. Travel costs: A significant portion of this has already been used for the Keystone (USA) meeting participation in March-April 2015. The remaining fund would be used for the 2016 Keystone meeting (Canada) which the applicant is planning to attend.
3. Others: Will be used for paying the costs of mice facilities etc.
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