Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site

Alyshia Newhart, Ilona U. Rafalska-Metcalf, Tian Yang, Lucy M. Joo, Sara Lawrence Powers, Andrew V. Kossenkov, Melissa Lopez-Jones, Robert H. Singer, Louise C. Showe, Emmanuel Skordalakes, Susan M. Janicki

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Abstract

Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replicationindependent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34Rand K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation.

Original languageEnglish (US)
Pages (from-to)19882-19899
Number of pages18
JournalJournal of Biological Chemistry
Volume288
Issue number27
DOIs
StatePublished - Jul 5 2013

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Single-Cell Analysis
Chromatin Assembly and Disassembly
Transcription
Cytomegalovirus
Histones
Chromatin
RNA
Nucleosomes
Euchromatin
Antisense RNA
Pediatrics
Double-Stranded RNA
Heterochromatin
Telomere
Glioblastoma
DNA Replication
Assays
Cell Cycle
Cells
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Newhart, A., Rafalska-Metcalf, I. U., Yang, T., Joo, L. M., Powers, S. L., Kossenkov, A. V., ... Janicki, S. M. (2013). Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site. Journal of Biological Chemistry, 288(27), 19882-19899. https://doi.org/10.1074/jbc.M113.473181

Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site. / Newhart, Alyshia; Rafalska-Metcalf, Ilona U.; Yang, Tian; Joo, Lucy M.; Powers, Sara Lawrence; Kossenkov, Andrew V.; Lopez-Jones, Melissa; Singer, Robert H.; Showe, Louise C.; Skordalakes, Emmanuel; Janicki, Susan M.

In: Journal of Biological Chemistry, Vol. 288, No. 27, 05.07.2013, p. 19882-19899.

Research output: Contribution to journalArticle

Newhart, A, Rafalska-Metcalf, IU, Yang, T, Joo, LM, Powers, SL, Kossenkov, AV, Lopez-Jones, M, Singer, RH, Showe, LC, Skordalakes, E & Janicki, SM 2013, 'Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site', Journal of Biological Chemistry, vol. 288, no. 27, pp. 19882-19899. https://doi.org/10.1074/jbc.M113.473181
Newhart, Alyshia ; Rafalska-Metcalf, Ilona U. ; Yang, Tian ; Joo, Lucy M. ; Powers, Sara Lawrence ; Kossenkov, Andrew V. ; Lopez-Jones, Melissa ; Singer, Robert H. ; Showe, Louise C. ; Skordalakes, Emmanuel ; Janicki, Susan M. / Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 27. pp. 19882-19899.
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