Fig 2: DUSP1 modulates LINE-1 mRNA expression. A qPCR of LINE-1 mRNA levels in E006AA-hT cells expressing shRNA to DUSP1 or non-targeting control (NS). B qPCR of relative LINE-1 levels in E006AA-hT cells that were treated with vehicle (DMSO) or DUSP1 inhibitor BCI. C qPCR of relative LINE-1 mRNA levels in E006AA-hT or LNCaP cells overexpressing vector only (VO) or DUSP1. D qPCR of DUSP1 mRNA levels in panel of prostate cancer cell lines (including E006AA-hT renal cell carcinoma line). E qPCR of relative LINE-1 mRNA levels (left) and western blots of LINE-1 ORF1p, DUSP1, and hsp90 (loading control) protein levels (right) in PC3 cells that were treated with siScramble or siDUSP1 siRNA. ORF1 protein levels relative to hsp90 were quantified, siScramble was set to 1

Fig 3: HEXIM1 and AFF1 colocalize to directly suppress rapid-response targets.a Venn diagram showing the significant overlap between AFF1 and HEXIM1 ChIP-seq peaks (Fisher’s exact test, two-tailed, P = 1×10−268). b Violin plot showing the significant enrichment of total Pol II counts at ChIP-seq categories: AFF1 unique peaks, HEXIM1 unique peaks, and peaks with Pol II but no HEXIM1 or AFF1 relative to overlapping AFF1 and HEXIM1 peaks (****P < 0.0001, two-tailed, unpaired t test). c Pie chart showing the peak distribution of AFF1 and HEXIM1 ChIP-seq data in promoters (transcription start site (TSS) ± 1 kb), gene bodies (TSS ± 1 kb through transcription end site), or intergenic regions. d Average profile plot showing the overlapping enrichment of AFF1 and HEXIM1 ChIP-seq signal near the promoters of their shared target genes. e Shared target genes among AFF1 ChIP-seq, HEXIM1 ChIP-seq, and the significantly upregulated genes in keratinocytes with 3-hr KL treatment. f Relative expression of the 92 AFF1-HEXIM1-KL(3 hr) shared target genes, ranked from low to high, in keratinocytes treated with KL versus DMSO for 3 hours. The top two upregulated genes ATF3 and DUSP1, as well as RND3, are highlighted in orange. g Heatmap showing differential expression of rapid-response and differentiation-activating genes with 1-hour and 3-hour nascent RNA-seq. h, i qRT-PCR showing the upregulation of ATF3, DUSP1, and RND3 with AFF1 or HEXIM1 knockdown (n = 3 technical replicates, data are presented as mean values ± standard deviation). j–l Genome browser tracks showing the enrichment of AFF1, HEXIM1, CDK9, undifferentiated (UD) Pol II, and differentiated (DF) Pol II ChIP-seq signal at ATF3, DUSP1, and RND3. Source data are provided as a Source Data file.

Fig 4: SEC mediates the rapid initiation of differentiation in response to PKC signaling.a qRT-PCR comparing the relative mRNA expression of differentiation-activating TFs as well as ATF3, DUSP1, and RND3 with 1 hour or 3 hours of TPA treatment relative to DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). b qRT-PCR shows that the PKC inhibitors, gouml6983 (Gouml) or bisindolylmaleimide I (Bis), consistently suppress the upregulation of representative genes induced by TPA treatment (1 hour) as compared with the DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). c, d qRT-PCR comparing gene expression induced by TPA alone or in combination with KL where panel c is TPA relative to a DMSO control and d is KL + TPA relative to KL alone. (n = 3 technical replicates, data are presented as mean values ± standard deviation). e qRT-PCR showing gene upregulation by TPA (1 hour) in combination with CDK9 inhibitors, flavopiridol (Flav) or NVP2, relative to DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). f Venn diagram (left) and heatmap (right) comparing 3-hour KL RNA-seq and 1-hour TPA RNA-seq differentially expressed (fold change ≥2, P < 0.05, two-tailed, Wald test) genes (Fisher’s exact test, two-tail, P = 2×10−5). Source data are provided as a Source Data file.

Fig 5: The SEC rapid-response gene ATF3 drives keratinocyte differentiation.a–c Western blot showing the overexpression (OE) of ATF3, DUSP1, or RND3 in keratinocytes using the pTRIPZ vector post DOX induction. d–f qRT-PCR showing the mRNA expression levels of differentiation-activating TFs with ATF3, DUSP1, or RND3 overexpression (n = 3 technical replicates, data are presented as mean values ± standard deviation). g–j Representative images and quantification of clonogenic assays comparing human keratinocytes expressing ATF3, DUSP1, or RND3 versus the empty-vector control. Colonies >1 mm2 are included for quantification (n = 3/group, ****P < 0.001, **P < 0.05, ns = not significant, ATF3 P < 0.0001, DUSP1 P = 0.3489, RND3 P = 0.0406, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). Source data are provided as a Source Data file.