The repressive and alleviating nature of FACT shapes the transcriptional landscape in ES 1 cells

both, efficient elongation of polymerases and nucleosome integrity. In cells, is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in mouse embryonic stem cells (mESCs), depletion of FACT leads to up- regulation of pro-proliferative genes and key pluripotency factors concomitant with hyper-proliferation of mES cells. Using MNase-, ATAC-, and Nascent Elongating Transcript Sequencing (NET-seq) we show that up-regulation of genes coincides with loss of nucleosomes upstream of the TSS and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to control expression of these On the hand, a large of down- regulated are controlled Tfap2c suggesting a cooperation this and we

similar binding pattern to each other over the transcription start site (TSS) of all the annotated 120 genes and were tightly linked to H3K4me3 (Fig. 1c). 121 122

Regulation of gene expression by FACT 123
To investigate how FACT orchestrates transcriptional regulation in ESCs, we depleted SSRP1 124 levels using short hairpin RNAs (shRNA - Supplementary Fig. 2a). Surprisingly, we observed 125 an increase in mESC proliferation following Ssrp1 knock-down (KD) as measured by 126 proliferation rate using MTT assays using independent shRNAs ( Fig. 2a & Supplementary Fig.  127 2b). This is in contrast to previously published data from tumour cell lines, in which proliferation 128 rates decrease and also terminally differentiated cells, where FACT depletion has no effect on 129 proliferation 8 . Subsequently, we sequenced the whole transcriptome (RNA-seq). In total, we 130 characterised 3,003 differentially expressed genes; 1,655 down-regulated and 1,348 up-131 regulated (Fig. 2b). Down-regulated genes were over-represented for pathways involved in 132 development, while up-regulated genes were involved in metabolic processes and positive 133 regulation of proliferation (Fig. 2c), indicating that the change in the transcriptome accounts for 134 the faster proliferation rates. These results suggest that FACT impacts developmental 135 processes and negatively controls cell proliferation in mES cells by controlling gene expression 136 patterns. Importantly, these findings suggest that FACT can act as activator, but also as 137 repressor of gene expression. Given the surprising finding that FACT seems to serve as a 138 repressor on a large set of genes and the fact that it serves as a transcription elongation factor, 139 depletion of FACT might cause transcription to decrease genome-wide. Such a scenario would 140 influence the RNA-seq results. We therefore repeated the experiment using ERCC spike-in 141 controls (Supplementary Figure 3), which confirmed the repressive role of FACT. Next, we 142 wanted to determine if the presence of FACT predicts whether genes are up-or downregulated 143 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint upon knock-down. A low correlation (Pearson's R = 0.11) was observed between the coverage 144 of SSRP1 (ChIP-seq) and the gene fold change (RNA-seq) of those genes in the Ssrp1 KD 145 (Fig. 2d), demonstrating that the presence of FACT is not a predictor for gene expression 146 changes. Taking these findings together, FACT can work directly as an enhancer or repressor 147 of transcription in mES cells. 148 We also observed in the course of the analysis that depletion of FACT leads to differential 149 splicing of genes and identified 356 Exon skipping/inclusion and 97 Intronic retention events 150 following FACT depletion (Supplementary Fig. 4b,c). Interestingly, a fraction of the differential 151 gene isoforms generated in the Ssrp1 KD group is over-represented in limbic system and 152 dendrite development pathways (Supplementary Fig. 4d), suggesting that genes involved in 153 neuronal development might be influenced by FACT.  Fig. 5a). Nucleosome occupancy was plotted for four different gene classes 163 according to the presence of SSRP1 in the control group (ChIP-Seq) and their relative gene 164 Fold Change (RNA-seq) in the Ssrp1 KD state. Overall, we observed little changes in 165 nucleosome occupancy genome-wide (Fig. 3a). Genes that are down-regulated in the Ssrp1 KD 166 ("Down-regulated" class) and bound by FACT exhibit a global mononucleosomal shift by a few 167 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint nucleotides right after the +1 nucleosome. Up-regulated genes showed a loss of nucleosome 168 occupancy in the gene body area regardless of FACT-bound status (Fig. 3a,b), potentially 169 reflecting the higher transcription rate through these genes. However, specifically in up-170 regulated genes bound by FACT ("Up-regulated" class), we observed a significant loss of 171 nucleosomes at the promoter (Fig. 3a,b). This difference in nucleosome occupancy at the 172 promoter region is highly reproducible among the different replicates. As there is a high 173 correlation between FACT occupancy and H3K4me3 (Fig. 1a,b & Supplementary Fig. 4a), we 174 decided to split this gene class by H3K4me3 levels. We identify three gene clusters with diverse 175 H3K4me3 levels that display distinctive nucleosomal patterns (Supplementary Fig. 5b). 176 The observed nucleosome depleted regions (NDRs) were different between up-and down-177 regulated genes. Such architectural differences have been previously attributed to different 178 levels of GC frequency. Indeed, GC frequency over SSRP1 targets was higher and broader in 179 the "Down-regulated" class corroborating a more open chromatin state (Fenouil et al., 2012) 180 ( Fig. 3a & Supplementary Fig. 5c). To confirm this difference in chromatin accessibility, we 181 performed ATAC-seq in Control and Ssrp1-depleted ES cells (Supplementary Fig. 5d-f). In line 182 with the observations of the MNase-seq experiments, we observe an increase in chromatin 183 accessibility in the absence of FACT at the promoter region of up-regulated genes. In 184 combination with the RNA-seq data, this reduction in nucleosome occupancy suggests that 185 FACT might act as a repressor by enabling higher nucleosome density upstream of the TSS. 186 Over the last decade, it has become apparent that promoter proximal pausing of RNA Pol II 187 plays an important role in regulating gene expression (Jonkers, Kwak, & Lis, 2014). In vitro, 188 FACT has been demonstrated to facilitate transcription through chromatinized templates 189 (Orphanides et al., 1999) and reduces pausing of the elongating polymerase when it encounters 190 nucleosomes (Hsieh et al., 2013). To explicate the interplay between the Pol II -FACT 191 complex, RNA Pol II transcription and to integrate this with the observed changes in 192 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint Fig. 6b). Initially, we sought to determine whether nascent transcription positively correlates 194 with mRNA levels. A higher correlation of nascent RNA -mRNA expression and a significantly 195 higher slope (P < 10 -5 ) was observed over the SSRP1-target regions in the Control state 196 suggesting higher levels of Pol II pausing and mRNA levels in the presence of FACT (Fig. 3c). 197 Nevertheless, in the Ssrp1 KD state the SSRP1-bound regions maintained a higher slope, 198 suggesting that pausing and elongation speed of Pol II are not controlled entirely by FACT 199 (Supplementary Fig. 6a). To confirm this, we measured the travelling ratio of Pol II over down-200 regulated and up-regulated genes. Indeed, "up-regulated" FACT-bound genes show a lower 201 travelling ratio, but no significant difference was observed among SSRP1-bound genes 202 following FACT depletion, indicating that FACT is not involved into the release of Pol II towards 203 successive elongation (Fig. 3d). Nevertheless, FACT-bound up-regulated promoters seem to 204 exhibit an intrinsic, different type of transcriptional pausing. One piece of evidence that these 205 genes might be regulated differently at the step of pausing comes from differences in Pol II 206 S2ph (ChIP-seq) density profiles (Supplementary Fig. 6c). In conclusion, up-and down-207 regulated genes upon FACT depletion show different nucleosome occupancy pattern and GC-208 content. FACT depletion leads to specific alterations in nucleosome density, and does not 209 greatly influence RNA Pol II release from promoters. 210 211

Tfap2c cooperates with FACT to enhance transcription in ES cells 212
Next, we sought to understand how FACT can act as activator and repressor. We started off by 213 interrogating whether up-and down-regulated genes might be controlled by different sets of 214 transcription factors. Indeed, motif analysis of the 500bp upstream of the TSS using the 215 iRegulon (Janky et al., 2014) extension for Cytoscape revealed that differentially expressed 216 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. investigate if there is a direct interaction between TBP and FACT in ES cells. We did not 222 observe any expression changes for RARB or TBP neither at an mRNA ( Fig. 2b) nor proteome 223 level (Supplementary Table 8). We then performed immunoprecipitations (IPs) against SSRP1 224 or SUPT16 followed by Mass spectrometry to fully interrogate protein-protein interactions (Fig.  225   4b). However, no direct interaction between FACT and RARB or TBP was observed. 226 Intriguingly, in the promoter of 308 out of 453 FACT-bound down-regulated genes we identified 227 a binding motif for Tfap2 (Fig. 4a, NES=3.903). Tfap2 transcription factors regulate a variety of 228 developmental processes (Kuckenberg et al., 2012). To investigate whether components of the 229 Tfap2 transcription factor family might mediate FACT-dependent downregulation, we examined 230 the RNA-seq dataset. We identified Tfap2c (also known as AP2-) mRNA levels as being 231 significantly downregulated (Fig. 2b). However, in total proteome analysis (Supplementary 232  (Fig.4b). We therefore sought to determine whether binding of Tfap2c might be 236 compromised on active promoters upon FACT depletion. IP for H3K4me3 followed by mass 237 spectrometry, in both Control and SSRP1 KD conditions, revealed that apart from both FACT 238 subunits, also Tfap2c exhibits reduced binding to H3K4me3 (Fig. 4c). ChIP-qPCR over 239 promoters of predicted Tfap2c target genes (Prdm16, Runx1, Wnt10a) confirmed the reduced 240 binding of Tfap2c in the Ssrp1 KD state (Fig. 4d). These data support a co-operative model 241 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. Intriguingly, we observed that only the FACT-bound, high H3K4me3 regions exhibit a broad 252 distribution of the chromatin mark (Fig. 5a); a result consistent with Set1 (responsible for 253 H3K4me3 establishment) distribution over the gene body area. Key pluripotency factors (e.g. 254 Oct4, Sox2, Nanog, and Klf4) and genes involved in embryogenesis belong to this gene cluster. 255 No broad H3K4me3 and Set1 patterns, were identified in the "unchanged" or "down-regulated" 256 gene classes (Supplementary Fig. 7a,c). In addition, we examined the distribution of several 257 nucleosome remodellers (de Dieuleveult et al., 2016) around the promoter of all three gene 258 classes ("Down-regulated", "Unchanged", "Up-regulated") to determine whether they might 259 cooperate with FACT and potentially be a predictor for the change in gene expression upon 260 FACT depletion. No change was observed in occupancy for Chd1, Chd2, or Chd4, but we 261 detected a striking difference in Ep400, Smarcad1, and Chd6/8/9 (Supplementary Fig. 8). This 262 implicates that changes in the presence and occupancy degree of the latter ones might 263 influence the transcriptional activity upon depletion of FACT levels. 264 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint We then focused more on the high and low H3K4me3 gene clusters in order to understand the 265 consequences of Pol II directionality. H3K4me3 serves as a recruitment platform for TFs and 266 RNA Pol II (Sims et al., 2007). NET-seq density plots identified that FACT targets displayed 267 higher levels of promoter-recruited RNA Pol II at the TSS compared to FACT-unbound 268 promoters, but also a strong and wide Pol II distribution similar to the H3K4me3 mark with little 269 signal of divergent Pol II in the anti-sense strand (Fig. 5b). The broad distribution in the sense 270 strand was also confirmed by employing published GRO-seq data (Supplementary Fig. 7e). 271 Non-SSRP1 target regions exhibited an equal distribution both in convergent and divergent Pol 272 II (H3K4me3 High - Fig. 5b). Upon knock-down of FACT, SSRP1 targets display higher levels 273 of convergent transcription, and even more strikingly, an increase (P < 10 -27 and P < 10 -20 ) in 274 divergent transcription compared to the non-SSRP1 targets. This occurred precisely at the 275 locations in which nucleosomes were depleted upon knock-down of FACT (P < 10 -8 , P < 10 -6 ) 276 suggesting that the presence of FACT inhibits antisense transcription by maintaining higher 277 nucleosome density upstream of the TSS. It is of note here as well that genes up-regulated and 278 targeted by FACT exhibit significantly increased levels of nucleosome occupancy (P < 10 -4 ) 279 upstream of the TSS compared to genes not bound by FACT (Fig. 5b). Neural Precursor Cells (NPCs -3 days into the differentiation process) that express key 304 neurogenesis markers (Pax6, Nes, Tubb3) but still maintain FACT and key pluripotency factors 305 at a high level (Fig. 6a). A quarter of the up-regulated genes in ES cells after Ssrp1 KD overlaps 306 with the up-regulated genes instigated by neuronal differentiation (P < 10 -13 , Fisher's exact test; 307 these genes bound by FACT show a significant increase (P < 10 -15 ) in divergent anti-sense 313 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint transcription concomitantly with loss of a nucleosomal impediment (P < 10 -46 ) (Fig. 6c). This 314 mechanism is also evident in lowly expressed/repressed genes that are up-regulated upon 315 FACT depletion (Supplementary Fig. 9). 316 We then depleted Ssrp1 levels at the onset of neuronal differentiation and performed 317 immunofluorescence for neurogenesis (β3-Tubulin) and dendritic (MAP2) markers at the same 318 time point as the RNA-seq experiment. SSRP1 KD caused a substantial increase in the 319 expression of those markers, indicating that loss of FACT function primes ES cells for the 320 neuronal lineage and enhances early neuronal differentiation (Fig. 6d).  Interestingly, a comparison of expression patterns in the early developing mouse brain identified 334 a set of only 13 genes, including Ssrp1 with high correlation of expression in the proliferating 335 cells of the VZ of the neocortex at early stages of development (Vied et al., 2014). This is a 336 transient embryonic layer of tissue containing neural stem cells (Rakic, 2009) and a place for 337 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. deletion of SSRP1 suggesting that it is involved in the regulation of balancing neuroblast self-341 renewal and differentiation 29 . A very recent report also highlights the role of FACT in assisting 342 cell fate maintenance. Using a genetic screen in C. elegans, all FACT subunits were identified 343 as barriers for cellular reprogramming of germ cells into the neuronal lineage (Kolundzic et al., 344 2017). Comparable to our results, the authors did not observe major chromatin architecture 345 alterations, but observed larger colonies during reprogramming assays in the absence of FACT, 346 indicative of higher proliferation rates. In agreement with these reports, our data demonstrate 347 that FACT-depleted ES cells differentiate much more efficiently into early neuronal precursors. that FACT has no effect on the release of paused Pol II and its successful progression towards 361 transcriptional elongation. 362 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. whether this is driven by FACT alone, or in combination with RNA polymerase and/or chromatin 386 remodellers. In order to address these questions, rapid depletion of FACT is required in 387 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint combination with time course experiments addressing loss of nucleosomes vs. upregulation of 388 antisense transcription. However, it is clear that this observed effect is very specific to FACT-389 bound genes, in which the histone chaperone operates as a repressor, suggesting that FACT is 390 required to maintain the observed high level of nucleosome occupancy and to inhibit antisense 391 transcription. One should note however, that this gene class shows low levels of antisense 392 transcription (Fig. 5b). Therefore, one plausible model would be that FACT is required on these 393 promoters to reinstate nucleosomes after initiation of antisense transcription. Depletion of FACT 394 would lead to loss of this function and loss of nucleosomes, which in turn would drive higher 395 levels of antisense transcription. Such a scenario would lead to a wider NFR and allow more 396 efficient recruitment of TFs and RNA polymerase. In addition, the torque generated by two 397 divergently elongating RNAPII molecules can create sufficient negative supercoiling density in 398 the DNA between the two promoters, which is known to increase RNAPII transcription efficiency 399 involved in embryogenesis, particularly in early neuronal differentiation, which is accelerated 404 when FACT is depleted (Fig. 6e). Therefore, the elevated expression of both FACT subunits 405 observed in undifferentiated cells might be required to fine-tune the transcriptional landscape, 406 along with other nucleosome remodellers, in order to maintain this undifferentiated state.         Ssrp1 KD among the 1 st (Library size normalisation) and 2 nd RNA-seq (Library size or spike-in 544 normalisation) runs. c, Same as b, but for down-regulated genes. d, Correlation plot between 545 the average gene expression of the 1 st run vs 2 nd run. Spearman correlation is displayed in red. 546 The red abline represents the slope of the plot. A high overlap in differential expression is 547 observed between library size (both 1 st and 2 nd run) and spike-in normalisation. However, spike-548 in normalisation defines many genes (n=4,254) as being up-regulated in the Ssrp1 KD state 549 which probably arises due to the variance among replicates. We conclude that spike-in 550 normalisation is not the most appropriate method for normalising RNA-seq data. whereas the shading represents either the SE of the mean (MNase-seq) or the 95% confidence 639 interval (NET-seq). Significant changes in Pol II/ nucleosome density were calculated using a 640 Wilcoxon rank test. 641 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint 43 642 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint

675
A combination of two different Ssrp1 shRNAs was used (1&2, 3&4) at a time and depletion was 676 quantified via western blotting using a monoclonal anti-Ssrp1 antibody (Biolegends). Anti-alpha 677 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted January 22, 2018. . https://doi.org/10.1101/251611 doi: bioRxiv preprint experiments as it yielded higher depletion of SSRP1 levels ( Supplementary Figure 2a,b). 679 Fourty-eight hours (48h) after transfection, puromycin (2 µg /ml) selection was applied for an 680 additional 24h period, before cell collection and RNA preparation. Total RNA was obtained via ExFold RNA spike-in mixes) were added during library preparation. An assessment of spike-in 699 normalisation can be viewed in Supplementary Fig. 10. All of the RNA-seq data presented in 700 this manuscript have been normalised to the total library size. Significant genes with an absolute 701 logFC > 1 and Adjusted P.Value < 0.01 were considered as differentially expressed 702 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.