Fig 2: TFIIIC-Associated HAT Activity Acetylates H3K18 at Nucleosomes over AEs in Response to Serum Depletion(A) ATAC-seq signal enrichment in T47D grown in the presence (+S) or in the absence of serum (–S) across TFIIIC-bound AEs detected in the absence of serum (–S) (–log10 of the Poisson p value). p value, two-tailed t test comparing each condition to its random control.(B) Average profile of H3K18ac enrichment in T47D grown in the presence (+S,) or absence of serum (–S) across all TFIIIC-bound AEs (–log10 of the Poisson p value). p value, two-tailed paired t test.(C) Sitepro average plot of nucleosome signals derived from ATAC-seq by selecting sequenced fragments of 180–250 bp (Buenrostro et al., 2013) spanning a 1 kb region over all the AEs bound by TFIIIC in T47D grown in the presence (+S) or absence (–S) of serum. Note the presence of a peak in the center of the region representing the AE (gray rectangle). SS causes a decrease in the nucleosome signal for both the AE-nucleosome and the surrounding ones (two-tailed unpaired t test).(D) Genome browser view of representative cell-cycle-related TFIIIC-associated promoters with normalized counts of ATAC-derived nucleosome signal for T47D grown in the presence (+S) or absence (–S) of serum. The position of the AE bound by TFIIIC positive for H3K18ac is indicated by a yellow rectangle. AE nucleosomes reduced upon SS are highlighted by a gray rectangle.(E) Venn diagram showing the total number of AEs bound by TFIIIC and those acetylated in H3K18 in serum-starved T47D.(F) Venn diagram showing the total number of TFIIIC-associated Pol II promoters and those enriched in H3K18ac in T47D grown in the absence of serum.(G) Plot for p300 occupancy across all TFIIIC-bound AEs spanning a 6 kb region in T47D grown in the presence (+S) or absence (–S) of serum.(H) H3K18ac immunostaining (red) in serum-starved T47D cells knocked down for GTF3C1 (siGTF3C1) or control (siCTRL). DAPI was used to stain nuclei (blue). Two different fields are shown. Scale bar, 100 µm. GTF3C1 depletion levels are shown in Figure S3H and quantification in Figure S3I.

Fig 3: Serum Depletion Induces TFIIIC and CTCF Interaction to Allow Long-Range DNA Looping and H3K18 Hyperacetylation of Distal TFIIIC-Looped Genes(A) Boxplot of CTCF binding events within a 10 kb region around the TSSs of TFIIIC-looped genes or of a random dataset of TSS of the same size (p value, Friedman X2 test).(B) Genome browser view of representative cell-cycle-related TFIIIC-activated genes CCND1, CCNF, CENPE, and POLQ. ChIP-seq data of CTCF and TFIIIC in T47D in the presence (+S) or absence (–S) of serum. Note that multiple CTCF binding sites (gray boxes) are present at the 5' end of these genes but no TFIIIC is detected. Transcription directionality is indicated with an arrow.(C) Immunoprecipitation of CTCF in soluble extracts from T47D cell grown in the presence (+) or absence (–) of serum (“beads” are used as a specificity control). Input lysates (10%) and immunoprecipitates were probed with CTCF and GTF3C2 antibodies (as a marker for TFIIIC).(D) Hi-C analysis of TFIIIC and CTCF contacts represented as log2 FC (O / E*; 95% CI) of the specific CTCF-TFIIIC contacts (O = observed) compared to the expected (E*) CTCF and TFIIIC additive effect for both +S and –S conditions. The PPr value indicates a high probability of an increase in TFIIIC/CTCF Hi-C contacts in the absence versus presence of serum.(E) Genomic representation of a 1 Mb region of chromosome 17 containing an AE-TFIIIC-CTCF loop induced by SS (framed with a circle in the Hi-C heatmaps). Color scale bar is reported with red representing values from 200 and above, white values equal to 100, and blue values equal to 0. ChIP-seq data for CTCF and TFIIIC in the presence (+S) or absence (–S) of serum is included at the top and bottom, respectively. Matrix heatmaps with Hi-C data are also included and aligned with the ChIP-seq data. Gray rectangles highlight the position of the AE-TFIIIC and the CTCF interacting regions.(F) Heatmap representation of H3K18ac spanning a 20 kb region of all human promoters in T47D cells grown in the presence or absence of serum. Biased clustering shows promoters with increased H3K18ac in the absence of serum, and the cluster contains several TFIIIC-looped genes (selected examples are listed).(G) Stacked plot representing changes in H3K18ac induced by SS in TFIIIC-looped genes. Note that around 70% of them display increased acetylation.

Fig 4: TFIIIC Controls Gene Expression in Response to SS via AEs-Mediated Long-Range Chromatin Looping(A) Hi-C analysis of intra-TAD contacts represented as log2 fold change (Observed / Expected: O/E) and 95% confidence interval (CI) of the contact enrichment between TFIIIC-bound AEs and Pol II promoters for genes differentially expressed (changed) or not affected (unchanged) by SS in T47D cells. Posterior probability (PPr) = 0.93.(B) Heatmap matrix of Hi-C interactions of PLIN4/5-UHRF1 loci (left panel) and CCNF locus (right panel) for TFIIIC-bound AEs in T47D in the presence or absence of serum. Color scale bar is reported with red representing values from 200 and above, white values equal to 100, and blue values equal to 0. The loops representing the higher frequency of interactions of the AEs and the corresponding genes are framed (Hi-C data, top panel). TADs are indicated by blue rectangles. The regions with changes in their interactions upon SS are framed with gray rectangles and zoomed out to help the visualization of the genome browser tracks of mRNA-seq (A and B indicate biological replicates; the two strands are represented separately), and ChIP-seq data for the protein/histone marks are indicated. The gene structure and transcription direction are reported at the bottom of the figure. Gray bold rectangles highlight the mRNA-seq regions with significant changes for the PLIN4/5, UHRF1, and CCNF loci. Red bold rectangles highlight the position of TFIIIC binding to the AEs.(C) Heatmaps of differential gene expression for siGTF3C5 and siCTRL T47D cells in the absence of serum compared to mock-transfected cells in the absence of serum. Color bar scale stands for log2FC of normalized RNA expression in each condition. Only the genes that changed their expression significantly in siGTF3C5 and not in siCTRL are shown. Two classes of genes were designated as TFIIIC-activated or TFIIIC-repressed genes. Cell-cycle-related genes whose expression was drastically reduced by siGTF3C5 are indicated, including UHRF1 and CCNF further analyzed in (B).(D) TFIIIC contact enrichment for the TFIIIC-activated and TFIIIC-repressed genes using Hi-C data in the absence of serum (p value for logistic regression comparing the two groups).(E) Venn diagram showing the overlap between the TFIIIC-activated genes and those bound by TFIIIC either directly within a 10 kb region or via DNA looping (TFIIIC-looped genes).

Fig 5: Impaired DNA Looping at TFIIIC-Looped Genes by TFIIIC Depletion or AEs Deletion Abrogates the Reactivation of Gene Expression upon Serum Re-exposure(A) Changes in specific intra-TADs contacts made by TFIIIC in T47D treated with siGTF3C5 or siCTRL in the absence of serum. Data are the log2 FC of observed versus expected (95% CI) of Hi-C data. The changes in siGTF3C5 versus siCTRL show significant (PPr = 0.99) decrease of total intra-TADs contacts compared to mock-transfected (–S) versus siCTRL.(B) Hi-C matrix heatmaps of the UHRF1 locus for siCTRL and siGTF3C5 cells in the absence of serum. Color scale bar is reported with red representing values from 200 and above, white values equal to 100, and blue values equal to 0. The looping between the AE bound by TFIIIC and its respective targets (PLIN4/5 and UHRF1) is indicated by a box when detected and an arrow when lost. Note that even in the absence of serum the Alu-FEM1A loops back to the PLIN4/5 locus upon siGTF3C5 (as reported for the condition of presence of serum in Figure 4B).(C) Venn diagram showing the overlap between the TFIIIC-looped and the serum-dependent genes compared to the regions showing compartment switch (Figure S4B). Note the higher overlap of the regions showing compartment changes with the serum-dependent genes (Fisher’s exact test).(D) RNA-seq expression analysis in T47D cells of TFIIIC-activated genes after serum re-exposure for the indicated times in conditions of siCTRL and siGTF3C5. Expression data are reported as RPM (**p < 1x10-12, two-tailed paired t test in comparisons for each time-point). The comparison between 0 to 3 h in siCTRL cells is shown to highlight the rapidity of gene activation after serum addition (***p < 1x10-20). GTF3C5-depleted levels were maintained during the time course (Figure S6B).(E) Schematic representation of the CRISPR-Cas9 approach to delete the TFIIIC-bound AE located between the PLIN4/5 and UHRF1 loci in chromosome 17. The wild-type (WT) and the deleted alleles are shown. The targeted AE is shown as a purple box, the position of the guide RNAs (gRNA1 and gRNA2) is marked with triangles, and the primers used for the screen (Figure S6C) are indicated with blue arrows. Arrows indicate the chromatin interactions in +S (red) and –S (black) conditions based on Figure 4B.(F) UHRF1 expression by qRT-PCR in T47D parental cells (WT) and AE-deleted (Clone11) in the absence of serum (mean ± SEM of biological duplicates with data in WT cells normalized to 1; one-tailed t test).