Analysis of ENCODE long RNA-Seq and ChIP-seq (Chromatin Immunoprecipitation Sequencing) datasets for HepG2 and HeLa cell lines uncovered 1647 and 1958 transcripts that interfere with transcription factor binding to human enhancer domains. TFBSs (Transcription Factor Binding Sites) intersected by these ‘Enhancer Occlusion Transcripts’ (EOTrs) displayed significantly lower relative transcription factor (TF) binding affinities compared to TFBSs for the same TF devoid of EOTrs. Expression of most EOTrs was regulated in a cell line specific manner; analysis for the same TFBSs across cell lines, i.e. in the absence or presence of EOTrs, yielded consistently higher relative TF/DNA-binding affinities for TFBSs devoid of EOTrs. Lower activities of EOTr-associated enhancer domains coincided with reduced occupancy levels for histone tail modifications H3K27ac and H3K9ac. Similarly, the analysis of EOTrs with allele-specific expression identified lower activities for alleles associated with EOTrs. ChIA-PET  and 5C (Carbon Copy Chromosome Conformation Capture) uncovered that enhancer domains associated with EOTrs preferentially interacted with poised gene promoters. Analysis of EOTr regions with GRO-seq  data established the correlation of RNA polymerase pausing and occlusion of TF-binding. Our results implied that EOTr expression regulates human enhancer domains via transcriptional interference. Transcriptional interference (TI) encompasses cis-regulatory processes involving two adjacent promoters, where the actual regulation is exerted via the act of transcription itself . Active High throughput RNA deep sequencing identified different types of non-protein coding transcription within eukaryotic enhancers . ArguablRecently, we identified TFbiTrs (Transcription Factor binding interfering Transcripts) within proximal promoter regions (PPRs: 1 kb upstream regions from the transcription start site [TSS]) of human protein-coding RefSeq hg19) genes. These transcripts modulate TF/DNA binding via transcriptional interference within intersected PPRs . Enhance genes. TLocal enrichments of H3K27ac or H3K9ac histone tail modifications indicate augmented activities of eukaryotic enhancer domains . EnhanceInteractome analysis for regions intersected by EOTrs via ChIA-PET  or 5C (Carbon Copy Chromosome Conformation Capture) revealed that enhancer domains associated with EOTrs preferentially interacted with poised gene promoters ,27. Theshttp://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf) R-packages   and DiffBind  . Pre-procis-element annotation system, v 1.0.2) and RefSeq/HAVANA exon annotations, to exclude cDNA contigs, which overlap with known human mRNAs (The identification of transcripts with unknown function (GENCODE v2 GTF-files) was performed with CEAS (an mRNAs  32)..cis-elemRNA datasets for the identification of EOTrs were the result of two initial filtering steps to provide enrichment for npcRNAs (non-protein coding RNAs) intersecting with human enhancer domains:Intronic and intergenic regions corresponding to polyadenylated and non-polyadenylated long RNAs (GSE26284) were overlaid with domains of H3K36me3/H3K4me3 (GSE29611) enrichment, using BEDTools’ intersectBed command ,34. ResuSimilarly, local intersections of these preselected transcript datasets with domains of H3K4me1 enrichments were corroborated with the aid of the BEDTools’ intersectBed command; resulting datasets were analyzed with the GSC to test the significance of H3K4me1 (GSE29611) compared to H3K4me3 (GSE29611) enrichments for the analyzed domains . TranscrGENCODE cDNA datasets representing polyadenylated and non-polyadenylated total RNA were used to quantify EOTr and RefSeq (hg19) mRNA (messenger RNA) expression levels. For all deep sequencing analysis we utilized RNA-seq datasets, which represented RNA prepared from whole cell lysates. RNA polyadenylation and splicing were identified according to GENCODE GTF files . BiologiLong putative ORFs (open reading frames) are absent from non-protein coding sequences ,11. Therhttp://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeRikenCage)  cluster datasets. CAGE clusters were preselected according to HMMs  ,41. ThiskenCage) ,43. JacckenCage) .URLs to all ChIP-seq datasets are provided in q-value ≤ 0.01 and mfold = 10, 100   was utilized for the analysis of genome-wide broad peaks representing histone tail modifications GSE31477) in EOTr and control regions . Replicat 5, 10) . The mfot 5, 10)  (31).477 in EOProximal promoters for EOTr candidates were identified within 1 kb upstream regions relative to the representative CAGE cluster peak via the analysis of H3K4me3 enrichments ,36,45 S. Intersehttps://www.encodeproject.org/chip-seq/transcription_factor/) and CEAS v 1.0.2 .,47.httpsWe utilized edgeR (v 3.24.0) and DiffBind (v 2.10.0) for the analysis of differential TF-binding, histone modifications and RNA expression . edgeR iActive enhancers are defined as genomic domains that display combined enrichment for H3K4me1, H3K27ac and/or H3K9ac along with p300 . They arActive promoters display combined enrichments for H3K4me3, H3K4me2, H3K27ac and RNAPII (RNA polymerase II) ,37. ReveEnrichments of histone tail modification H3K27me3 (GSE29611) were investigated (i.e. as representative heterochromatin mark) for intronic and intergenic EOTr-associated enhancer regions within HepG2 and HeLa cell lines ,50 Supp.Individual ChIP-seq datasets for TF-binding were retrieved from GEO  were utilized to quantify the effects of EOTr expression on TF-binding . STAP prExtraction of binding motifs and calculations of PWMs from ChIP-seq peaks via PscanChIP (v 1.3) and WebLogo were used to generate logos for the resulting TF-binding motifs  53,54).,54.53,54Quantification of relative TF–DNA binding affinities in EOTr and non-EOTr regions via STAP.Extraction of binding motifs computed for identical loci across HepG2 and HeLa cell lines  by PscanChIP(54). Pre-computed cell line specific background files representing HepG2 and HeLa cell lines were utilized for the calculation of binding motifs in EOTr and non-EOTr loci for the probed TFs. Resulting motifs were converted into PWMs and considered for further analysis via STAP tools .STAP output data were displayed graphically with TRAP (Transcription Factor Affinity Prediction) v 3.05 to summarize affinity predictions in EOTr and non-EOTr regions, respectively .STAP command line option ‘-dt’ aided in the identification of EOTrs associated with sites of favorable or unfavorable TF-binding affinities ,18. Sorthttp://hannonlab.cshl.edu/fastx_toolkit/), and resulting GRO-seq cDNAs were aligned to the human genome assembly (hg19) using Bowtie version 0.12.9 (-v 2 -k 3 -m 1 –best)  . BAM fil1 –best)  (Supplem1 –best) .GATK  (v 4.0.1.0) was used for the analysis of allele-specific EOTr expression in HepG2 and HeLa cell lines . ENCODE P < 0.05 test for EOTr+ and EOTr– alleles. Active allelic EOTrs were defined by H3K4me1+H3K27ac over-representation. Analysis of differential binding for H3K27ac within EOTr+/EOTr– allelic variants was performed via DiffBind (v 2.10.0)  .URLs to all datasets are provided in For HeLa, ChIA-PET analysis comprises: (i) linker filtering, (ii) short read mapping, (iii) PET (paired-end tag) classification, (iv) binding site identification and (v) interaction cluster analysis with the ChIA-PET tool v2 . The intIndividual FASTQ replicates representing the 5C interactome for HepG2 cells were aligned to the human genome (hg19) via Bowtie (v 2–2.2.5). SAM output files were converted into sorted BAM with samtools version 1.3.1 (samtools view -bS input.sam | samtools sort -file repX) . BAM fil2-tests are detailed in Results were analyzed via the Genome Structure Correction tool  35). Fu. Fu35). n. IQR is the interquartile range or distance between the first and third quartiles, where n is the number of cells (All boxplots were drawn with DiffBind (v 2.10.0). Notches indicate 95% confidence intervals (CI) for the median, calculated as ±1.58 × IQR/√of cells . The firof cells  within EDeep sequencing datasets for polyadenylated and non-polyadenylated RNAs in HepG2 and HeLa cell lines were assembled with GENCODE v2 GTF files and quantified with edgeR  Figure –C. For eP-value < 0.01). The comparison revealed the specific association of transcripts (as observed within the 200 bp upstream flanks from the TTS) with H3K36me3 domains  vs. non-EOTr regions (EOTr–) underscored our results  and 3.8–4.2 (HeLa) CPM did not cause the occlusion of TF-binding. Interestingly, for the non-EOTr datasets (eRNAs) expression values ranged between log2 0.42–1.70 (HepG2) and 0.53–1.21 CPM (HeLa)  for intronic and intergenic transcripts underscored that the intersection of H3K27ac and EOTr PPRs was specific .,42.15,42RNA deep sequencing captures transcript steady state levels within living cells . ConcentEnrichments of H3K9ac and/or H3K27ac modifications signify active enhancer domains ,37,81. AIn summary, reduced relative TF-binding affinities were accompanied by diminished enhancer activities . As anti2-tests. We analyzed the number of H3K27ac starting peaks for the same enhancer domains within EOTr and non-EOTr regions (i.e. domains downstream from EOTr TTS but contained within the same enhancer). The results underscored the significance (P < 0.01) of lower activities associated with EOTr-intersected domains compared to surrounding regions devoid of interfering transcription  to 70–73% (intergenic: HepG2 and HeLa) of the entire domain were intersected by our transcript datasets. For the same set of EOTr-associated enhancers, H3K27ac and H3K9ac enrichments were consistently lower for transcript-overlaid domains compared to immediate flanking regions (which are devoid of RNA expression). In line with the mechanism of TI, effects related to EOTr expression were locally confined and barely detectable outside the transcribed regions. The significance of this finding was statistically evaluated with the aid of χcription .Histone acetyltransferase p300 deposits H3K27ac marks on histones of active enhancers ,36,81,82These findings agreed with the fact that eRNA transcription did not occlude TF-binding within intersected domains .Chromatin interactomes for EOTr loci were investigated in order to identify potential effects related to TI on the formation of enhancer-promoter (E/P) networks. Enhancer domains associated with allele-specific EOTr expression and the corresponding non-EOTr alleles (i.e. enhancer domains devoid of EOTrs for control) were input for this analysis . This approach bypassed cell line specific influences on enhancer activities that potentially hamper the analysis across cell lines, and revealed more interacting loci for enhancer alleles devoid of EOTrs . As a consequence of TI, the resulting interactome complexities, i.e. the number of corresponding E/P interactions, were compromised . ChIA-PET and 5C analysis allowed the identification of interacting gene promoters for HeLa and HepG2 cell lines. Corresponding PPRs were scanned for histone tail modification H3K4me3 in combination with either H3K27ac or H3K27me3, indicative of active or poised promoter states, respectively . For proFor intronic EOTr-associated enhancers, ChIA-PET/5C (in HeLa and HePG2 cell lines) analysis revealed that subsets of these intronic enhancers interacted with host gene (host genes are genes with introns containing EOTr-regulated enhancer domains) promoters (63% intronic enhancers in HeLa and HepG2). Interestingly, even these host gene promoters displayed preferentially poised characteristics . The resEnhancer domains within multicellular eukaryotes often are transcribed, leading to different classes of potentially regulatory RNAs ,12,14,88Recently, we uncovered TFbiTrs, transcripts that occlude TF/DNA binding in proximal promoter regions (PPRs) and, in turn, inhibit gene expression . Key feaEnhancer activities are positively correlated with H3K27ac enrichments ,24. The Pervasive transcription within genomes generates sites of overlapping or interleaved transcription ,92. The We propose that EOTrs, as enhancer regulating transcripts, are highly flexible molecular tools and as such could serve many functions depending on the regulated enhancer module or occluded TF. Future analysis with more comprehensive datasets will enable to address their functional relevance in broader detail. Also, gene expression in HepG2 and HeLa cell lines is substantially different from non-cancer tissues ,96. The In case of TI, the act of transcription itself is accompanied by the local competition between TFs and RNAPII for DNA binding within overlapping regions. This mechanism could be the underlying cause of lower TF-binding affinities in EOTr regions. To test these assumptions, we inspected the correlation of RNA polymerase pausing and TF-binding within EOTr-intersected domains ,79–80. TDecreased activities for EOTr-associated enhancers, were correlated with reduced enhancer/promoter interactions as revealed by ChIA-PET and 5C. Fewer interacting loops were identified for EOTr-intersected enhancers per regulated promoter than for enhancer domains devoid of the occluding transcription. Therefore, even enhancer/promoter interactions are reduced in number and might be inhibited by interfering EOTr expression . Notablygkaa026_Supplemental_FilesClick here for additional data file.


S. cerevisiae, here we show that the majority of histone acetylation is dependent on transcription. This dependency is partially explained by the requirement of RNA polymerase II (RNAPII) for the interaction of H4 histone acetyltransferases (HATs) with gene bodies. Our data also confirms the targeting of HATs by transcription activators, but interestingly, promoter-bound HATs are unable to acetylate histones in the absence of transcription. Indeed, HAT occupancy alone poorly predicts histone acetylation genome-wide, suggesting that HAT activity is regulated post-recruitment. Consistent with this, we show that histone acetylation increases at nucleosomes predicted to stall RNAPII, supporting the hypothesis that this modification is dependent on nucleosome disruption during transcription. Collectively, these data show that histone acetylation is a consequence of RNAPII promoting both the recruitment and activity of histone acetyltransferases.Histone acetylation is a ubiquitous hallmark of transcription, but whether the link between histone acetylation and transcription is causal or consequential has not been addressed. Using immunoblot and chromatin immunoprecipitation-sequencing in  Histone acetylation is a ubiquitous hallmark of transcription. Here the authors provide evidence that the majority of histone acetylation is dependent on transcription, specifically due to the requirement of RNAPII for the recruitment and activity of histone acetyltransferases. More recently, genome-wide localization studies across eukaryotes, including yeast and mammals, revealed that histone tail acetylation primarily occurs at the promoters and 5′ ends of transcribed genes2. Although some forms of acetylation have been referred to as “global” and “non-targeted”3, genome-wide occupancy studies show that histone acetylation levels correlate strongly with transcription, suggesting a causal relationship between the two.Lysine acetylation of histone amino-terminal tails has been linked to gene expression for many decades4. Most HAT complexes have relatively low substrate specificity and modify multiple lysine residues within either H3 or H4. Thus, histone acetylation sites within H3 and H4 generally show similar distributions2, and mutations of histone lysine residues, with the exception of H4K16, result in comparable changes in gene expression5. Histone acetylation is a dynamic mark due to the activity of histone deacetylase complexes (HDACs). Similar to HATs, HDACs generally exist as multi-protein complexes with catalytic subunits that can deacetylate multiple lysines on one or more histones6.Histone acetylation is catalyzed by conserved histone acetyltransferases (HATs), generally consisting of a catalytic subunit complexed with auxiliary proteins required for enzymatic activity and targetingSaccharomyces cerevisiae, the most well-characterized proteins with lysine acetyltransferase activity are Gcn5 and Esa1, which are the catalytic subunits of multiple HAT complexes, including the H3-specific HATs, SAGA, and ADA for Gcn5, and the H4-specific HATs, NuA4, and Piccolo for Esa14. SAGA and NuA4 are targeted to gene promoters via an interaction between a shared subunit, Tra1, and DNA-bound transcription activators9, which is thought to target acetylation of nucleosomes flanking promoters. This, together with the observation that Gcn5 and Esa1 are required for transcription of multiple genes13, has led to the widely accepted model that histone acetylation acts upstream of transcription initiation. It should be noted however that, in addition to histones, HATs acetylate many non-histone proteins involved in transcription initiation14. As such, whether SAGA and NuA4 activate transcription primarily through acetylation of core histones remains uncertain.In 18 and the phosphorylated carboxy-terminal domain (CTD) of RNAPII19. In addition, co-transcriptional histone exchange mediates incorporation of acetylated histones into nucleosomes within transcribed regions20, and recent work has shown that RNA can promote the activity of CBP at enhancers21. Taken together these observations suggest that histone acetylation can also be a consequence of the transcription process. However, the relative contribution of these pathways to histone acetylation patterns remains unknown.While the current model for targeting HATs by transcription activators upstream of transcription is widely accepted, there are several examples of histone acetylation being deposited as a consequence of transcription. Numerous HATs interact with co-transcriptional H3K4me3In this study, we sought to determine the relative contributions of the “causal” vs. “consequential” pathways for targeting histone acetylation to transcribed genes. We first found that inhibition of transcription results in rapid histone deacetylation in both yeast and mouse embryonic stem cells (mESCs), demonstrating that a significant portion of histone acetylation is a consequence of transcription. Loss of RNAPII also results in depletion of Epl1, a subunit of the yeast NuA4 and Piccolo HATs, from gene bodies, consistent with HAT targeting by RNAPII. Residual, transcription-independent Epl1 binding was observed over promoter regions, in agreement with the targeting of HATs by transcription activators, but surprisingly, acetylation in these regions is still transcription-dependent. Thus, although these results are consistent with previous models for targeting HATs, we found that HAT recruitment alone is insufficient to mediate acetylation of the associated nucleosomes, indicative of post-recruitment regulation of acetyltransferase activity. One model that is consistent with our data is that the acetylation of histones is dependent on nucleosome disruption by RNAPII. In agreement with this, we see increased acetylation at nucleosomes predicted to impede RNAPII passage.S. cerevisiae. Previous studies have used the rpb1-1 temperature-sensitive mutant to disrupt transcription22. However, more recent experiments have suggested that this mutant does not directly inhibit RNAPII, as shifting the mutant to the restrictive temperature has minimal effects on transcript synthesis23 and does not lead to rapid dissociation of RNAPII from gene bodies24. To achieve effective inhibition of RNAPII, we, therefore, treated cells with 1,10 phenanthroline monohydrate , which has been shown to rapidly inhibit transcript synthesis23. Confirming efficient transcription inhibition by 1,10-pt, we observed a global loss of RNAPII serine 5 CTD phosphorylation by immunoblot analysis  is primarily a cause or consequence of transcription has not been definitively tested. We, therefore, sought to assess the dependence of histone acetylation on transcription by inhibiting RNAPII activity in sis Fig.  and rapisis Fig. . Immunobsis Fig. . Similarsis Fig. , or degrsis Fig. . Notablysis Fig.  and coulsis Fig. , confirmsis Fig. . HATs arsis Fig. , and tranes Fig. .Fig. 1ThS. cerevisiae cells with 1,10-pt for 15 m and performed ChIP-seq for H3K23ac, H4K8ac, and H4K12ac. Consistent with previous studies27, we used non-transcribed regions to account for global changes in ChIP-seq experiments (see “Methods”). While no major changes to nucleosome occupancy or position were observed following the short transcription inhibition performed here, large decreases in histone acetylation were observed , disrupts the incorporation of the HAT module into NuA431, and thus cells expressing Epl11–485 are thought to have Piccolo, but not NuA4. We used this information to determine whether bound Epl1 was in the form of NuA4 or Piccolo by repeating ChIP-seq in cells expressing Epl11–485. Analysis of all and just unidirectional promoters , but not untagged ChIP-sequence fragments, showed increased abundance on highly expressed genes  and associated with more highly expressed genes  than all NDRs. Importantly, these peaks overlapped regions with transcription factor binding sites38, and Epl1 occupancy in these regions was reduced following Epl1 truncation ]. Each bin was further split into quintiles based on the predicted ability to strongly or weakly form nucleosomes48. This approach enabled the identification of nucleosomes with similar levels of RNAPII but differing predicted nucleosome occupancies. Data for the middle quintile for RNAPII occupancy are shown in Fig. 2(ChIP/input)], intrinsically stable nucleosomes were enriched for H4K12ac, but not Epl1, both under steady-state and TSA-treated conditions , 20 mM HEPES, 1 mM l-glutamine, 100 U/ml penicillin–streptomycin, 1 mM nonessential amino acids, ~10–50 ng/ml of recombinant LIF, 1 mM sodium pyruvate, and 0.1 mM β-mercaptoethanol on 0.2% type A gelatinized tissue culture plates.FUCCI reporter mESCs63.All strains used in this study were isogenic to S288C and are listed in Supplementary Table Yeast drug treatments were performed in YPD media at the following concentrations: 400 μg/ml 1,10 phenanthroline monohydrate , 10 μg/ml thiolutin , 1 mM 1-naphthalene acetic acid , 20 μg/ml doxycycline , 5 μM 1-Naphthyl PP1 , 25 μM trichostatin A (dissolved in DMSO), 10 μM α factor . mESCs were treated with Actinomycin D at 25 μg/ml .Whole-cell lysates or cellular fractions were analyzed by SDS-PAGE using the antibodies listed in the key resource table  and sonicated  to produce an average fragment size of 250 bp. The lysate was spun down at 90002, 1 mM CaCl2). Samples were incubated with 100 units of MNase for 10 m at 37 °C. Lysates were clarified by centrifugation at 9000g for 10 m. To extract insoluble chromatin, pellets were resuspended in 200 μl of lysis buffer with 0.2% SDS and sonicated in a Diagenode Bioruptor at the medium output for 30 s on and 30 s off for four cycles, before centrifugation at 9000g for 10 m. The second supernatant was pooled with the first, and the buffer composition of the lysate was adjusted to that of the original lysis buffer . The supernatant was precleared by rotating with Protein G Dynabeads for 1 h at 4 °C, 10% of the lysate was reserved for input, and immunoprecipitations were performed using α-HA, α-H3K23ac, α-H4K12ac, or α-H4K8ac antibodies.For MNase ChIP-seq, the pellet was resuspended in MNase digestion buffer , twice with LiCl wash buffer , and once with TE. Synthetic spike-in DNA was added to eluates, to aid in quantification. Following proteinase K digestion, DNA was purified by phenol, chloroform, isoamyl alcohol extraction and RNase A treated.64. Briefly, 2–10 ng of ChIP material was end-repaired and A-tailed before being ligated to TruSeq PE adaptors. The adaptor-ligated material was subject to 8–11 rounds of PCR amplification, and an aliquot of each library was run on an Agilent Tape Station to check the size distribution and molarity of the PCR products. Equimolar amounts of indexed, amplified libraries were pooled, and fragments in the 200–600 bp size range were selected on an agarose gel. An aliquot (1 μl) of the library pool was run on an Agilent Tape Station to confirm proper size selection. In between each reaction, the material was purified using NucleoMag solid-phase reversible immobilization paramagnetic beads.Libraries for paired-end sequencing were constructed using a custom procedure for paired-end sequencinghttp://cutadapt.readthedocs.io/en/stable/), before aligning to the saccer3 genome using BWA (version 0.7.15-r1140)65. For analysis of epl1(1–485) data, reads of chrXII were removed from all data sets, as this chromosome appeared to be unstable in this mutant (1.5× coverage). Coverage tracks represent reads per genome coverage, calculated using the Java Genomics Toolkit  scripts, ngs.BaseAlignCounts and wigmath.Scale. Log2 transformed ChIP over input tracks were calculated using the Java Genomics Toolkit and regions without signal in the input were removed to avoid division by 0. Replicates were pooled for subsequent analysis, and figures were generated in R. FASTQ files from Weiner et al. (2015) were mapped to the saccer3 genome using BWA version 0.7.15-r114065. Reads were extended to 146 bp and read per genome coverage and log2 transformed ChIP over input files were calculated using deepTools version 3.0266. FASTQ files from Steunou et al. (2016) were similarly mapped, but extended to 350 bp.Adapter sequences were removed from paired-end FASTQ files using cutadapt  cells were normalized to silent regions. The genome was divided into 250 bp bins, bins outside the interquartile range for coverage in the input were discarded, the 100 regions with the lowest Rpb3 signal were defined as silent regions, and these silent regions were used to normalized ChIP-seq datasets for cross-condition comparisons . RefSeq mm9 TSSs were downloaded from the UCSC Genome Browser (https://genome.ucsc.edu/). To identify mouse genes with active, non-divergent, promoters, transcribed genes were defined as those with greater than the median PRO-seq signal over 1 kb downstream of the TSS, and transcribed genes in the lowest quintile of PRO-seq signal upstream of the TSS were designated as having unidirectional promoters (3035 genes).Yeast transcription start and end sites were downloaded from the supplemental files of Chereji et al.47 with Rpb3 signal greater than the median were classified as transcribed. Nucleosomes, where Rpb3 changed by less than 10%, were classified as “Rpb3 stable”, while those decreasing by at least 3× were classified as “Rpb3 lost”. Boxplots represent the first to third quartiles, with whiskers extending to 1.5 times the interquartile range or to the extreme of the data. Notches are equal to ±1/58 IQR /sqrt(n), giving an approximation of the 95% confidence interval for the difference in 2 medians.For transcribed nucleosomes classified by Rpb3 change upon 1,10-pt treatment  and the peak position was selected. Peak positions with an IP minus input greater than 0.5 RPGC in the Epl1 ChIP-seq but not in the untagged control were selected as Epl1 peaks. NDRs in close proximity to tRNA genes or centromeres were removed from further analysis due to the binding of Epl1 to these elements.To find promoter peaks of Epl1, the Epl1 MNase ChIP-seq was compared to its input within the NDR for each gene68 to find enriched motifs from the JASPAR nonredundant core fungi motifs69. For the top two hits, Rap1 and Aft2, CentriMo70 was used to plot the distance from the best motif site to the Epl1 peak center and the motif probabilities around the best motif site for the regions containing target motifs.For motif analysis, Epl1 peaks were compared to 1958 NDR regions depleted for Epl1 binding (maximum IP minus input less than 0.1 RPGC). The 500 bp regions around peak centers were then inputted into the MEME-ChIP Differential Enrichment algorithm71 and matrices aligned to other features were produced using the visualization.MatrixAligner script from the Java Genomics Toolkit. Heatmaps were generated using deepTools66, and for 2D heatmaps, plot2DO (version 1) was used72.Metaplot matrices centered on TSSs were generated using the sitepro script from the CEAS package 1.0.2Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Drosophila developmental timing. The model accurately recapitulates published studies of PRE/TRE mediated epigenetic memory of both silencing and activation. With minimal parameter changes, the same model can also recapitulate experimental data for a different PRE/TRE that allows dynamic regulation of its target gene. The model predicts that both cell cycle length and PRE/TRE identity are critical for determining whether the system gives stable memory or dynamic regulation. Our work provides a simple unifying framework for a rich repertoire of PRE/TRE functions, and thus provides insights into  genome-wide Polycomb/Trithorax regulation.Polycomb and Trithorax group proteins maintain stable epigenetic memory of gene expression states for some genes, but many targets show highly dynamic regulation. Here we combine experiment and theory to examine the mechanistic basis of these different modes of regulation. We present a mathematical model comprising a Polycomb/Trithorax response element (PRE/TRE) coupled to a promoter and including  Polycomb (PcG) and Trithorax (TrxG) group regulate several hundred target genes with important roles in development and disease. Here the authors combine experiment and theory to provide evidence that the Polycomb/Trithorax system has the potential for a rich repertoire of regulatory modes beyond simple epigenetic memory. Polycomb/Trithorax response elements (PRE/TREs) are cis-regulatory elements that can maintain epigenetic memory of repressed gene expression states over many cell generations. In transgenic reporter assays using PRE/TREs from Homeobox (Hox) genes, maintenance of repression depends on the Polycomb group (PcG) proteins5. Several Drosophila Hox PRE/TREs have also been shown to maintain the memory of transiently activated gene expression states, in a manner dependent on the Trithorax group (TrxG) proteins6. Several different Drosophila PRE/TREs are interchangeable in these assays, suggesting that epigenetic memory is a general property of PRE/TREs10. The above examples from Drosophila have several features in common: First, the responsiveness of the system to reporter gene expression state decreases as development proceeds6. Second, the initial repressors or activators disappear during development, and expression status is maintained in their absence. Finally, the state maintained by the PRE/TRE is either on or off, and is stable over the whole of development. This has given rise to a paradigm in which PRE/TREs are thought to be switchable elements that maintain stable epigenetic memory of both silent and active states, and do so more stably as development proceeds11.Epigenetic memory is essential to many biological systems, allowing maintenance of gene expression states over multiple cell generations in the absence of the initiating signalsHox genes, many of which do not conform to the above criteria16. These genes include many that switch late in development, or that switch dynamically several times. This raises the question of how these genes overcome the restrictions imposed by a memory system that gains stability as development proceeds. In addition the expression patterns of many of these PcG/TrxG target genes are far more complex than a simple on or off state20. Finally for a large number of these genes, the transcription factors that regulate them do not disappear but are present throughout the time window of their expression or repression. Thus these genes do not appear to be subject to epigenetic memory in the classic sense, raising the intriguing question of how the PcG and TrxG proteins are involved in their regulation (reviewed in ref. 13).However, there are also results that argue against classifying all PRE/TREs as epigenetic memory elements. Genome-wide studies of PcG and TrxG target genes in flies and vertebrates have identified several hundred targets beyond the 4. Understanding the mechanistic basis for these different modes of regulation will be essential for understanding genome-wide PcG/TrxG function in health and disease. A large gap in our understanding of PcG/TrxG regulation has been the lack of a coherent theoretical framework that links developmental timing and transcriptional regulation to PRE/TRE activity. We are working to bridge this gap by using simple mathematical models.In summary, although some PcG/TrxG target genes are subject to epigenetic memory in the strict sense, many are regulated in a far more dynamic manner, suggesting a rich repertoire of PcG/TrxG regulatory modes. How a given gene responds to PcG/TrxG regulation may depend on developmental timing, transcriptional status of the associated gene, and inherent PRE/TRE properties, determined by differences in their nucleic acid sequencesDrosophila development. We combine theory and experiment to quantitatively dissect the contributions of developmental timing, transcriptional input and PRE/TRE identity to the output of the system. We show that this simple model can accurately recapitulate published studies of PRE/TRE mediated epigenetic memory of both silencing and activation in Drosophila, and that cell cycle length is an essential component of memory. Furthermore with minimal parameter changes, the model can also precisely recapitulate our own experimental data for a different PRE/TRE that allows late switching and highly dynamic regulation of its target gene. In summary, we show that a single simple model can account for profoundly different regulatory modes, and we identify parameters that govern those differences. Thus, this work has broad implications for understanding the molecular nature of locus-specific and developmental differences in stability and flexibility of genome-wide PcG/TrxG regulation.Here we present a mathematical model consisting of a PRE/TRE coupled to a promoter. The system is subjected to replication cycles whose length and number reflect those that occur during Drosophila development, we used minimal stochastic models for a promoter and a PRE/TRE, and introduced coupling between them. The model, its implementation and the assumptions used are described in detail in To examine the regulatory interactions between PRE/TREs and their target promoters during p1 and p2, respectively, which can be modified to reflect different promoter strengths , neutral (U) or active (A) configuration. The PRE/TRE state (active or silent) is given by the proportion of nucleosomes that are in the A or M configurations, respectively. Each of the A and M configurations represents all histone modifications and other bound molecules, such as TrxG and PcG proteins or non-coding RNAs that contribute to activation or silencing. Thus the model makes no assumptions about the molecular nature of memory. We use whole nucleosomes as the minimum unit, as in refs. 22  gives the probability of interconversions between A, U and M configurations that are independent of feedback ) that are adjusted at each iteration ). Model 1 is used for all results shown in the main figures, models 1 and 2 are compared in Supplementary Figs. C  tha. C Fig. , which dDrosophila development, we curated published data on cell cycle timing for all developmental stages as described in detail in 26 and that transcription is actively and globally repressed during mitosis28. For the PRE/TRE, cell division was modelled as described previously in refs. 22, by setting each nucleosome to U with a probability of 0.5, at the end of each cell cycle. Thus on average, half of the nucleosomes are set to U at the end of each cell cycle. This is based on the observation that parental histones and their modifications are partitioned randomly to the two daughter chromosomes after replication29. In summary, this simple model comprises several essential features of PRE/TRE mediated gene regulation during Drosophila development, namely a regulatable promoter coupled to a bistable PRE/TRE, and the known timing of cell cycles throughout development.To take account of the changes in cell cycle length during Drosophila reporter assay3 ) is linked to a β-galactosidase (LacZ) reporter gene, with or without a PRE/TRE  or activation (large p1)  Fig. . To ask p1) Fig. .p3 and p4 (for silencing and activation respectively) were kept equal to each other. The coupling strength C, of PRE/TRE to promoter and promoter to PRE/TRE was kept equal in both directions, and this strength was kept constant during the initiation and maintenance phases  (feedback); p5 (feedback-independent transition) and Ci,m (coupling during initiation and maintenance phases). An additional constraint in the parameter search was the requirement for a uniform and spatially precise memory of the initiated pattern until the end of the maintenance phase under the influence of the PRE/TRE , p5, and Ci,m were found, under which the system gave a precise memory of the promoter expression pattern established during the initiation phase, namely repression in the anterior and uniform high expression in the posterior  and p5. However, these parameters also showed upper and lower limits, due to the stringent criteria that must be fulfilled for memory to be effective  affects the stability of the system. If p5 is low, then feedback dominates, and the system has a high capacity for memory but is unable to respond to changes in the promoter state during early initiation (cycle 14). If p5 is high, the opposite is true: the PRE/TRE responds well to the promoter but is unable to maintain memory  was required for optimum memory  state at any given time, Fig. This analysis revealed that longer initial cycles allowed the PRE/TRE to stabilise more often into random A or M states before being coupled to the promoter, causing variegation  reporter gives a red pigment in adult eyes  (feedback) p5 (feedback independent transitions) Ce (early coupling) and Cim (late coupling) as used above  protein and of histone modifications in stage 2–14 nce Fig. . We analNeither histone modifications nor the PC protein showed any detectable signal in interphase nuclei during cycles 1–13 Fig. . All acc37, marked ‘m’ in Fig. 38 also detected H3K27me3 in early mitotic nuclei and in pole cell nuclei, however, immunofluorescence data on accumulation in interphase nuclei were not presented, and time points beyond stage 5 were not analysed in that study.In somatic cells, whilst PC, H3K4me1 and H3K4me3 were detectable in interphase nuclei at stages 5–6, the other modifications (H3K36me and H3K27me3) first became detectable in a subset of interphase nuclei at stage 9 Fig. . The sam38. However, these studies did not address time points later than stage 5  gene  reporter gene under control of eya regulatory sequences by generating the eya1::GFP reporter construct. The reporter contains 9.2 kb of genomic eya sequence, including the eya enhancer, promoter, putative TSS PRE/TRE, exon 1 and the intronic PRE/TRE. A TurboGFP reporter gene is fused in frame to the first three codons of eya exon 2, and a second mw reporter enables analysis in adults . Constructs containing the enhancer were highly expressed and showed no further activation in a ph410 mutant background  PRE/TRE, which shows memory of both silencing and activation in the assays described in Figs. 310. Surprisingly, the bxd PRE/TRE caused strong variegation of GFP expression  were reduced from 0.25 to 0.2, and the feedback-independent transition parameter, p5, was increased approximately four-fold to 0.17. The large increase in p5 and concomitant reduction in  renders the PRE/TRE more flexible, allowing it to respond continuously to the promoter state. Thus with specific parameter changes the simple model is able to quantitatively recapitulate the observed behaviour of the eya PRE/TRE in the developing eye disc.This analysis showed that the model can indeed recapitulate the smooth and sharpened gradient with specific parameters Fig. . Interesbxd PRE/TRE in this assay we found that an optimal fit was obtained with the same coupling parameters as for eya, and for all other parameters , identical values to those determined for the memory assay  for the best fit of the bxd PRE/TRE model to the data (p5 = 0.04) was four-fold lower than for the eya PRE/TRE model (p5 = 0.17). The best- fit values of the feedback parameters  were higher for the bxd PRE/TRE  = 0.25) than for the eya PRE/TRE  = 0.2). Low values of p5 and high values of  tend to make the system more bistable and inflexible, leading to variegation, caused by the perpetuation of random active and silent PRE/TRE states that were adopted before coupling. The gradient imposed by the promoter was nevertheless discernable in the bxd model at intermediate coupling strengths . This suggests that in the living animal, coupling and decoupling between PRE/TREs and promoters or enhancers may be developmentally regulated, locus-specific and biologically important. Several candidate mechanisms for coupling exist, including spreading of marks from a nucleation site49, specific PRE/TRE-promoter communication50, regulation by non-coding RNAs51, long-range chromatin interactions52 and trans-regulation by homologous pairing54  (feedback) and p5 (feedback-independent transitions).We have shown both experimentally and theoretically that the identity of the PRE/TRE itself is decisive in determining the regulatory properties of the system. We show experimentally that when two different PRE/TREs are placed in an otherwise identical regulatory setting, the eya PRE/TRE with high p5 and low  is flexible and able to respond continuously to promoter inputs, but unable to maintain memory. In contrast, the bxd PRE/TRE with a low p5 and high  is more bistable, less flexible and more able to maintain memory of initial states. In the model, if such a strongly bistable PRE/TRE is coupled to a promoter during the initiation phase, it responds to the promoter and later gives stable memory. However, if this same strongly bistable PRE/TRE is coupled to a promoter at later stages, it causes the promoter to variegate. Thus the model predicts that variegation  is a result of the late coupling of a strongly bistable PRE/TRE to a promoter. These observations provide a coherent hypothesis for the mechanistic and developmental relationship between variegation and epigenetic memory.In the model, the eya and bxd PRE/TREs? Interestingly, one of the largest differences in known motifs between the bxd and eya PRE/TREs is the configuration of binding sites for the TrxG protein GAGA factor (GAF) , the eya PRE/TRE contains a run of five consecutive GAF motifs  were collected, by allowing flies to lay for 2 h and ageing collections for the appropriate time period at 25 °C. For each collection, 1 g of embryos was dechorionated in 3% bleach (2.8% hypochloride) in eggwash  and washed extensively with eggwash, then once with PBS, 0.01% Triton. Embryos were transferred to crosslinking solution  and incubated with vigorous horizontal shaking for 15 minutes at room temperature. Embryos were transferred to stop solution , then subsequently washed for 10 min with rotation in wash solution A , and wash solution B . Crosslinked embryos were resuspended in 5.5 ml sonication buffer , and 1 ml aliquots were sheared using a Covaris S220 ultra sonicator with the following settings: 65 Watt, 20% DF, 200 cycles per burst. The time of sonication was optimised to obtain chromatin fragments of ca. 500 bp as follows: 0–2 h or 2–4 h old embryos: 45 min; 4–6 h old embryos: 30 min; 6–8 h old embryos: 25 min; 8–10 h old embryos: 20 min. The DNA content of a 50 μL aliquot of each chromatin preparation was measured by spectrophotometry (Denovix) after reverse crosslink and DNA extraction. The chromatin preparations were diluted to equivalent DNA concentrations. Two independent chromatin preparations and two to three independent ChIP assays were performed for each time point. IPs were performed using 50 μg DNA in 300 μl volume with 4.5 μg of each primary antibody as listed in Supplementary Table ChIP and chromatin preparation on whole embryos was performed as in refs. 61): 7 μL IP or input material was rendered blunt-ended in 25 μL total volume using the Quick Blunting Kit (NEB). Double-stranded linkers were prepared by annealing 20 mer and 24 mer oligos  and the 20 mer oligo as primer at 150 nM final concentration, with the following program parameters: 2 min 50 °C, 2 min 94 °C, 40 × .As a control for linker ligation, a gel-purified 448 bp blunt-ended PvuII fragment of pBluescriptIIKS+ was ligated to linkers under the same conditions as the ChIP ligations, and a dilution series of known quantities was subjected to qPCR, showing that linker ligation is efficient and amplification is linear with fragment concentration. Primer efficiency was calculated on the basis of plasmid and input dilution series to be 2.0 ± 0.3 (meaning that the product is amplified 2.0 ± 0.3 times per cycle). To further control for uniform amplification by qPCR we compared input chromatin samples before and after blunting, ligation and qPCR by agarose gel electrophoresis. This showed a similar size distribution of fragments around the mean of 500 bp, thus the linker-mediated qPCR does not appear to introduce bias for a particular fragment size.ChIP and input samples from two independent chromatin preparations were subjected to qPCR in the same plate, each in a 5-fold dilution series . We compared technical replicates by evaluating the correlation between data series from ChIP and inputs from independent chromatin preparations from each time point. We observed a high correlation between independent ChIP assays performed with the same antibody from the same time point . . This gives enrichments as a proportion of input, where an enrichment of 1 would be obtained if the quantity of IP material were equivalent to 1% of input (because the input samples were diluted 100-fold prior to qPCR). This dilution step was necessary to give input Ct values in a similar range to those of the ChIP samples). The ChIP vs. input calculation gave a measure of the reproducibility of ChIP assays )/(2(Ct input – Ct H3 ChIP)). However, this expression simplifies to 2(Ct ChIP H3 – Ct ChIPx), and thus the input is not required in this case. Indeed we found that including the input in the former calculation led to amplification of errors. Thus for the data shown in Fig. 35 the absolute numbers of modifications are calculated as a percentage of total H3 molecules, which is a different calculation than the one we perform here. To avoid confusion, we use the term “enrichment relative to Histone H3 ChIP” on the axis of Fig. (Ct ChIP H3 – Ct ChIP x).We calculated enrichments for each modification and for unmodified histone H3 relative to (input/100) for each dilution using the following formula: enrichment relative to input = 2eya1::GFP reporter construct was generated using primers and templates as shown in Supplementary Table eya locus  contains the eye specific enhancer, the first exon and the eya PRE/TRE , carrying NheI, AvrII, KpnI and SacI sites in place of the PRE/TRE sequence. The bxd PRE/TRE  with mutations as shown in Supplementary Fig. The see Fig.  were eacsee Fig. . Fragmeneya1::GFP or variants were generated by ΦC31 mediated integration by the IMBA fly injection service. The landing site used here corresponds to site 2 at position 46E1 in ref. 41 (chr. 2R) at genomic location 5,965,083 . All fly stocks are available on request. To test the effects of the ph410 mutation on miniwhite expression, flies of the following genotypes were crossed for each transgene: females: w-, ph410/ w-, ph410;Transgene/CyO x males: w-; Transgene/CyO. Female progeny lacking the CyO balancer were heterozygous for the ph410 mutation and homozygous for the transgene. These flies were aged for four days before taking photographs. The ph410 allele  is a hypomorphic allele of ph. It disrupts the proximal ph transcription unit but leaves the closely related distal transcription unit intact.Transgenic fly lines carrying 3rd instar larval eye discs were stained in 5 µM SYTO Red Fluorescent Nucleic Acid Stain (Invitrogen) and imaged on a confocal microscope LSM 700/Axioimager (Zeiss).eya exon 1 and GFP) using the primers and templates shown in Supplementary Table TM, Invitrogen) according to manufacturers instructions. Tissues were mounted in Vectashield with DAPI (Vector Laboratories) on microscope slides. Images were taken using confocal microscopy, with LSM 700 Axioimager (Zeiss).PCR products were generated for two in situ probes . The line scans were placed in anterior-posterior orientation at 90° to the morphogenetic furrow as shown in Fig. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary SoftwareReporting Summary


Plasmodium falciparum has an unusually euchromatic genome with poorly conserved positioning of nucleosomes in intergenic sequences and poorly understood mechanisms of gene regulation. Variant histones and histone modifications determine nucleosome stability and recruit trans factors, but their combinatorial contribution to gene regulation is unclear.The malaria parasite Here, we show that the histone H3 acetylations H3K18ac and H3K27ac and the variant histone Pf H2A.Z are enriched together at regulatory sites upstream of genes. H3K18ac and H3K27ac together dynamically mark regulatory regions of genes expressed during the asexual life cycle. In contrast, H3K4me1 is depleted in intergenic sequence and dynamically depleted upstream of expressed genes. The temporal pattern of H3K27ac and H3K18ac enrichment indicates that they accumulate during S phase and mitosis and are retained at regulatory sequences until at least G1 phase and after cessation of expression of the cognate genes. We integrated our ChIPseq data with existing datasets to show that in schizont stages H3K18ac, H3K27ac and Pf H2A.Z colocalise with the transcription factor PfAP2-I and the bromodomain protein PfBDP1 and are enriched at stably positioned nucleosomes within regions of exposed DNA at active transcriptional start sites. Using transient transfections we showed that sequences enriched with colocalised H3K18ac, H3K27ac and Pf H2A.Z possess promoter activity in schizont stages, but no enhancer-like activity.P. falciparum regulatory sequences and contribute to gene activation. These findings expand the knowledge of the chromatin landscape that regulates gene expression in P. falciparum.The dynamic H3 acetylations define  Plasmodium falciparum is the cause of most global malaria mortality. P. falciparum differs from other eukaryotes in the unusually high AT content of its genome (90% in intergenic regions) [P. falciparum nucleosomes confirmed the unique nature of P. falciparum chromatin [P. falciparum genes are specifically regulated during its haploid, 48-h intra-erythrocytic developmental cycle (IDC) [Plasmodium genes are specifically activated by stage-specific transcription factors [P. falciparum bromodomain protein 1 (PfBDP1) to activate a subset of erythrocyte invasion genes [P. falciparum genes.The parasite regions) , its uniregions)  and the regions) . Structuhromatin , which mle (IDC) , 6 and g factors . In at lIncreased nucleosome occupancy can directly hinder transcription and is dependent on nucleosome stability and thus nucleosome composition. Histone modifications can also recruit factors that facilitate or repress transcription. Associations between combinations of variant histones and histone modifications with depletion of nucleosomes at regulatory elements have been shown in yeast and for the human Encyclopedia of DNA Elements (ENCODE) Project. Yeast and human active promoters are enriched in H3K9ac, H3K18ac, H3K27ac, H3K4me3 and the histone variant H2A.Z –20, wherMetazoan enhancers can be many kb distant from the cognate gene, but yeast have upstream activating sequences (UAS) that are typically located within 450 bp of the transcriptional start site (TSS) . AnalogoP. falciparum intra-erythrocytic cycle gene expression correlates with intergenic, upstream, dynamic enrichment of several euchromatic histone modifications [Throughout the ications , 36–39. ications , 41.P. falciparum intergenic regions. P. falciparum intergenic sequence is maintained in a nucleosomal array aside from a short, nucleosome depleted region (NDR) of variable length that precedes the well positioned + 1 nucleosome at TSSs [P. falciparum is too short to resolve enhancer and promoter interactions within the same intergenic region using Hi-C approaches [P. falciparum [P. falciparum enhancer-like elements identified by relative H3K4me1 enrichment [P. falciparum. Indeed H3K27ac is enriched in intergenic sequence of euchromatic genes in P. falciparum oocysts and sporozoites, but is not clearly associated with gene expression in these mosquito stages [Multiple regions of increased chromatin accessibility , 41 and  at TSSs . Whetherproaches  and morelciparum –47. Putarichment  did not richment . H3K18aco stages .P. falciparum gene expression we investigated the genome-wide correlation of promoter and enhancer associated histone modifications with cognate gene expression profiles as well as with published DNA accessibility and transcription factor binding data. We demonstrate that dynamic enrichment of H3K18ac and H3K27ac at Pf H2A.Z containing nucleosomes at TSSs correlates with gene expression levels, whereas H3K4me1 is generally absent from intergenic sequences and thus depleted from these putative regulatory elements. Acetylations of H3K18 and H3K27 accumulate during mitosis and appear to mark regulatory sequences of genes for expression rather than as a consequence of expression. From transient transfection analysis, we concluded that histone modifications that identify active enhancers and promoters in metazoan and yeast cells do identify P. falciparum sequences with schizont stage promoter activity but not enhancer-like elements.To get better insight into the complex regulatory network underlying P. falciparum genome and how this is associated with regulatory sequences and gene expression, we analysed genomic enrichment of the variant histone H2A.Z and the histone modifications H3K4me1, H3K18ac and H3K27ac. These markers of chromatin structure are all enriched at metazoan enhancers and, with the exception of H3K4me1, at yeast and metazoan promoters [To investigate chromatin structure of the romoters –24.https://compbio.med.harvard.edu/antibodies/)) [https://www.histoneantibodies.com)) [P. falciparum histones by showing that each antibody bound only the single H3 band in western blots of P. falciparum histones ) , (Histones.com)) . We confFluorescence microscopy confirmed the predicted nuclear localisation of all three H3 modifications Fig. a. The diThe abundance of Pf H2A.Z and the H3 modifications throughout the intra-erythrocytic lifecycle was assessed by western blot using histone H3 as a loading control. The levels of H3K4me1 and H2A.Z were constant throughout the lifecycle. The levels of both H3K18ac and H3K27ac were low in ring- to mid-trophozoite-stages and peaked at late-trophozoite- to early-schizont-stages (32–40 hpi) prior to dropping at mid- to late-schizont stage  of the libraries were all acceptable , but depleted within transcripts determined from the cognate RNAseq . Interestingly, GC content was very high for intergenic sequence.Although on average intergenic H3K4me1 was depleted, 76 intergenic peaks of H3K4me1 were detected in schizont stages. Bidirectional transcription originated from the H3K4me1 peaks and the peaks also colocalised with relative depletion of H3K4me3 in a published schizont dataset  p = 0.0009) than all of the other protein coding genes in the P. falciparum genome in schizonts. Overall, the H3K4me1/input and H3K4me1/H3K4me3 data suggested that intergenic H3K4me1 enrichment was not marking transcription regulating sequences. Indeed, the AT content and chromatin characteristics of these peaks were more similar to coding than intergenic sequence.To further investigate whether the H3K4me1/H3K4me3 ratio was informative of putative enhancer function, we identified peaks of H3K4me1 calculated relative to published H3K4me3  in nuclP. falciparum was consistent with their collective marking of regulatory sequences, as they do, together with H2AZ, in yeast and metazoans. To investigate this possibility, Pf H2A.Z peaks that intersected H3K18ac and H3K27ac peaks were filtered to retain only the 2196 ring stage and 2645 schizont stage, intergenic, colocalised peaks (ICPs). Existing datasets of nucleosomal occupancy  [The dynamic, enrichment of H3K27ac and H3K18ac upstream of expressed genes in romatin) , DNA accromatin) , 41 and romatin)  were exaThe inferred nucleosomes at the Pf H2A.Z summits of the 2645 ICPs in schizont stages Fig. a. Thus, p < 0.0001)  Fig. b.P. falciparum TSS has been previously noted [The association between the TSSs and the ICPs suggested an association with regulatory sequences. ATACseq reveals exposed DNA presumed to be available to bind transcription factors. The 2645 schizont-stage ICPs overlaid ATACseq peaks  and regily noted .p < 0.00005 Welch’s t test with Bonferroni correction)  whilst ring-stage ATACseq peaks were not closer to schizont ICPs compared to schizont intergenic non-ICPs. These differences are consistent with a stage-dependent proximity of the ICPs to the stage-matched ATACseq peaks.In schizont stages the Pf H2A.Z summits of the ICPs were on average 1.3-fold closer 4400 bp) to schizont-stage ATACseq peaks [0 bp to son) Fig. c. The trn = 151) , its binding sites (ATACseq) and co-activator (PfBDP1), the well characterised PfAP2-I/PfBDP1-regulated invasion genes were analysed , 41. Pf n = 1371) and then filtered for the presence of an adjacent downstream gene within 2500 bp that was in the top quartile by expression in schizonts and upregulated at least threefold in schizonts compared to ring stages.To investigate whether ICPs marked promoter or enhancer-like regulatory sequences, the ICPs were filtered by a set of stringent criteria to generate a set of sequences for further analysis . In both stages, the putative promoters, but not enhancers, intersected with more TSS tags [p < 0.0001, Dunn’s multiple comparisons test all p < 0.0003). The putative promoters also intersected more sense strand TSS tags than antisense , but the putative enhancers did not  of matched DNA samples. We used qPCR for normalisation because firefly luciferase activity was too low for accurate detection. Luminescence was determined for parasites expressing nanoluciferase driven by (1) each of three schizont- and three ring-stage putative promoters, selected randomly from the 54 putative promoter sequences; (2) the hsp86 promoter as a positive control; (3) an AT content-matched P. falciparum intergenic sequence that lacked any chromatin modifications indicative of regulatory activity and that was located upstream of a non-expressed gene to control for non-sequence specific effects and (4) a no DNA control  and the trend to greater activity approached significance for the third schizont-stage putative promoter (PF3D7_1460600 p = 0.0908), but none of the three ring-stage putative promoters had greater activity than the AT content-matched control sequence  in 1 × NuPAGE® MES SDS running buffer  at 150 V for 70 min. Proteins were transferred to an Amersham HYBOND-ECL membrane  in cold transfer buffer  at 400 mA for 1 h and successful transfer was verified by Ponceau S  staining. The membranes were incubated with primary and secondary antibodies and bound antibodies were detected using Immobilon Western Chemiluminescent HRP Substrate .Parasites at 5–10% parasitemia were harvested at 8-h intervals, erythrocytes were lysed in 0.075% saponin and parasite pellets then dissolved in 2 × reducing Laemmli buffer  at a concentration of 5 × 10P. falciparum peptides PfH3 unmodified 1 (residues 1–21), PfH3 unmodified 2 (residues 21–43), PfH3 unmodified 3 (residues 47–65), PfH3K4me1 (residues 1-21), PfH3K9ac, PfH3K14ac, PfH3K18ac, PfH3K27ac, PfH3K23,27ac and PfH3K56ac and biotinylated human peptides HuH4 (residues 1–21) , HuH4K20me1 , HuH4K20me3  and nonbiotinylated human peptides HuH3K4me3 , HuH3K36me3  and HuH3K9me3  were used for testing antibody specificity. All P. falciparum peptides and nonbiotinylated human peptides were used at 0.25 mg/ml except for HuH4K20me1 and HuH4K20me3 which were used at 0.07 mg/ml while HuH4 was 0.065 mg/ml. All peptides were then tested at further dilutions of 1:4, 1:8, 1:16 and 1:32. One microlitre of each dilution was loaded on an Amersham HybondTM HYBOND-ECL membrane . One membrane was blocked with 5% w/v skim milk powder in TBS-T, washed, incubated with streptavidin–HRP diluted 1:50,000 in TBS-T, washed, and the membrane bound streptavidin–HRP was detected using Immobilon Western Chemiluminescent HRP Substrate . The other membranes were blocked with 5% w/v skim milk powder in in TBS-T, each was incubated with a primary antibody, washed, incubated with secondary antibody conjugated to HRP, washed again then bound antibodies were detected using Immobilon Western Chemiluminescent HRP Substrate .Custom-made, biotinylated Smears of asynchronous parasite cultures were prepared on glass slides and air-dried overnight. Cells were fixed and permeabilised by a mixture of ice cold 10% methanol/90% acetone for 5 min at room temperature and air-dried. Cells were rehydrated in PBS, stained with primary antibodies, washed with PBS, stained with secondary antibody and DAPI , washed, mounted in ProLong® Gold Antifade Reagent  and left to cure overnight. Imaging was performed using a Zeiss Fluorescence Upright Microscope (ZEISS Axioskop 2 mot plus) with the 100 × oil objective.g for 10 min at 4 °C and nuclei from 2 × 109 ring-stage parasites, 1 × 109 trophozoite-stage parasites or 4 × 108 schizont-stage parasites were resuspended in 200 μl SDS lysis buffer . Chromatin was sheared into 200–600 bp fragments by sonication in a Bioruptor UCD-200 (Diagenode) for 28 min  and diluted 1:10 in ChIP dilution buffer .Chromatin was isolated from ring-stage (8–14 h post-invasion), trophozoite-stage (30–36 hpi) and schizont-stage (38–44 hpi) parasites as previously described . Briefly8 ring-stage or 2.5 × 108 trophozoite-stage or 1 × 108 schizont-stage parasites were immunoprecipitated overnight at 4 °C with 8 μg antibody and 30 μl prewashed protein A/G beads. 1/10th of the volume of immunoprecipitated chromatin was also incubated overnight at 4 °C for use as the input control. Beads were washed once with low-salt immune complex wash buffer , once with high-salt immune complex wash buffer , once with LiCl immune complex wash buffer  and twice with TE buffer  and co-immune-precipitated DNA was then eluted twice with freshly made elution buffer . Reversal of cross-linking was performed by incubating the eluate at 45 °C overnight with 500 mM NaCl. DNA was purified using the MinElute PCR purification kit . All ChIP experiments were performed in three biological replicates.For each immunoprecipitation, diluted chromatin was pre-cleared using protein A/G beads  at a beads-to-chromatin volume ratio of 1:200 for 1 h at 4 °C. Approximately 470 μl pre-cleared chromatin from 5 × 10ChIPed DNA libraries were prepared using the NEBNext® ultra DNA library prep kit for Illumina® . 6 ng input or chromatin-immunoprecipitated DNA was used and adaptor-ligated DNA was purified without size selection using Agencourt AMPure XP beads . The PCR amplification step was performed using 1 unit of high-fidelity KAPA HiFi DNA polymerase , 0.3 mM dNTPs, 0.5 μM NEBNext Universal PCR Primers for Illumina and 0.5 μM NEBNext Index Primer for Illumina (NEBNext® Multiplex Oligos for Illumina® Index Primers Set 1 (E7335) and Set 2 (E7500)) in 1 × KAPA HiFi buffer containing tetramethylammonium chloride for 12 PCR cycles [P. falciparum hsp70 gene. Total RNA (2.5 μg) was depleted of haemoglobin mRNA using the GLOBINclear™ Kit  and mRNA then purified using magnetic oligo d(T) beads (NEBNext® Poly(A) mRNA Magnetic Isolation Module, New England BioLabs, E7490S). Indexed mRNA libraries were prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® . PCR enrichment was performed as per the ChIPseq method described above. The PCR conditions were 98 °C for 1 min for the initial denaturation followed by 12 cycles of 98 °C for 10 s and 65 °C for 1 min and a final extension at 65 °C for 5 min. Library clean-up was performed using 0.8 × AMPure beads and RNA libraries were eluted with nuclease-free water.Stage-matched RNA from at least two biological replicates was extracted, gDNA digested and RNASeq libraries constructed as described previously . BrieflyInput and chromatin-immunoprecipitated DNA libraries and mRNA libraries were subjected to quantification and quality check for purity and fragment size range using the Agilent Bioanalyzer and Qubit Fluorometer analyses prior to sequencing by the HiSeq2500 (Illumina) at the Melbourne Translational Genomics Platform. 126-bp paired-end reads were generated.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adaptor sequences were trimmed and reads were quality filtered using Trim Galore! . ChIP-seq and RNA-seq reads from biological replicates from each stage mapped to P. falciparum 3D7 genome were visualised using IGV [Bioinformatic analysis was performed on Ubuntu 14.04.5 LTS (GNU/Linux 3.13.0–105-generic x86_64). Quality control of all raw sequencing data was performed using FastQC  using Subread [q = 0.01) [Input DNA and chromatin-immunoprecipitated DNA library sequencing reads were mapped to the  Subread . The res Subread . H2A.Z,  = 0.01) . NGSplot = 0.01)  was usedSchizont and ring-specific candidate enhancers and promoters and their cognate genes were identified using BEDTools . BrieflyP. falciparum MNase-seq read coverage normalised per site in BEDGRAPH format at time points 15 h post-invasion and 40 h post-invasion was retrieved from the Gene Expression Omnibus GSE66185 [P. falciparum 3D7 v12 were retrieved from PlasmoDB (https://plasmodb.org). Coordinates of total 5567 intergenic regions were obtained using “bedtools complement”. The average score of BEDGRAPH records that intersected with P. falciparum intergenic regions and colocalised H2A.Z, H3K18ac and H3K27ac peaks was determined using “bedtools map”. Mann–Whitney U test was performed using GraphPad Prism version 6.GSE66185 . CoordinP. falciparum TSS tag coverage at 10 and 42 h post-invasion were retrieved from the Gene Expression Omnibus GSE68982 [Stranded P. falciparum-3D7-t40 replicate 1 GSM2789028 was mapped to the P. falciparum 3D7 strain genome release 12 version 3 (PlasmoDB) using BWA-MEM [ATACseq data from  BWA-MEM  as per [ BWA-MEM  and peak BWA-MEM  in bioco BWA-MEM . The res BWA-MEM .www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and reads then mapped to the P. falciparum 3D7 strain genome release 12 version 3 (PlasmoDB) by BWA-MEM v 0.7.17 [The schizont-stage MNase-seq T40A sample from SRX885818: GSM1616491: comprised 5 paired-end fastq libraries SRR1813391 to SRR1813395 . Adapterv 0.7.17  and filtv 0.7.17  for aligv 0.7.17 . The bamv 0.7.17 .www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and then reads were mapped to the P. falciparum 3D7 strain genome release 12 version 3 (https://plasmodb.org) by BWA-MEM v 0.7.17 [The schizont-stage AP2I ChIPseq and input data  from GEOv 0.7.17  and filtv 0.7.17  for aligv 0.7.17 . The bamv 0.7.17 . The dupv 0.7.17  to generv 0.7.17  called pIn order to compare ICP peaks with chromatin accessibility across asexual stages, ATACseq data were acquired and mean peak positions derived from the start and end of the reported peaks . The disNcoI and XbaI restriction sites to create the plasmid pNluc. An AT content-matched P. falciparum intergenic sequence that lacked any chromatin modifications indicative of regulatory activity and that was located upstream of a non-expressed gene was used to control for non-sequence specific effects , and synthesising the sequence with GeneArt (Life Technologies), and also cloned into the MluI and XhoI restriction sites of the promoter-containing pNluc constructs. The promoter-only constructs were made by digesting promoter-containing constructs using MluI and XhoI restriction enzymes, removing overhangs using Klenow fragment and ligating to form the final plasmid.The putative promoter and enhancer sequences were amplified from TM 4D-Nucleofector™ System (Lonza), using program FP158. The transfected parasites were immediately cultured in medium containing fresh erythrocytes and harvested 6–8 days after transfection. The parasites transfected with putative ring-stage regulatory elements were harvested at ring stage and the parasites transfected with putative schizont-stage regulatory elements were harvested at schizont stage.5 µg of plasmids containing test sequences driving Nluc, along with 15 µg of pPf86  were ethParasites were treated with 0.1% (w/v) saponin in PBS and washed in PBS. Freed parasites were added to white 96-well plates and treated with NanoDLR™ Stop & Glo reagent, to measure nanoluciferase activity. In addition, the levels of firefly luciferase DNA and the number of parasite genomes were measured by quantitative PCR using SYBR™ Green PCR Master Mix (ThermoFisher). The level of nanoluciferase activity was calculated relative to the amount of firefly luciferase DNA per parasite genome to determine the expression of nanoluciferase per plasmid per parasite for all transfections.Additional file 1: Fig S1. Specificity of ChIPseq antibodies for P. falciparum histones. Western blot of P. falciparum whole-cell lysate probed with the antibodies used in this study and with antibody to H4K12ac as an additional control showing the presence of multiple histones on the western blot.Additional file 2: Fig S2. Specificity of ChIPseq antibodies for P. falciparum histone modifications. Dot blot of biotinylated histone peptides probed with rabbit antibody to a) H3K18ac , b) rabbit antibody to H3K27ac , c and f) rabbit antibody to H3K4me1 and d and e) streptavidin as a loading control. Peptides indicated on the left were diluted from right to left as shown above the figure. Starting concentrations (neat) were 0.25 mg/ml for all P. falciparum (Pf) peptides and Human (Hu) H3K4me3, H3K36me3 and H3K9me3 peptides; 0.07 mg/ml for Human H4K20me1 and H4K20me3; 0.065 mg/ml for Human H4. Human H3K4me3, H3K36me3 and H3K9me3 were not biotinylated and their loading was confirmed by ponceau red staining shown for neat peptides in lanes to the left. P. falciparum H3 unmodified 1, 2 and 3 peptides represented residues 1–21, 21–43 and 47–65, respectively. A minor cross reaction of anti-H3K18ac antibody with a P. falciparum histone H3 unmodified peptide (residues 47–65) was observed, but the antibody was at least 32-fold more sensitive for H3K18ac than H3 (residues 47–65). The rabbit anti-H3K27ac antibody strongly bound both the P. falciparum H3K27ac peptide and the H3K23acK27ac peptide. The anti-H3K27ac antibody also bound the P. falciparum H3K9ac peptide but by densitometry it generated no more than 1/32 of the signal of H3K9ac.Additional file 3: Table S1. Quality summary of ChIPseq data.Additional file 4: Table S2. Quality summary of RNAseq data.Additional file 5: Fig S3. Reproducibility of ChIPseq. PCA plots and associated scree plots for all ChIPseq and input replicates for ring stages, trophozoite stages and schizont stages.Additional file 6: Fig S4. Individual sequencing tracks. Read coverage for ChIP, input and RNA sequencing replicates across 78 kb within chromosome 14.Additional file 7: Fig S5. Correlations between expression level and histones and their modifications across transcriptional units. Log2 (ChIP/input) plotted from 2500 bp upstream (−2500) to 2500 bp downstream (2500) of the transcriptional start (TSS) and stop (TES) sites for 7612 ring stage transcripts and 7711 trophozoite stage transcripts assembled by Cufflinks ranked in descending order by transcript abundance (fpkm). The Spearman r correlation value (r) for the upstream and downstream enrichment of each histone or histone modification is indicated below the plots.Additional file 8: Fig S6. Average enrichment profiles of histones and their modifications across genes binned by expression level. The average profile of H2A.Z, H3K18ac, H3K27ac, H3K4me1 and H3 over the coding sequences ± 2500 bp for genes grouped into terciles by expression level  and for silent genes (pink). The enrichment profile of each chromatin mark was presented as the log2 ratio over the input control. In rings, trophozoites and schizonts gene expression categories were, respectively, the top , middle  and bottom  terciles, and silent genes . 5′End: the start codon of a gene; 3′End: the stop codon of a gene.Additional file 9: Fig S7. A) Average enrichment profiles of chromatin features and RNA levels from this study across the summits ± 1.8 kb of intergenic, peaks of H3K4me1 ChIP from this study relative to H3K4me3 from [n = 259) [me3 from  B) Averame3 from . Averagen = 259)  orientedn = 259) , and then = 259) . D) Spean = 259)  and for Additional file 10: Table S3. Number of highly expressed genes differentially expressed between stages.Additional file 11: Comparison with nascent RNA.Additional file 12: Table S4. Comparison between lifecycle stages of ChIP enrichment upstream of highly expressed genes differentially expressed between stages.Additional file 13: Fig S8. Comparison of enrichment profiles of histones and their modifications across genes dynamically expressed in rings and trophozoites. Average log2 ratio of ChIP enrichment of Pf H2A.Z (green), H3K18ac (orange), H3K27ac (purple) and H3K4me1 (pink) all relative to ChIP enrichment of H3 plotted over the coding sequences ± 2500 bp of: gene set 3 (n = 299) that were expressed at least threefold more in trophozoite stage than in ring stage and that were in the top quartile by trophozoite-stage expression and: gene set 4 (n = 455) that were expressed at least threefold more in ring stage than in trophozoite stage and that were in the top quartile by ring-stage expression. 5′End: the start codon of a gene; 3′End: the stop codon of a gene.Additional file 14: Fig S9. Comparison of enrichment profiles of histones and their modifications across genes dynamically expressed in trophozoites and schizonts. Average log2 ratio of ChIP enrichment of Pf H2A.Z (green), H3K18ac (orange), H3K27ac (purple) and H3K4me1 (pink) all relative to ChIP enrichment of H3 plotted over the coding sequences ± 2500 bp of: gene set 5 (n = 433) that were expressed at least threefold more in schizont stage than in trophozoite stage and that were in the top quartile by schizont-stage expression and: gene set 6 (n = 173) that were expressed at least threefold more in trophozoite stage than in schizont stage and that were in the top quartile by trophozoite-stage expression. 5′End: the start codon of a gene; 3′End: the stop codon of a gene.Additional file 15: Fig S10. Enrichment of histones and their modifications across ATACseq peak summits. Log2 ChIP H3K4me1 (this study)/H3K4me3 [/H3K4me3 , Log2 Ch/H3K4me3  plotted Additional file 16: Fig S11. Flow chart for the bioinformatic strategy used to identify candidate proximal and distal regulatory sequences upstream of schizont stage-expressed genes.Additional file 17: Table S5. Coordinates of tandem ICPs tested for regulatory activity.


Pharmacological inhibition of ribosome biogenesis is a promising avenue for cancer therapy. Herein, we report a novel activity of the FDA-approved antimalarial drug amodiaquine which inhibits rRNA transcription, a rate-limiting step for ribosome biogenesis, in a dose-dependent manner. Amodiaquine triggers degradation of the catalytic subunit of RNA polymerase I (Pol I), with ensuing RPL5/RPL11-dependent stabilization of p53. Pol I shutdown occurs in the absence of DNA damage and without the subsequent ATM-dependent inhibition of rRNA transcription. RNAseq analysis revealed mechanistic similarities of amodiaquine with BMH-21, the first-in-class Pol I inhibitor, and with chloroquine, the antimalarial analog of amodiaquine, with well-established autophagy-inhibitory activity. Interestingly, autophagy inhibition caused by amodiaquine is not involved in the inhibition of rRNA transcription, suggesting two independent anticancer mechanisms. In vitro, amodiaquine is more efficient than chloroquine in restraining the proliferation of human cell lines derived from colorectal carcinomas, a cancer type with predicted susceptibility to ribosome biogenesis stress. Taken together, our data reveal an unsuspected activity of a drug approved and used in the clinics for over 30 years, and provide rationale for repurposing amodiaquine in cancer therapy. Ribosome biogenesis has emerged in recent years as a potent therapeutic target against cancer. Fast-dividing malignant cells require enhanced ribosome production to meet the increased needs for protein synthesis to sustain the elevated metabolism and proliferation . High raTranscription of ribosomal RNA (rRNA) is carried out exclusively by the RNA polymerase I (Pol I) inside the nucleolus. This process requires the presence of the upstream binding factor UBF on the active ribosomal DNA (rDNA) genes, enabling the recruitment of the pre-initiation complex (PIC) and Pol I components to the rDNA gene promoter. Each rDNA gene codes for the 47S rRNA precursor, which is further processed to form the 18S, 5.8S and 28S mature rRNAs. In parallel, RNA polymerase III transcribes the 5S rRNA in the nucleoplasm, while RNA polymerase II transcribes the mRNAs of the ribosomal proteins (RPs). Mature 5S rRNA and RPs are imported to the nucleolus and preassembled together with the 18S, 5.8S, and 28S RNAs to form the 60S and 40S ribosomal subunits. Subsequently, mature ribosomes are formed in the cytoplasm through a series of export and maturation processes phenyl}quinolin-4-amine : A mixture of 3-[(diethylamino)methyl]aniline , 4,7-dichloroquinoline , ethanol (2 mL), and 12 M HCl  was heated in the micro wave oven at 150 °C for 30 min. The solvent was removed in a rotavapor. Water (3 mL) and 5 M NaOH (0.5 mL) were added to the crude material and the mixture was extracted with dichloromethane (2 mL). The organic phase was dried over Na2SO4 and the product was purified by column chromatography on silica  to generate 7-chloro-N-{3-[(diethylamino)methyl]phenyl}quinolin-4-amine  [M + H] + m/z 340; 1H NMR  ppm 0.98 , 2.44–2.50 , 3.54 , 6.91 , 7.07–7.11 , 7.20–7.26 , 7.30–7.39 , 7.56 , 7.89 , 8.40–8.47 , 9.10 . Synthesis of 7-chloro-N-[3-(pyrrolidin-1-ylmethyl)phenyl]quinolin-4-amine : A mixture of 3-(pyrrolidin-1-ylmethyl)aniline , 4,7-dichloroquinoline , acetonitrile (1 mL), water (1 mL), and 12 M HCl  was heated in a sealed tube at 110 °C for 4 h. The reaction was cooled to r.t., and 5 M NaOH  was added to the stirred solution. The product was collected by filtration and ½ of this crude material was purified by column chromatography on silica  to generate 7-chloro-N-[3-(pyrrolidin-1-ylmethyl)phenyl]quinolin-4-amine . LCMS [M + H] + m/z 338; 1H NMR  ppm 1.64–1.74 , 2.39–2.48 , 3.59 , 6.92 , 7.06–7.11 , 7.22–7.27 , 7.29–7.34 , 7.35 , 7.56 , 7.89 , 8.43 , 8.46 , 9.09 .Starting materials and intermediates used in the synthesis of compounds described herein are commercially available from Sigma-Aldrich, Enamine, and Combi-Blocks. Flash column chromatography was performed in a ISCO combi flash system using Merck silica gel 60 Å (40−63 mm mesh). 1H NMR spectra were recorded on a Bruker DRX-400. Chemical shifts are expressed in parts per million (ppm) and referenced to the residual solvent peak. Analytical HPLC-MS was performed on an Agilent MSD mass spectrometer connected to an Agilent 1100 system with: Method acidic pH, Column ACE 3 C8 (50 mm × 3.0 mm), HWhile searching for Pol I inhibitors among FDA-approved drugs, we assessed the impact of AQ on levels of 47S rRNA, using a set of primers targeting mature 18S, 5.8S and 28S rRNAs and six primers for regions present exclusively in the 47S precursor rRNA. Precursor-specific regions showed a dose-dependent decrease in 47S synthesis Fig. . As expePOLR1D, POLR1E, and TAF1B components of the Pol I PIC. BMH-21 downregulated UBFT, TWISTNB, and POLR1A and upregulation of POLR1B, POLR1C, TAF1C, and TAF1D  can be reproduced by its analog CQ; and (ii) are comparable with those induced by BMH-21. For this, we employed electron microscopy in order to detect subtle changes. AQ induced dramatic condensation of the nucleolar chromatin along with loss of the distinctive nucleolar regions FC, DFC, and GC, similarly to BMH-21Fig. . In contAQ is more efficient than CQ in stabilizing p53 at equimolar concentrations , 19, howTranscription of rDNA becomes inhibited upon DNA damage by ATM kinase-mediated signaling , 34. We +-ATPase that maintains acidic pH inside the lysosomes. Increase in pH inhibits hydrolases and autophagy [AQ, along with other members of the 4-aminoquinoline family, including CQ, hydroxychloroquine (H-CQ), and Lys-05 (dimeric CQ) inhibit autophagy . While 4utophagy . Althougutophagy . Howeverp value < 0.001 and gene expression fold changes >0.6 and <−0.6. Under equimolar concentrations, AQ induced more gene expression changes than CQ . Notably, BMH-21 induced the most dramatic perturbation, with 3316 affected genes. Differentially expressed genes were further sorted among the three compounds using Venn diagrams to identify common and unique gene perturbations.Transcriptome analysis can identify shared and unique mechanisms of action among compounds, pointing to links between biological activities and diseases . Unlike Among the CQ-regulated genes, 37% (85 out of 228) were also regulated by AQ, including transcripts enriched for cholesterol biosynthesis, response to hypoxia and blood vessel development Fig. . Indeed,Furthermore, 1041 genes were specifically affected by AQ, enriched in angiogenesis/VEGF, autophagy and cell adhesion pathways. Among all genes perturbed by the three compounds, 33.9% overlapped between AQ and BMH-21, including those related to cell-cycle arrest, DNA replication stress, and p53 signaling, consistent with our results and previous studies , 25. AmoThese results reveal that AQ induces transcriptional changes that overlap with those induced specifically either by CQ or BMH-21, thereby overall consistent with the concept that AQ impacts cells simultaneously through two mechanisms of action: autophagy inhibition and ribosome biogenesis stress.R2 of 0.22 was found  . The humThese results provide an additional mechanistic feature of AQ, showing that the nucleolar activity is retained in the absence of the reactive intermediates, and supporting feasibility of chemical optimization of AQ to enhance the nucleolar disruption capacity while eliminating idiosyncratic side effects.To investigate whether chemical optimization of AQ could increase its efficiency of rDNA transcription inhibition, we first tested amopyroquine (ApQ), a commercially available structural analog of AQ. In ApQ, the 2-diethylamino side chain is replaced by a cyclic pyrrolidone one Fig. . NotablyOur findings show that the FDA-approved antimalarial drug AQ inhibits ribosome biogenesis, an activity not shared by other 4-aminoquinoline family members. AQ triggers proteasome-dependent degradation of RPA194 (POLR1A), a feature reported for the experimental drugs BMH-21  and BMH-Targeting ribosome biogenesis has been proposed as a therapeutic strategy for hematological malignancies , ovarianOngoing clinical trials are addressing the potential of CQ and H-CQ in combinatorial treatments for various cancer types. Most such trials were designed with the rationale to increase the efficacy of other anticancer therapies through inhibition of treatment-induced autophagy. Although it is too early to draw any conclusions, data from first clinical trials and additional preclinical data suggest a potential for implementing these drugs in anticancer treatment . To the In conclusion, we have demonstrated that AQ inhibits ribosome biogenesis and disrupts nucleolar structure, triggering degradation of RNA polymerase I. Moreover, AQ exerts high cytotoxic activity against CRC, a neoplasia with predicted sensitivity to ribosome biogenesis inhibitors. Our study supports the rationale for repurposing an old and inexpensive drug to target an emerging vulnerability in cancer, which may translate into more affordable and accessible therapies.Supplementary material


Candida albicans, given its multifaceted role in cellular processes, including GlcNAc scavenging, import and metabolism, morphogenesis (yeast to hyphae and white to opaque switch), virulence, GlcNAc induced cell death (GICD), etc. During signaling, the exogenous GlcNAc appears to adopt a simple mechanism of gene regulation by directly activating Ngs1, a novel GlcNAc sensor and transducer, at the chromatin level, to activate transcriptional response through the promoter acetylation. Ngs1 acts as a master regulator in GlcNAc signaling by regulating GlcNAc catabolic gene expression and filamentation. Ndt80-family transcriptional factor Rep1 appears to be involved in the recruitment of Ngs1 to GlcNAc catabolic gene promoters. For promoting filamentation, GlcNAc adopts a little modified strategy by utilizing a recently evolved transcriptional loop. Here, Biofilm regulator Brg1 takes up the key role, getting up-regulated by Ngs1, and simultaneously induces Hyphal Specific Genes (HSGs) expression by down-regulating NRG1 expression. GlcNAc kinase Hxk1 appears to play a prominent role in signaling. Recent developments in GlcNAc signaling have made C. albicans a model system to understand its role in other eukaryotes as well. The knowledge thus gained would assist in designing therapeutic interventions for the control of candidiasis and other fungal diseases.The amino sugar, N-acetylglucosamine (GlcNAc), has emerged as an attractive messenger of signaling in the pathogenic yeast  Candida albicans lives as a commensal on healthy individuals and can cause life-threatening systemic infections, contributing substantially to morbidity and mortality in immunocompromised patients . B. BNRG1,  of Nrg1 ,46 , which are involved in GlcNAc metabolism, and which play a crucial character in morphological switching as well as its virulence ,49,50. Talbicans ,52. The albicans . Some ofic genes . It recric genes . Initiallux pump . In C. aApart from Ngs1-Brg1 mediated signaling, GlcNAc also triggers hyphal transition through the pH sensing pathway, where Rim101 acts as transcriptional regulator that is known to induce hyphal formation in response to alkaline pH, and also provides adaptation to high ambient pH . Here, GC. albicans . T. TE. colrganisms . Eukaryorganisms .E. coli, chromatin exists in a simple non-condensed and non-restrictive state; so, once repressor NagC is removed from the promoter DNA, the promoter is accessible for basal transcriptional machinery to initiate transcription , GICD, virulence, and inter-species communication, etc. [C. albicans to respond rapidly and appropriately to the host niche environmental conditions for maximal use of nutrients and competing over other microbes [BRG1 expression and inhibiting the inhibitory effect of Nrg1 for promoting filamentation (14). Interestingly, it appears that GlcNAc triggers white/opaque switching and yeast/hyphal transition contrastingly through two different pathways; while white/opaque switching occurs through the Ras1-cAMP/PKA dependent pathway, yeast/hyphal transition occurs independently [Accumulating evidences revealed that, in on, etc. ,48. Thusmicrobes . Therefomicrobes ,31. GlcNmicrobes . Most suentation . Interesendently ,40,48,55NGS1 has been shown to be important for the growth on maltose.Although it appears that Ngs1-Rep1 complex is directly involved in the induction of GlcNAc catabolic gene expression by acetylation of cognate gene promoters, possible involvement of other regulatory factors that might act as either repressors and/or activators in the induction mechanism (mentioned in open questions), cannot be ruled out. In support of this argument, the negative role of Hxk1 in GlcNAc induction mechanism can definitely gain considerable attention, for which the molecular mechanism is yet to be revealed . FurtherAs GlcNAc sensor Ngs1 and associated Ndt80 transcription factor Rep1 are constitutively bound to promoter regions, it is important to determine the genome wide occupancy of these factors. Either ChIP-seq or ChIP-on-chip can be used to find out genome-wide binding regions, as well as sequence of the binding elements . This anAs it is known that GlcNAc induced filamentation is different from GlcNAc induced metabolic gene expression, how does GlcNAc differentiate these two cellular processes separately; what is the molecular mechanism, and are there any environmental factors influence these processes?Is there any role of Ngs1 in GlcNAc induced white to opaque switching?What is the mode of regulation for Hxk1 mediated GlcNAc signaling to stimulate metabolic gene expression?What are the genome-wide binding sites for Ngs1 and Rep1, and is there any role of these transcription factors for the growth on other carbon sources, such as, galactose, mannose, etc.?BRG1?What is the DNA-binding transcription factor responsible for the recruitment of Ngs1 at the promoter region of


Human Cytomegalovirus (HCMV) is a highly prevalent herpesvirus, persistently infecting between 30 and 100% of the population, depending on socio-economic status (Fields et al.,  Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that belongs to the β-herpesvirus subfamily. It has the largest DNA genome among the human herpesviruses with ~235 kb pairs, and encodes at least 173 annotated genes based on the low-passage strain Merlin, the current reference strain for wild-type HCMV , proteomics and high-throughput RNAi screens  has revolutionized our ability to monitor gene expression levels on a genome wide basis. Techniques such as purification of poly-A transcripts or depletion of ribosomal RNA (rRNA) have improved the sensitivity and accuracy of RNA-seq , providing cells with “heavy” lysine  at the time of infection to identify newly synthesized peptides, and labeling peptides with TMT for mass spectrometry. pSILAC results showed that 24 of 46 proteins rescued at 12 HPI with MG132 exhibited increased degradation in HCMV-infected cells compared to mock infection, whilst 12 proteins did not exhibit instability or degradation. Some of these proteins had extremely high turnover rate, making it difficult to observe a difference between mock and HCMV infection , whereas some of these proteins were also rescued by the application of MG132 during mock infection . In summary, proteins that were rescued by MG132 but did not exhibit instability by pSILAC may still be degraded, and post-transcriptional and post-translational regulations may have worked in combination to regulate the gene expression. In addition, RNA-seq analysis was performed to identify proteins for which expression was determined primarily by post-transcriptional regulation rather than mRNA transcription. Several candidates were shown to have upregulated transcription but downregulation of protein expression, including the HCMV restriction factor Sp100, an E3 ubiquitin ligase ANAPC1 and the helicase-like transcription factor HLTF. The authors further characterized HLTF, which was not previously reported to be targeted for proteasomal degradation by HCMV, and found that UL145 is necessary and sufficient to degrade HLTF. UL145 was also found to recruit the Cullin 4 (CUL4) E3 ligase complex to target HLTF to proteasomal degradation. Finally, HLTF was shown to act as a restriction factor similar to Sp100, at an early stage of virus replication.mRNA levels don't always correspond to protein levels due to various factors, including post-transcriptional modification, mRNA stability, and regulation by miRNAs   using small interfering RNA (siRNA) and short hairpin RNA (shRNA), expression of host factors can be systematically silenced and the effect of individual gene depletion on virus replication can be investigated. In addition, the recently developed and advanced CRISPR genome editing technology allows mutations to be introduced to target genes and disrupt protein expression of host factors that are important for virus replication.siRNA screens are usually performed in arrayed fashion where each well in a microplate has a unique RNAi reagent targeting one gene. On the other hand, shRNA screens are often performed as pooled screens where all RNAi reagents are pooled together and introduced randomly to cells, where shRNAs can be detected by next generation sequencing (NGS) or barcode microarray following phenotypic selection. High-throughput RNAi screens have contributed greatly to our understanding of basic cell biology as well as complex host-virus interactions which can lead to development of novel therapeutics.siRNAs are regulatory RNA duplexes, usually 21–23 nucleotides long, with 2 nucleotide overhangs on the 3′ ends. They bind to complementary mRNA transcripts, causing degradation  on HCMV replication. However, CD4 and WNK4 were not found to be expressed in HFFs infected with HCMV; thus, they were excluded from further analysis. PI3K-C2A was further characterized since it had not been previously linked to HCMV replication at the time of the study. The PI3K family is important for nearly all aspects of cell biology, acting at nearly all cellular membranes to regulate a wide range of signaling, membrane trafficking and metabolic processes  was developed by inserting an SV40 promoter-driven GFP cassette into the intragenic region between TRS1 and US34   pathway. A high-coverage shRNA library consisting of around 55,000 sequences  that target all known human protein-encoding genes was generated  library has been used in a screen to identify host factors required for HCMV replication. The library targeted 19,050 human genes with over 120,000 sgRNA sequences  with one or more sequences that can hybridize and form hairpin structures  analysis to identify host factors targeted by HCMV-encoded miRNAs  was performed in HCMV strain TowneSystems virology, involving high-throughput molecular profiling and bioinformatics, has highlighted the complexity of host-virus interactions and contributed to our understanding of how HCMV works, novel cellular functions and potential new drug targets (Law et al., in vivo prior to proceeding to clinical trials. The development of host-oriented antivirals will have lower rates of the emergence of drug-resistant virus, thereby providing a better option for therapeutics. However, drug toxicity in the host is a major challenge most novel antivirals need to overcome because some host factors are required for normal cellular functions.With advances in sequencing, mass spectrometry and genome editing, high-throughput studies become more efficient and accurate in the identification of host-virus interactions. Despite the discovery of novel host factors important for virus replication, the precise functions and roles of these factors remain to be fully elucidated. The potential of these factors as antiviral targets will also require extensive examinations Integrating the various high throughput approaches, from RNA-seq to proteomics to phenotypic screens, represents an attractive future goal of systems approaches in HCMV research as well as systems research in general. With improving pathway analysis, integrated systems approaches may lead to major advances in our understanding of basic virus biology, cell biology and the discovery of novel antiviral therapies.C-HL wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.