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.

Automated summary

The heterodimeric histone chaperone FACT, composed of SSRP1 and SPT16, is involved in dynamic nucleosome rearrangements during DNA-dependent processes. In this study, post-translational modifications of SSRP1 and SPT16 were investigated in Arabidopsis cells. Acetylation of lysine residues in SSRP1's C-terminal region and phosphorylation of serine/threonine residues in SPT16's acidic C-terminal region were identified. Mutational analysis showed that acetylation of SSRP1 had minimal effects, but phosphorylation of SPT16 by protein kinase CK2 was crucial for histone interactions. Plants expressing a non-phosphorylatable version of SPT16 had reduced histone binding and exhibited growth and developmental defects. These findings suggest that phosphorylation of SPT16 is required for proper nucleosome occupancy at the transcriptional start sites of active genes.