Fig 2: A mechanistic model of the suppression of divergent antisense transcription by ZWC complex-mediated SPT5 phosphorylation. (A) The ZWC complex is recruited to the TSSs of active genes through direct, simultaneous binding of ZC3H4 and WDR82 to the S5p RNAPII CTD. This allows CK2 to phosphorylate eight newly identified serine and tyrosine residues in the N-terminal acidic domain of SPT5, a subunit of DSIF, which may facilitate the transcriptional transition from early to productive elongation through nucleosomes (not depicted) in the sense transcription direction. How SPT5 N-terminal phosphorylation supports this transition needs to be further investigated. Although not shown, the ZWC complex may phosphorylate additional transcriptional co-regulators that also participate in the transition from early to productive transcription elongation. (B) In the absence of ZC3H4, the ZWC complex is destabilized, and CK2 is unable to phosphorylate SPT5. Failure to phosphorylate the N-terminal acidic domain of SPT5 prevents SPT5 from fully supporting the transition from early to productive elongation in the sense transcription direction; this increases the RNAPII population that moves in the opposite direction and results in increased divergent antisense transcription and decreased sense transcription. This phenomenon is more profound at promoters that naturally produce divergent antisense transcripts. Failed phosphorylation also increases S2p RNAPII CTD and H3K36me3 marks upstream of the TSS (not depicted). Note that the total number of RNAPII molecules around the TSS is unchanged.

Fig 3: SPT6 is necessary for human epidermal differentiation.a Eight elongation factors were knocked down greater than 75% by siRNAs. Knockdown cells were placed in high confluence and calcium for 3 days to induce differentiation. RT-qPCR was used to quantify the knockdown levels of each elongation factor shown on the X-axis. Two separate siRNAs (SPT6-1i and SPT6-2i) targeting different regions of SPT6 were used. qPCR values were normalized to L32. n = 3 independent experiments. b Western blot for knockdown of SPT5 and ELL. N = 3 independent experiments which all showed similar results. c, d RT-qPCR quantifying the relative mRNA expression levels of epidermal differentiation genes TGM1 and GRHL1 in differentiated cells as described in a. n = 3 independent experiments. e Immunofluorescent staining of late differentiation markers FLG (red) and LOR (green) in day 6 regenerated human epidermis treated with control (CTLi) or two distinct siRNAs targeting SPT6 (SPT6-1i and SPT6-2i). Merged image includes Hoechst staining of nuclei. n = 3 independent experiments, scale bar = 50 μm. f Immunofluorescent staining of early differentiation marker K10 (red) and proliferation marker ki67 (green) in day 6 regenerated human epidermis treated with control (CTLi) or two distinct siRNAs targeting SPT6 (SPT6-1i and SPT6-2i). Merged image includes Hoechst staining of nuclei. n = 3 independent experiments. Scale bar = 50 μm. g Quantification of the %ki67 positive cells in the basal layer from images taken in f. n = 3 independent experiments. At least 100 basal layer cells were quantitated per experiment. h Hematoxylin and eosin staining of CTLi or SPT6i knockdown regenerated human epidermis (day 6). N = 3 independent experiments which all showed similar results. Scale bar = 50 μm. i Quantification of the tissue thickness from images taken from e, f. n = 3 independent experiments. j RT-qPCR quantifying the relative mRNA expression levels of epidermal differentiation genes in CTL and SPT6 knockdown regenerated human epidermis (day 6). N = 3 independent experiments. k RNA-Seq analysis of CTL and SPT6 knockdown regenerated human epidermis (day 6). N = 3 independent experiments. In total, 4275 genes were upregulated (red) and 1465 genes were downregulated (blue) upon SPT6 depletion. Heatmaps are shown in Log2 scale. l Gene ontology (GO) terms for the 4275 genes upregulated in SPT6i tissue using hypergeometric distribution. m GO terms for the 1465 genes reduced in expression upon SPT6 loss using hypergeometric distribution. n Overlap of the SPT6 knockdown gene expression signature with the differentiation signature. p value was calculated using hypergeometric distribution. Mean values are shown with error bars = SD. ****p < 0.0001 (two-sided t-test for c, d, g, i, j).

Fig 4: ZWC complex-mediated phosphorylation of eight serine and tyrosine amino acid residues in the N-terminal acidic domain of SPT5. (A) A schematic diagram of SPT5 and derived fragments with acidic, NGN (NusG N-terminal), KOW (Kyrpides, Ouzounis and Woese), and CTR domains. SPT4 associates with the NGN domain to form DSIF. Eight serine and tyrosine amino acids phosphorylated by the ZWC complex (identified in this study) are indicated in the enlarged N-terminal acidic domain of SPT5. Note that the N-terminal acidic domain contains many serine/threonine/tyrosine residues in addition to the eight amino acids phosphorylated by the ZWC complex. Numbers indicate amino acid residues. FL, full-length. (B and C) ZWC complex-mediated SPT5 phosphorylation in vitro. Purified DSIF (composed of FLAG-tagged SPT5 and untagged SPT4) was subjected to in vitro kinase assays with ZWC complexes containing wild-type (WT) or catalytically-dead mutant (K68A) CK2α in the presence and absence of quinalizarin, as indicated. Phosphorylation status was monitored by autoradiography (B) and immunoblotting with an anti-phospho-CK2 substrate antibody (C). Note that no autoradiographic signal was observed at the SPT4 position in (B). (D–F) ZWC complex-mediated phosphorylation of eight serine and tyrosine amino acid residues in the N-terminal acidic domain of SPT5 in vitro. In vitro kinase assays with the ZWC complex were performed on DSIFs composed of the indicated SPT5 fragments and SPT4 (D), WT and mutant (S6Y2→A, harboring alanine substitutions at eight ZWC complex-mediated phosphorylation sites) SPT5 1–270 fragments (E), and DSIFs containing WT and S6Y2→A mutant SPT5 (F). The phosphorylation status of SPT5 fragments was monitored by autoradiography. Phosphorylated SPT5 fragments are marked by asterisks in (D). (G–I) ZWC complex-mediated SPT5 phosphorylation in cells. HEK293T cells were treated with quinalizarin (G) or ZC3H4-targeted siRNA (H), as indicated, and whole-cell extracts were immunoprecipitated with anti-SPT5 antibody. Whole-cell extracts prepared from FLAG-tagged WT and S6Y2→A mutant SPT5 HEK293T cell lines were immunoprecipitated with an anti-FLAG antibody (I). SPT5 phosphorylation status was monitored by immunoblotting with an anti-phospho-CK2 substrate antibody. SPT5 is indicated by an arrow. A non-specific band recognized by the anti-phospho-CK2 substrate antibody is indicated by an empty circle.

Fig 5: The ZWC complex is localized at TSSs of active genes. (A) Representative snapshot image of ChIP-seq results generated for ZWC complex subunits, RNAPII, SPT5 and histone modifications. Read densities were visualized in four genomic regions with different levels of transcriptional activity. (B) Heat map analyses of ChIP-seq peaks from ZWC complex subunits, RNAPII, SPT5 and histone modifications. ZC3H4 peaks (n= 10 237) were aligned with those of other ZWC complex subunits, RNAPII, SPT5 and modified histones. Read intensities were visualized from −1 kb relative to the TSS to +1 kb relative to the TES (transcription end site). (C) Transcription-dependent association of the ZWC complex with chromatin. Peak intensities are shown for the top 10% highly transcribed genes and all transcriptionally active genes (n= 24 640, RPKM >0.1). RPM, reads per million.