2021 Fiscal Year Research-status Report
Measurement of Chromatin Architecture, and its Function in Regulating Neuronal Activity
| Project/Area Number |
20K15909
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
徐 方可 国立研究開発法人理化学研究所, 脳神経科学研究センター, 基礎科学特別研究員 (60867216)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | neuronal activity / transcription factor |
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| Outline of Annual Research Achievements |
In the original proposal, the major goal was to investigate chromatin structure and gene expression changes in neurons at different activity states and/or neurons from mutants showing abnormal neuronal activities. The neurons of interest is specifically a subgroup of somatic sensory neurons, C4da, which located in between muscles and epidermis. It is difficult to acquire pure and abundant DNA or RNA from those neurons for sequencing. I have optimized a protocol working for RNA samples as low as 250pg for sequencing. Some test samples of manipulating a transcriptional factor potentially important for C4da neuron development will be sequenced soon. I am currently working on further optimizing our cell dissociation protocol to increase the starting DNA material could be used for DamID or ChIP-seq.
I have narrowed down to some interesting genes that might specifically involved in regulating neuronal activity. Based on our data, the level of a C3 and C4da specific transcription factor, cut (ct), increased after either physical or genetical activation of C4da. Manipulation of ct expression in C4da neurons resulted in similar behavior output changes as manipulating the activity of C4da neurons. The data indicates ct is potentially related to neuronal activity. I am planning on performing RNA-seq on samples with genetically silenced or activated activity along with running DamID or ChIP-seq for CT. We are hoping to explain the roles ct plays in response to neuronal activity changes and to identify genes regulated by CT that participated in the downstream behavioral changes.
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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
There are extra technical issues needed to be solved for getting abundant pure and healthy neurons from FACS for the downstream sequencing experiments. Therefore, although I have acquired more functional data from the target genes of interest, the sequencing experiments have not been completed. There are also additional delays for reagents orders and shipments.
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| Strategy for Future Research Activity |
The biggest technical challenge of this project is to pure but abundant number of neurons of our interest in order to analyze their DNA and RNA. While I have made progress to acquire enough cells for RNA sequencing, more trials and optimizations will be needed for the DNA related sequencing experiments. The main goal will be firstly optimizing the current cell dissociation and sorting protocol to enhance the efficiency of acquiring enough neurons of interest. DamID or ChIP-seq will be performed for a transcription factor that showed changes after C4da activation. RNA-seq will be done after knocking down this transcription factor transitory during development.
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Research Products
(1 results)
