CLAMP regulates heat-stress-induced transcriptional repression more strongly than activation

A-B. MA plots showing significant changes (FC ≥ 1.5 and padj ≤ 0.05) in the levels of nascent transcripts (red dots) after HS (heat stress) compared to the control NHS (No heat stress) condition in control (gfp RNAi) cells (A) and after HS (heat stress) in the absence of CLAMP (clamp RNAi) compared to control (gfp RNAi) cells (B). Dotted horizontal lines in A and B denote ±1 log2 FC change cutoffs. C. Immunofluorescence images of HS (37⁰C for 1 Hr) salivary gland polytene spreads showing the distribution of active RNA polymerase II (red) on chromatin (DAPI: blue) in the presence (undriven clamp RNAi) and absence (fkh-GAL4>clamp RNAi) of CLAMP (green). The scale bar (100 μM) applies to both panels in C. D-E. Venn diagrams comparing HS-activated transcripts (deep red circle in D) and HS-repressed transcripts (light red circle in E) from control cells (gfp RNAi) to clamp RNAi HS Up-regulated (dark green circle) and clamp RNAi HS Down-regulated (light green circle) transcripts in clamp RNAi-treated cells.

Heat stress induces widespread local 3D chromatin changes at the loop anchors

A-B. MA plots for differential loop anchors gained upon HS (HS induced Up loop anchors) or loop anchors lost upon HS (HS induced Down loop anchors) in control cells (A, Up loops=24,291, Down Loops=4,265) and clamp RNAi cells (B, Loops Up-regulated=8,274, Loops Down-regulated =2,000). p<0.01 C. Bar plots show the average number of genes associated with (top box) and present at (bottom box) the HS-induced genome-wide chromatin loop anchors and HS-induced CLAMP-dependent chromatin loop anchors. D. Box plots showing average loop size of HS-induced genome-wide changing chromatin loops and CLAMP-dependent HS-induced differential chromatin loops with significant difference between average chromatin HS-induced up and down loop sizes, both in case of HS-induced genome-wide changing chromatin loops (Mann–Whitney Test, FDR corrected p=5.63E-186) and CLAMP-dependent HS-induced differential chromatin loops (Mann–Whitney Test, FDR corrected p=3.97E-70), and also between HS-induced genome-wide changing and CLAMP-dependent HS-induced differential chromatin loops themselves (Mann–Whitney Test, FDR corrected p=0) E. Upset Plot showing genes associated with differential chromatin loop changes and cardinality of every category combination seen as chromatin loop changes after HS in control (top two rows) and clamp RNAi-treated (bottom two rows) cells. The first four columns show chromatin loop-associated genes only in each category, and the following six columns show those in each combination of exactly two named sets, the four columns after that combination of three named sets, and the last column shows genes associated with chromatin loop changes in all four categories. HS-associated CLAMP-dependent chromatin loop changes are mostly represented in the last three columns/bars. F-I. Venn diagrams comparing HS-associated chromatin loop changes (genes within the loop) and HS-activated and repressed genes (F and G); as well as CLAMP-dependent chromatin loop changes during HS with CLAMP-dependent HS-induced transcriptional changes (H and I), showing significant (Fisher’s Test) overlap between HS-associated Up loops (F) and Down loops (G) with HS-repressed genes; and CLAMP-dependent HS-associated Down loops (H, clamp RNAi up-regulated HS-loops) and Up loops (I, clamp RNAi down-regulated HS-loops) with CLAMP-dependent HS-repressed genes (clamp RNAi HS up-regulated genes). FDR corrected p-values are 7.487 E-09 (F), 2.53 E-4 (G), 2.0 E-6 (H), and 4.290 E-15 (I), respectively. J. 350 kb region on 3L showing differential contact interaction results of selfish algorithm on HS and NHS micro-C contact map (I) with associated chromatin loops that are formed (II) and inhibited after HS (III). CLAMP-dependent differential contact interaction results of the selfish algorithm are shown on clamp RNAi-treated HS and control HS micro-C contact maps (IV) with CLAMP inhibited (V) and favoured (VI) chromatin loops. The color bar in I and IV indicates the q-value (BH adjusted p-value) produced from the DCI analysis. Darker color means this interaction has a lower q-value; that is to say, two contact maps are more diverse at this location. Bed files for differential nascent RNA-seq (SLAM-seq) data from HS and NHS clamp RNAi-treated and control cells show the CLAMP-dependent HS-repressed genes (VII) among the corresponding genes within the genomic region associated with the loops (VIII)

CLAMP regulates chromatin loop changes during HS, directly as well as indirectly, to repress gene transcription

A. 2D metaplot of CLAMP HiChIP data centered on significant interactions identified from CLAMP HiChIP under NHS or HS conditions for differential loop anchors gained upon HS (HS Up loop anchors, N=71) or loop anchors lost upon HS (HS Down loop anchors, N=980). The score indicates the enrichment of the center pixel compared with the top left corner. B. CLAMP-bound loop pile-ups for HiChIP data from heat stress (HS) and control cells showing loss and gain of chromatin structures marked by chromatin loops defined in HS and control cells. The color is average interaction frequency across the entire dataset surrounding the defined HS Up and Down loops. A high signal indicates strong interaction between loop anchors. ±20 kb padding was applied to the central 2.5 kb bin, displaying a 42.5 kb aggregated region. C. Barplots showing average number of genes associated within (top box) and present at (bottom box) the CLAMP-bound chromatin loop anchors up and down-regulated after Heat shock (HS) D. Box plots showing significant difference (Mann–Whitney Test, FDR corrected p=0.0354) between average loop size of CLAMP-bound chromatin loop anchors up and down-regulated after Heat shock (HS). E. Upset Plot comparing all (Micro-C data) and CLAMP-bound (HiChIP data) chromatin loops lost after HS with HS-repressed and CLAMP-dependent HS-repressed genes, showing significant (Fisher’s Test) overlap between CLAMP-bound HS-associated Down-loops with global genome-wide loss of HS-associated loops (p=1.26E-115), HS-repressed genes (p=9.59E-18), and CLAMP-dependent HS-repressed genes (clamp RNAi HS up-regulated genes, p=2.57E-19). F. 300 kb region on 2R showing CLAMP-regulated differential contact interaction results of selfish algorithm on clamp RNAi-treated and control HS micro-C contact map (I) with associated CLAMP suppressed (II) and induced (III) chromatin loops after HS. CLAMP-associated differential contact interaction results of the selfish algorithm are shown on HS and NHS CLAMP HiChIP contact maps (IV) with CLAMP-bound (V and VII) directly associated Up (VI) and Down (VIII) chromatin loops. The color bar in I and IV indicates the q-value (BH adjusted p-value) produced from the DCI analysis. A darker color means this interaction has a lower q-value; that is to say, two contact maps are more diverse at this location. Normalized reads and bed files for differential nascent RNA-seq (SLAM-seq) data from HS and NHS clamp RNAi-treated and control cells show the CLAMP-dependent HS-repressed genes (IX and X) among the corresponding genes within the genomic region associated with the loops (XI)

HS-associated chromatin loops directly and indirectly regulated by CLAMP have different chromatin-bound factors under NHS conditions

A. Upset plot comparing all CLAMP-dependent (Micro-C data, clamp RNAi up-regulated HS Loops) and CLAMP-bound (HiChIP data) chromatin loops lost after HS with CLAMP-dependent HS-repressed genes, showing a significant (Fisher’s Test) number of CLAMP-dependent HS-repressed genes are associated with CLAMP-dependent loss of chromatin loops after HS (p=1.55E-04). Notably, a significant proportion of these are directly CLAMP-bound (p=8.78E-32). B. Schematic showing three categories of CLAMP-dependent HS-repressed genes based on 3D chromatin organisational changes associated with HS. C. Average profile for chromatin-bound factors across CLAMP-dependent HS-repressed genes present at direct and indirect CLAMP-dependent HS-induced Down-loop anchors, genes not associated with any loop anchors, and a random set of genes (negative control). D. 35 kb region on 2L showing distribution of chromatin bound factors at CLAMP-dependent HS-repressed genes dock and drongo and associated CLAMP-bound chromatin loops lost after HS

Model for CLAMP-dependent HS-induced transcriptional gene repression