Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Shigeki Suzuki, Hiroaki Hoshino, Kazuma Yoshida, Jun Nakanishi, Shizu Tsuchiya-Hirata, Seiji Kobuke, Naoto Haruyama, Fusanori Nishimura, Hideki Shiba

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Abstract

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.

Original languageEnglish
Pages (from-to)2303-2309
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume495
Issue number3
DOIs
Publication statusPublished - Jan 15 2018

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Cell proliferation
Dentin
Chromatin
Squamous Cell Carcinoma
Genes
Cell Proliferation
Genome
RNA
Messenger RNA
Proteins
Genetic Promoter Regions
Gene expression
Epithelial Cells
Cells
Genetic Epigenesis
Gene Expression
cdc Genes
Untranslated RNA
Fractionation
Small Interfering RNA

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma. / Suzuki, Shigeki; Hoshino, Hiroaki; Yoshida, Kazuma; Nakanishi, Jun; Tsuchiya-Hirata, Shizu; Kobuke, Seiji; Haruyama, Naoto; Nishimura, Fusanori; Shiba, Hideki.

In: Biochemical and Biophysical Research Communications, Vol. 495, No. 3, 15.01.2018, p. 2303-2309.

Research output: Contribution to journalArticle

Suzuki, Shigeki ; Hoshino, Hiroaki ; Yoshida, Kazuma ; Nakanishi, Jun ; Tsuchiya-Hirata, Shizu ; Kobuke, Seiji ; Haruyama, Naoto ; Nishimura, Fusanori ; Shiba, Hideki. / Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma. In: Biochemical and Biophysical Research Communications. 2018 ; Vol. 495, No. 3. pp. 2303-2309.
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AU - Hoshino, Hiroaki

AU - Yoshida, Kazuma

AU - Nakanishi, Jun

AU - Tsuchiya-Hirata, Shizu

AU - Kobuke, Seiji

AU - Haruyama, Naoto

AU - Nishimura, Fusanori

AU - Shiba, Hideki

PY - 2018/1/15

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N2 - Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.

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