Phosphorylation of the FACT histone chaperone subunit SPT16 affects chromatin at RNA polymerase II transcriptional start sites in Arabidopsis.

The heterodimeric histone chaperone FACT, consisting of SSRP1 and SPT16, contributes to dynamic nucleosome rearrangements during various DNA-dependent processes including transcription. In search of post-translational modifications that may regulate the activity of FACT, SSRP1 and SPT16 were isolated from Arabidopsis cells and analysed by mass spectrometry. Four acetylated lysine residues could be mapped within the basic C-terminal region of SSRP1, while three phosphorylated serine/threonine residues were identified in the acidic C-terminal region of SPT16. Mutational analysis of the SSRP1 acetylation sites revealed only mild effects. However, phosphorylation of SPT16 that is catalysed by protein kinase CK2, modulates histone interactions. A non-phosphorylatable version of SPT16 displayed reduced histone binding and proved inactive in complementing the growth and developmental phenotypes of spt16 mutant plants. In plants expressing the non-phosphorylatable SPT16 version we detected at a subset of genes enrichment of histone H3 directly upstream of RNA polymerase II transcriptional start sites (TSSs) in a region that usually is nucleosome-depleted. This suggests that some genes require phosphorylation of the SPT16 acidic region for establishing the correct nucleosome occupancy at the TSS of active genes.

Elongation factor 1 is a component of the Arabidopsis RNA polymerase II elongation complex and associates with a subset of transcribed genes.

Elongation factors modulate the efficiency of mRNA synthesis by RNA polymerase II (RNAPII) in the context of chromatin, thus contributing to implement proper gene expression programmes. The zinc-finger protein elongation factor 1 (ELF1) is a conserved transcript elongation factor (TEF), whose molecular function so far has not been studied in plants. Using biochemical approaches, we examined the interaction of Arabidopsis ELF1 with DNA and histones in vitro and with the RNAPII elongation complex in vivo. In addition, cytological assays demonstrated the nuclear localisation of the protein, and by means of double-mutant analyses, interplay with genes encoding other elongation factors was explored. The genome-wide distribution of ELF1 was addressed by chromatin immunoprecipitation. ELF1 isolated from Arabidopsis cells robustly copurified with RNAPII and various other elongation factors including SPT4-SPT5, SPT6, IWS1, FACT and PAF1C. Analysis of a CRISPR-Cas9-mediated gene editing mutant of ELF1 revealed distinct genetic interactions with mutants deficient in other elongation factors. Moreover, ELF1 associated with genomic regions actively transcribed by RNAPII. However, ELF1 occupied only c. 33% of the RNAPII transcribed loci with preference for inducible rather than constitutively expressed genes. Collectively, these results establish that Arabidopsis ELF1 shares several characteristic attributes with RNAPII TEFs.