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Setd1a plays pivotal roles for the survival and proliferation of retinal progenitors via histone modifications of Uhrf1

鄧, 小月 東京大学 DOI:10.15083/0002006929

2023.03.24

概要

[課程-2]
審査の結果の要旨
氏名 鄧 小月
We have been studying epigenetic histone modifications during retinal
development. This study is intended to further figure out the roles of Setd1a, the
member of a Set/COMPASS complex that catalyzes H3K4me3, during retinal
development.
As results:
1) I examined the expression pattern of Setd1a transcripts during retinal
development at different developmental stages. Results showed that Setd1a was
expressed in proliferating cells, amacrine cells and retinal ganglion cells but not in
bipolar cells.
2) I examined the effects of shRNA-mediated downregulation of Setd1a at E17D3
and E14D7, and observed drastically changed phenotypes, which were increased
apoptosis and decreased proliferation, implying that Setd1a depletion affects the
survival and proliferation of late RPCs.
3) By prolong the culture time to E17D14, I confirmed that Setd1a depletion
damages late retinal progenitors and decreases the abundance of late-stage subtype
retinal cells.
4) Since it is known that the SET domain is responsible for catalyzing H3K4me3,
I found that the expression of wild-type SETD1A, but not SETD1A that lacked the
catalytic SET domain, reversed the shSetd1a-induced phenotype, indicating that

Setd1a contributes to the survival and proliferation of retinal cells through its
methyltransferase activity.
5) RNA Sequencing of shSetd1-expressing and control retinal cells revealed that
proliferation-related genes were downregulated upon shSetd1a expression. Based on
public available H3K4me3-ChIP Sequencing data of retinal development, we identified

Uhrf1 as a candidate target gene of Setd1a.
6) I confirmed that the expression of shSetd1a led to a decrease in Uhrf1
transcript levels and reduced H3K4me3 levels at the Uhrf1 locus in the retina.
7) Increased apoptosis and the suppression of proliferation in late retinal
progenitor cells were observed in retinal explants expressing shUhrf1, similar to the
outcomes observed in shSetd1a-expressing retinas. The overexpression of UHRF1 did

not rescue shSetd1a-induced apoptosis, but was able to reverse the suppression of
proliferation. These results indicate that Setd1a regulates Uhrf1 expression, and these
two molecules co-operate to regulate retinal progenitor cell survival and proliferation.
8) Uhrf1 is a hemi-methylated DNA-binding protein and facilitates DNA
methylation by recruiting Dnmt1, however, we did not observe marked changes in DNA
methylation in the absence of Uhrf1.
Taken together, we figured out the effects of shRNA-mediated knock-down of the
H3K4me3 methyltransferase Setd1a in vitro retinas, which were proliferation failure
and increased apoptosis, indicating that Setd1a contributes to the survival and
proliferation of retinal cells by regulating histone methylation. A potential downstream
effector, Uhrf1, on proliferation and apoptosis was also functionally validated. Taken
together, our results demonstrate that Setd1a regulates Uhrf1 expression, and these
two molecules co-operate to regulate retinal progenitor cell survival and proliferation.
Though some tissue-specific mechanisms that regulate Setd1a function remain to be
determined, Setd1a was proved to be strongly associated with cell proliferation and
survival, which enriched the knowledge of histone modifications involved in retinal
development.
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56

Acknowledgements

Firstly, I would like to express my utmost appreciation to my supervisor Professor Sumiko

Watanabe for offering me an opportunity to undertake this PhD study and providing generous

supports. Without her complete trust and continuous encouragement, I would not be able to

commence this PhD program, let alone having such great scientific experience. Always and

forever, I will remember Sumiko sensei and our laboratory.

Secondly, I am sincerely grateful to my advisor

Dr. Toshiro Iwagawa for his perfect

scientific knowledge teaching, effective guidance, and invaluable inspiration throughout the

whole process, all of which are essential for the completion of this thesis and my PhD

program.

Thirdly, I would like to thank Professor Yutaka Suzuki and Dr. Masaya Fukushima for

their contribution to this thesis, and all the other lovely lab members for their always generous

help.

I would also like to acknowledge my country China and the China Scholarship Council

(CSC) for providing sufficient scholarship that supports my living here in Japan.

Last but by no means least, my heartfelt thanks go to my parents

Deng yuanwen and

Luo lin for their deep love and wholehearted support, which inspired me to pursue my ideal,

and to be a better person.

57

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