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Super-enhancer Acquisition Drives FOXC2 Expression in Middle Ear Cholesteatoma

Abstract

Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in middle ear cholesteatoma. It is known that histone acetylation destabilizes the nucleosome and chromatin structure and induces gene activation. The association of histone acetylation with chronic inflammatory diseases has been indicated in recent studies. In this study, we examined the localization of variously modified histone H3 acetylation at lysine 9, 14, 18, 23, and 27 in paraffin-embedded sections of human middle ear cholesteatoma (cholesteatoma) tissues and the temporal bones of an animal model of cholesteatoma immunohistochemically. As a result, we found that there was a significant increase of the expression levels of H3K27ac both in human cholesteatoma tissues and the animal model. In genetics, super-enhancers are clusters of enhancers that drive the transcription of genes involved in cell identity. Super-enhancers were originally defined using the H3K27ac signal, and then we used H3K27ac chromatin immunoprecipitation followed by sequencing to map the active cis-regulatory landscape in human cholesteatoma. Based on the results, we identified increased H3K27ac signals as super-enhancers of the FOXC2 loci, as well as increased protein of FOXC2 in cholesteatoma. Recent studies have indicated that menin-MLL inhibitor could suppress tumor growth through the control of histone H3 modification. In this study, we demonstrated that the expression of FOXC2 was inhibited by menin-MLL inhibitor in vivo. These findings indicate that FOXC2 expression under histone modifications promoted the pathogenesis of cholesteatoma and suggest that it may be a therapeutic target of cholesteatoma.

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Availability of Data and Material

The datasets produced and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Code Availability

The study uses data obtained from the UCSC genome browser http://genome.ucsc.edu/index.html.

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Acknowledgements

We would like to thank Dr. Jeffrey S. Rubin with the National Cancer Institute/CCR/LCMB for providing the human KGF cDNA construct. We thank Dr. Masahiro Takahashi, Dr. Kazuhisa Yamamoto and Dr. Yutaka Yamamoto (Department of Otorhinolaryngology, Jikei University School of Medicine) for the harvesting of the human tissues. We also thank Miss Shiho Kondo and Mr. Daisuke Endo (Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences) for their excellent technical assistance in this work. We also would like to thank Prof. Takehiko Koji (Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences) for his comments regarding this work.

Funding

This study is supported by JSPS KAKENHI Grant Number JP25462647, JP16K11186, and JP19K09857 to T. Yamamoto-Fukuda, no. JP18K16908 to N. Akiyama).

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Yamamoto-Fukuda T, design of the work; acquisition, analysis, interpretation of data, drafting the work; Akiyama N, analysis and interpretation of data, drafting the work; Kojima H, experimental supports.

Corresponding author

Correspondence to Tomomi Yamamoto-Fukuda.

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Ethics Approval

All the experimental procedures were performed in accordance with the Nagasaki University with the approval of the Institutional Animal Care and Use Committee (Nos. 1209241015-2 and 1404011269), the Guidelines for Animal Experimentation of Jikei University with approval guidelines (No. 2015-139C4), the Human Ethics Review Committee of Jikei University School of Medicine (approval number is 27-344 8229).

Consent for Publication

This study protocol was approved by the Human Ethics Review Committee of Jikei University School of Medicine, and signed informed consent was obtained from all the patients or their guardians for this study (approval number is 27-344 8229).

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The authors declare no competing interests.

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Yamamoto-Fukuda, T., Akiyama, N. & Kojima, H. Super-enhancer Acquisition Drives FOXC2 Expression in Middle Ear Cholesteatoma. JARO 22, 405–424 (2021). https://doi.org/10.1007/s10162-021-00801-7

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Keywords

  • FOXC2
  • Epigenetics
  • H3K27ac
  • Menin-MLL inhibitor
  • Middle ear cholesteatoma