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

Abstract

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.

Distinct role of subunits of the Arabidopsis RNA polymerase II elongation factor PAF1C in transcriptional reprogramming

“…Chromatin immunoprecipitation (ChIP) was essentially performed as previously described ( Antosz et al., 2020 ; Michl-Holzinger et al., 2022 ). Plants (14-DAS in vitro grown) were crosslinked with formaldehyde and used for isolation of nuclei, before chromatin was sheared using a Bioruptor pico device (Diagenode).…”

Transcription elongator SPT6L regulates the occupancies of the SWI2/SNF2 chromatin remodelers SYD/BRM and nucleosomes at transcription start sites in Arabidopsis

“…In combination with our previous work, we found that plant SPT6L associates with TSS ( 18 ) and regulates the deposition of the nucleosome (Figure 4A ), suggesting that plant SPT6L, unlike its orthologs in yeast and Drosophila, may be involved in the transcription initiation stage. Interestingly, recent work has uncovered that the phosphorylation status of Arabidopsis SPT16, a subunit of the FACT complex, is involved in regulating nucleosome occupancy around TSS ( 67 ), suggesting the joint function of SPT6L and FACT on nucleosome regulation during early transcription stage. Additionally, we found an increased nucleosome occupancy around TSS in spt6l (Figure 4A ), which is inconsistent with reduced nucleosome occupancy after the mutation of SPT6 in yeast ( 68 ).…”

“…Both SYD and BRM interact with SPT6L (SYD/BRM-SPT6L), SPT6LΔtSH2 (SYD/BRM-SPT6LΔtSH2), and Pol II (SYD/BRM-Pol II) (Figure 2A - B and Supplementary Figure S6A-C ), but the two chromatin remodelers show enrichment over the TSS regions rather than gene bodies (Figure 1B , D ) ( 28 , 30 , 54 ), suggesting that on one side, SPT6L, as the core subunit of the transcription machinery, may precisely guide chromatin remodelers to active transcription sites and maintain proper nucleosome environment; on the other side, the association between chromatin remodelers and transcription machinery may gradually attenuate during early elongation to productive transcription. Within this transition, the phosphorylation of the Pol II CTD domain (phos-Pol II) plays a critical role and the activities of SPT6 and SPT16 are also regulated in the wave of phosphorylation in yeast and plants, respectively ( 67 , 72 ). By applying the P-TEFb inhibitor, we found that phos-Pol II is required to recruit chromatin remodelers to the transcription machinery (Figure 2D and Supplementary Figure S5A ).…”

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

“…Protein interaction assays were essentially performed as described previously (Michl‐Holzinger et al ., 2022), with recombinant ELF1 proteins fused to glutathione S transferase (GST) mixed with equimolar amounts of recombinant Arabidopsis H2A‐H2B or with bovine cytochrome C (CytC; Sigma Aldrich) in GST buffer (0.2–0.35 M NaCl, 25 mM HEPES pH 7.6, 0.05% (v/v) NP40, 5 mM DTT, 10% (v/v) glycerol, 2 mM MgCl 2 ). Following incubation (30 min at 30°C), glutathione‐sepharose beads were added and the samples were incubated for 3 h on a rotating wheel at 4°C.…”