Vertebrate Spt2 is a novel nucleolar histone chaperone that assists in ribosomal DNA transcription

Abstract

SummaryIn eukaryotes, transcription occurs in the chromatin context with the assistance of histone-binding proteins, such as chromatin/ nucleosome remodeling factors and histone chaperones. However, it is unclear how each remodeling factor or histone chaperone functions in transcription. Here, we identify a novel histone-binding protein, Spt2, in higher eukaryotes. Recombinant human Spt2 binds to histones and DNA, and promotes nucleosome assembly in vitro. Spt2 accumulates in nucleoli and interacts with RNA polymerase I in chicken DT40 cells, suggesting its involvement in ribosomal RNA transcription. Consistently, Spt2-deficient chicken DT40 cells are sensitive to RNA polymerase I inhibitors and exhibit decreased transcription activity, as shown by a transcription run-on assay. Domain analyses of Spt2 revealed that the C-terminal region, containing the region homologous to yeast Spt2, is responsible for histone binding, while the central region is essential for nucleolar localization and DNA binding. Based on these results, we conclude that vertebrate Spt2 is a novel histone chaperone with a separate DNA-binding domain that facilitates ribosomal DNA transcription through chromatin remodeling during transcription.

Structure–function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2

“…Furthermore, The C terminus (571-685) of hSpt2, but not its N-terminal counterpart (1-570), bound His-tagged H3/H4 under low-salt conditions as monitored by pull-down assays (Osakabe et al 2013). We independently confirmed that the C terminus (571-685) of GST-tagged hSpt2 bound H3/H4 under low-salt conditions as monitored by pull-down assays (data not shown).…”

“…Previous studies established that full-length hSpt2 bound to both H2A/H2B and H3/H4 under low-salt (0.15 M NaCl) conditions as monitored by gel shift assays (Osakabe et al 2013). Furthermore, The C terminus (571-685) of hSpt2, but not its N-terminal counterpart (1-570), bound His-tagged H3/H4 under low-salt conditions as monitored by pull-down assays (Osakabe et al 2013).…”

“…5A; Sternberg et al 1987;Osakabe et al 2013). Most of the hSpt2C residues involved in intermolecular recognition of the complex with H3/H4 are conserved in S. cerevisiae (ySpt2C) (designated by an asterisk in Fig.…”

“…Recently, several reports confirmed that the majority of H3/H4 tetramers are maintained, rarely split, and recycled back during transcription (Xu et al 2010;Katan-Khaykovich and Struhl 2011). Importantly, several classes of histone chaperones are involved in histone H3/H4 dynamics on transcribed genes, including Asf1, HIRA, FACT (facilitates chromatin transcription complex), Spt6 (encoded by Suppressors of Ty insertions gene family 6), and Spt2 (Orphanides et al 1999;Kaplan et al 2003;Nourani et al 2006;Jamai et al 2009;Avvakumov et al 2011;Osakabe et al 2013). Most of these conserved factors are essential to the maintenance of nucleosomal structure and the redeposition of H3/H4 within coding regions of genes (Avvakumov et al 2011;Thebault et al 2011;Smolle and Workman 2013).…”

“…The histone chaperones Asf1 (English et al 2006;Natsume et al 2007) and Daxx (Elsasser et al 2012;Liu et al 2012) have been cocrystalized with H3/H4, with their structures confirming earlier biochemical studies indicating that these histone chaperones interact with, exchange, and deposit H3/H4 dimers (English et al 2006;Natsume et al 2007;Elsasser et al 2012;Liu et al 2012). Here we focused on the newly discovered histone chaperone hSpt2 (Osakabe et al 2013) and its complex with histone H3/H4 from a combined structural and functional perspective. Spt2 protein functions in the remodeling and/ or maintenance of chromatin structure during transcription through its ability to bind chromatin in vivo (Nourani et al 2006).…”