O-235 ACLY entry into the nucleus to bind with EP300 and regulate the genomic DNA and ribosomal DNA transcription during early embryonic development

Abstract

Abstract Study question ATP citrate lyase (ACLY) is one of the key enzymes to produce acetyl coenzyme A. What is its role in early embryonic development? Summary answer ACLY affected by activation of T447, S451 and S455 sites into nucleus and band with EP300 to affect genomic DNA and ribosomal DNA transcription. What is known already ACLY is one of the key enzymes to produce acetyl coenzyme A, which participates in fatty acid metabolism and regulates acetylation. Studies have found that ACLY knockout cells can survive, while the cell proliferation capacity will be weakened. ACLY is highly expressed in the cytoplasm of Hela cells colon cancer cells and other somatic cells. When AKT phosphorylates S455 site, ACLY enters the nucleus and participates in DNA damage repair. Its localization and role in embryos have not been studied. The only thing we know is homozygous mice could not be detected at E8.5 days after implantation. Study design, size, duration Twenty-four human day 6 developmentally arrested embryos were used to detect ACLY localization. Dozens of ACLY heterozygous female mice were used to test the time points of ACLY homozygous lethality after mating with heterozygous male mice. After zygotes were taken from wild-type C57BL6 mice, microinjection technology was used to explore the effects of knocking down ACLY, changing ACLY location or mutating ACLY. HeLa cells were used to explore at the molecular level. Participants/materials, setting, methods In this experiment, we used human abandoned or donated embryos, gene knockout mice, wild-type C57BL6 mice and Hela cell. The experimental techniques used include in vitro embryo culture, EU staining, plasmid transfection, immunoprecipitation, western blot analysis, in vitro transcription, microinjection, immunofluorescence, RT-PCR and RNA-seq. Main results and the role of chance Firstly, our study found its special nuclear localization in early embryos for the first time and proved that its localization was related to embryonic development potential. Secondly, the specific period of ACLY homozygous death was identified by ACLY heterozygous mice. And by knocking down ACLY or adding inhibitor SB204990, It was found that ACLY played an important role in cell proliferation, blastocyst formation and lineage differentiation of early embryo development. Thirdly, we proved that ACLY nucleation played an important role in the development of early embryos. and the activated form of T447, S451 and S455 three sites led to ACLY nucleation. Finally, we found that ACLY affected the level of histone acetylation and UBF acetylation. And then genomic DNA and ribosomal DNA transcription level were decreased. We further explored how the acetyl group produced by ACLY regulated transcription. The interaction between ACLY and EP300 was confirmed by immunofluorescence and COIP experiments. After knocking down EP300 in embryos or inhibiting its function with inhibitor c646, the results are consistent with knocking down ACLY. The above experimental data prove that ACLY affects histone acetylation level and regulates gene opening through the interaction with EP300. Limitations, reasons for caution We need to further explore the factors that promote ACLY into the nucleus, so as to add it to in vitro culture to enhance the embryonic development potential. Wider implications of the findings The study provides a new mechanism for insulin or lysophosphatidic acid to promote embryo development, and helps to improve the quality of embryo development in the process of embryo culture in vitro. Trial registration number not applicable