KAP1 negatively regulates RNA polymerase II elongation kinetics to activate signal-induced transcription

Abstract

Signal-induced transcriptional programs regulate critical biological processes through the precise spatiotemporal activation of Immediate Early Genes (IEGs); however, the mechanisms of transcription induction remain poorly understood. By combining an acute depletion system with high resolution genomics approaches to interrogate synchronized, temporal transcription, we reveal that KAP1/TRIM28 is a first responder that fulfills the temporal and heightened transcriptional demand of IEGs. Unexpectedly, acute KAP1 loss triggers an increase in RNA polymerase II elongation kinetics during early stimulation time points. This elongation defect derails the normal progression through the transcriptional cycle during late stimulation time points, ultimately leading to decreased recruitment of the transcription apparatus for re-initiation thereby dampening IEGs transcriptional output. Collectively, KAP1 plays a counterintuitive role by negatively regulating transcription elongation to support full activation across multiple transcription cycles of genes critical for cell physiology and organismal functions.

Figure 1.. KAP1 is a positive regulator of serum-induced transcription.

(A) Scheme showing dTAG strategy targeting the C-terminus of KAP1 with the dTAG cassette. FKBPV: degron; Puro: Puromycin resistance cassette; BSD: Blasticidin resistance cassette. (B) Western blot showing dTAG-mediated KAP1 degradation kinetics in HCT116 KAP1^dTAG cells. (C) Scheme showing the experimental design alongside the cell treatments. (D) RT-qPCR assay highlighting gene expression of one representative IEG (FOS) in DMSO-and dTAG-treated cells during a serum stimulation time course. Data represent mean ± SEM (n=3, Student’s t-test comparing DMSO to dTAG at each of the indicated time points). *P< 0.05, **P<0.01, ***P<0.001. (E) RNA-Seq volcano plot identifying IEGs as upregulated genes upon 30 min of serum stimulation (n=3, false discovery rate [FDR] < 0.05). (F) Violin plot showing upregulation of IEGs organized by induction of expression: 2-Fold IEGs (n=236), 4-Fold IEGs (n=69), 8-Fold IEGs (n=34), and 16-Fold IEGs (n=16). Random (n=236) denotes random genes selected from non-DE genes (n=10,361). (G) Violin plot showing Log2FC values of dTAG versus DMSO in serum treated cells. Median % changes in expression is listed above the violin in each IEGs cluster and Random non-DE genes. (H) Western blot showing protein expression of two representative IEGs (c-Fos and ATF3) during a serum time course ± dTAG.

Figure 2.. KAP1 localizes to the gene bodies and 3’ ends of IEGs upon serum stimulation.

(A) HA (KAP1) ChIP-Seq metagene analysis at 16-Fold IEGs in the three indicated conditions. (B) HA (KAP1) ChIP-Seq browser track of FOS in the four indicated conditions. (C-D) HA ChIP-Seq quantitation’s of KAP1 density at 4-Fold IEGs and 16-Fold IEGs at (C) PP regions and (D) GB regions. The Log2FC value is plotted for the respective time point normalized to serum 0 min in DMSO-treated cells. Statistics were calculated between the dTAG plus serum treatment condition and the respective condition shown on the Tukey plot. Wilcoxon signed-rank test (*P<0.05, **P<0.01, ***P<0.001). (E) HA (KAP1) ChIP-Seq metagene analysis of all genome-wide KAP1 peaks (n=12,665) in the three indicated conditions. (F) HA (KAP1) ChIP-Seq browser track at the SFPQ locus in the four indicated conditions.

Figure 3.. KAP1 regulates Pol II occupancy during serum stimulation.

(A-B) Pol II ChIP-Seq metagene at 16-Fold IEGs showing occupancy of Pol II at: (A) 15 min and (B) 30 min serum stimulation time points. (C-E) Pol II ChIP-Seq quantitation’s of Pol II density at 4-Fold IEGs and 16-Fold IEGs: (C) PP regions, (D) GB regions, and (E) 3’ ends. The Log2FC value is plotted for dTAG versus DMSO at the respective serum time point and statistics calculated for the comparison between the early and late time points. Wilcoxon signed-rank test (*P<0.05, **P<0.01, ***P<0.001). (F-G) Pol II and HA ChIP-Seq browser track in multiple conditions at the (F) NR4A1 locus and (G) ATF3 locus. The arrow indicates the polyadenylation (pA) site.

Figure 4.. Acute KAP1 depletion leads to increased elongation kinetics at early serum stimulation.

(A-B) PRO-Seq metagene profile at 16-Fold IEGs showing active Pol II density at the (A) 5 min and (B) 10 min serum stimulation time points. (C) PRO-Seq quantitation of nascent transcription (GB density). The Log2FC value is plotted for each sample normalized to serum 0 min in DMSO-treated cells. Wilcoxon signed-rank test (*P<0.05, **P<0.01, ***P<0.001). (D) Pol II ChIP-Seq, PRO-Seq, and HA ChIP-Seq browser track in multiple conditions at the FOSB locus. (E) Rate of Change in Coverage (ROCC) calculation (Proxy Rate) for 4-Fold IEGs and 16-Fold IEGs calculated through the serum stimulation time course. Wilcoxon signed-rank test (*P<0.05, **P<0.01, ***P<0.001).

Figure 5.. Acute KAP1 depletion leads to decreased occupancy of regulators of transcription elongation and initiation at late serum stimulation.

(A-C) ChIP-Seq metagene profile of (A) SPT5, (B) CDK9, and (C) CDK7 at 16-Fold IEGs in the three indicated conditions. (D-E) ChIP-Seq quantitation of (D) SPT5, (E) CDK9, and (F) CDK7 in PP regions of 4-Fold IEGs and 16-Fold IEGs. Log₂FC value is plotted to compare each time point to no serum condition (0 min) in DMSO-treated cells. The median percent increase when compared to DMSO-Serum 0 min is listed below each bar in the Tukey plot. (G-H) ChIP-Seq browser tracks of all factors in the three indicated conditions at the (G) FOS locus and (H) ATF3 locus.

Figure 6.. KAP1 facilitates signal-induced transcription activation by negatively regulating Pol II elongation kinetics.

(A) Model of KAP1 facilitating signal-induced transcription by negatively regulating Pol II elongation kinetics during early stimulation to allow for the timely transition of elongation to termination and normal re-initiation events (including recruitment of the transcription apparatus to the promoter) during late stimulation. (B) KAP1 depletion increases Pol II elongation kinetics during early stimulation, consequently leading to defects in the recruitment of the transcription apparatus to the promoter during late stimulation thereby dampening IEGs expression.