MOESM3 of Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Additional file 3: Figure S3. Effect of flavopiridol on RNA polymerase II occupancy at the hsp70 locus upon heat shock. a N-ChIP-qPCR measurement of the occupancy level of Pho at the hsp70 locus in S2 DRSC cells during the heat shock response detailed in Fig. 1a. Distance of the location of the primers used from the hsp70 TSS is − 85 bp and has been depicted in the form of a cartoon below the data figure. b, c ChIP-qPCR measurements of occupancy levels of RNA polymerase II CTD and S2P form of RNA polymerase II, respectively, at the hsp70 locus in S2 DRSC cells. Distances of the location of the primers used from the hsp70 TSS are as follows: for a (+ 78 bp) and for b (+ 802 bp) and has been depicted in the form of a cartoon below the data figure. d qRT-PCR measurements of hsp70 transcript levels in third-instar Drosophila larvae under the conditions used for double polytene immunostaining. Distance of the location of the primers used from the hsp70 TSS is + 687 bp and has been depicted in the form of a cartoon below the data figure. For b–d, the control bar represents cells that were maintained at 25 °C for 15 min, the HS bar represents cells that were maintained at 37 °C for 15 min, the FP control bar represents cells that were treated with 500 nM flavopiridol for 40 min and then maintained at 25 °C for 15 min and the FP HS bar represents cells that were treated with 500 nM flavopiridol for 40 min and then maintained at 37 °C for 15 min. Data information: In (a–d), data are presented as mean ± SEM (n = 2)

Predicting the replication timing profile of the <i>Drosophila</i> S2 cells genome.

<p>(A) Predicted versus experimentally measured replication timing of the <i>Drosophila</i> S2 cells genome represented as smoothed color density scatter plot. Model predictions were generated using the Lasso model based on CBPs and second-order interactions between HMs (HMs²+CBPs) and trained at promoters. Prediction accuracy is Pearson correlation coefficient. The orange line indicates the model fit, whereas the dashed gray line indicates the bisector . (B,C) Measured (top track) and predicted (middle and bottom track, see Methods) replication timing profiles along 6 Mb and 12 Mb of chromosomes 3R (B) and 3L (C), respectively. A color gradient representation of feature signals is shown at the bottom for chromatin features within the bootstrap-Lasso simplified model (K8ac = H4K8ac; K36me1 = H3K36me1 and K79me1 = H3K79me1). The yellow rectangle in B highlights the genomic position of the Bithorax Complex.</p

MOESM2 of Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Additional file 2: Figure S2. RNA polymerase II binding dynamics at Act42A locus during the heat shock response. a, b ChIP-qPCR measurements of occupancy levels of RNA polymerase II CTD and S2P form of RNA polymerase II at the Act42A locus during the heat shock response detailed in Fig. 1a. The control line represents cells that were maintained at 25 °C for the entire duration of the time course detailed in Fig. 1a. Distance of the location of the primers used from the Act42A TSS is as follows: for a and b (+ 104 bp) and has been depicted in the form of a cartoon below the data figure. Data information: In (a, b), data are presented as mean ± SEM for n = 2

MOESM7 of Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Additional file 7: Table S1. List of the primer sequences used in the study

Histone modification levels predict replication timing across different cell types.

<p>(A) Predicted versus experimentally measured differences in replication timing between S2 and Bg3 cells unique promoters (S2-Bg3) represented as smoothed color density scatter plot. Model predictions were generated using differences in HMs levels and their pairwise interactions for a subset of HMs that were profiled in both S2 and Bg3 cell lines (CHMs). The orange line indicates the model fit, whereas the dashed gray line indicates the bisector . (B) Scatter plot of model features according to their <i>z</i>-scores and bootstrap-Lasso selection probabilities (<i>p</i>). Features with are colored in red (positive coefficient values) or blue (negative coefficient values) and their coefficient distributions are shown on the right as violin plots. Features are ranked by decreasing selection probabilities. (C, top) Replication timing of S2 cells promoters versus Bg3. Differentially replicating promoters are color-coded according to the quadrant (delimited by dashed blue lines) they belong to (red: early replicating in S2 and late replicating in Bg3; green: early in both S2 and Bg3; blue: late in S2 and early in Bg3, aqua: late in both S2 and Bg3). A total of promoters exhibit a log fold change greater than or equal to 1 (). (C, bottom) Experimentally determined replication timing in Bg3 versus predictions generated by a model based on pairwise interactions between CHMs in S2 cells. Prediction accuracy is Pearson correlation coefficient. The dashed gray line indicates the bisector .</p

MOESM4 of Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Additional file 4: Figure S4. Chromatin binding dynamics of Spt5 and the dissection of the protein–protein interaction domains of Pho. a ChIP-qPCR measurements of occupancy levels of FLAG-Spt5 at the hsp70 locus over the time course detailed in Fig. 1a. The control line represents cells that were maintained at 25 °C for the entire duration of the time course detailed in Fig. 1a. The cartoon at the bottom of the figure represents the distance of the location of the primers used from the hsp70 TSS. b–c co-IP assays of S2 DRSC cells transiently transfected with plasmids expressing FLAG-tagged dSfmbt or Spt5 (FLAG-dSfmbt, FLAG-Spt5) and HA-tagged Pho, N-Pho (a.a 1-351), 1-200N-Pho (a.a 1-200), 173-351N-Pho (a.a 173-351), C-Pho (a.a 352-520). Cell lysates were used for pull-downs using an anti-FLAG antibody and were later probed by Western blot using an anti-HA antibody. MW = molecular weight in kDa. d co-IP assays for S2 DRSC cells transiently transfected with plasmids expressing FLAG-tagged Pho and HA-tagged Spt5 or HA-tagged dSfmbt. S2 DRSC cells were either maintained at normal growth temperature (25 °C) or heat shocked at 37 °C for 15 min. Cell lysates were used for pull-downs using an anti-FLAG antibody and were later probed by Western blot using an anti-HA antibody. MW = molecular weight in kDa

MOESM5 of Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Additional file 5: Figure S5. The dynamic interaction switch between Pho and Spt5 is independent of DNA but dependent upon RNA. a co-IP assays of S2 DRSC cells transiently transfected with plasmids expressing FLAG-tagged Spt5 (FLAG-Spt5) and HA-tagged Pho (HA-Pho). S2 DRSC cells were either maintained at normal growth temperature (25 °C) or heat shocked at 37 °C for 15 min. Prior to performing the pull-down, the cell lysates were treated with either RNase A or DNase I. Thereafter, the treated cell lysates were used for pull-downs using an anti-FLAG antibody and later probed by Western blot using an anti-HA antibody. b co-IP assays of S2 DRSC cells transiently transfected with plasmids expressing FLAG-tagged Spt5 (FLAG-Spt5) and HA-tagged N-Pho (HA-N-Pho). S2 DRSC cells were either maintained at normal growth temperature (25 °C) or heat shocked at 37 °C for 15 min. Cell lysates were prepared and were used for pull-downs using an anti-FLAG antibody. They were later probed by Western blot using an anti-HA antibody

MOESM1 of Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Additional file 1: Figure S1. Schematic representation of the proteins involved in the silencing, activation and re-silencing of the hsp70 locus. a At optimal growth temperature, Pho, a DNA-binding PcG member, binds to promoter region of the hsp70 locus. Pho interacts with dSfmbt, which together form a recruitment platform for PRC1 to the hsp70 locus. In addition, RNA polymerase II is maintained in the paused state by NELF and Spt5, which act as pausing factors. Upon heat shock, HSF, along with P-TEFb, is recruited to chromatin and releases RNA polymerase II from the paused state. P-TEFb modifies Spt5 and converts into an elongation factor. It also modifies the CTD of RNA polymerase II to enable productive elongation. However, upon removal of the heat shock stimulus, the locus should eventually return to its paused state. Thus, hsp70 is an ideal model gene to study the eviction and recruitment of PRC1 upon activation and re-silencing, respectively. The colour code for the protein names is as follows: silencing in red, pausing factors in orange and activators in green

Signalling crosstalk during early tumorigenesis in the absence of Polycomb silencing

<div><p>In response to stress and injury a coordinated activation of conserved signalling modules, such as JNK and JAK/STAT, is critical to trigger regenerative tissue restoration. While these pathways rebuild homeostasis and promote faithful organ recovery, it is intriguing that they also become activated in various tumour conditions. Therefore, it is crucial to understand how similar pathways can achieve context-dependent functional outputs, likely depending on cellular states. Compromised chromatin regulation, upon removal of the Polycomb group member <i>polyhomeotic</i>, leads to tumour formation with ectopic activation of JNK signalling, mediated by <i>egr</i>/<i>grnd</i>, in addition to JAK/STAT and Notch. Employing quantitative analyses, we show that blocking ectopic signalling impairs <i>ph</i> tumour growth. Furthermore, JAK/STAT functions in parallel to JNK, while Notch relies on JNK. Here, we reveal a signalling hierarchy in <i>ph</i> tumours that is distinct from the regenerative processes regulated by these pathways. Absence of <i>ph</i> renders a permissive state for expression of target genes, but our results suggest that both loss of repression and the presence of activators may collectively regulate gene expression during tumorigenesis. Further dissecting the effect of signalling, developmental or stress-induced factors will thus elucidate the regulation of physiological responses and the contribution of context-specific cellular states.</p></div

Signalling-dependent activators are required for de-repression of a PRE in <i>ph</i> mutant clones.

<p><b>(A-A”)</b> The rpr(PRE)-GFP reporter is generally undetected in control eye discs with neutral clones. <b>(B-B”)</b> The reporter becomes activated in <i>ph</i> clones, but its expression is again reduced in clones expressing UAS-bsk^DN to block JNK signalling <b>(C-C”)</b>. <b>(D-D”)</b> Similar abrogation of reporter expression in <i>ph</i> clones was observed when using a version with mutated binding sites for the canonical JNK transcription factor AP-1. DAPI staining (cyan) is shown in the left panels, while the central panels show the reporters and the distribution of RFP-marked clones is shown on the right panels. Scale bar represents 200 μm.</p