The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, synchronizes and maintains daily cellular and physiological rhythms across the body, in accordance with environmental and visceral cues. Consequently, the systematic regulation of spatiotemporal gene transcription in the SCN is vital for daily timekeeping. So far, the regulatory elements assisting circadian gene transcription have only been studied in peripheral tissues, lacking the critical neuronal dimension intrinsic to the role of the SCN as central brain pacemaker. By using histone-ChIP-seq, we identified SCN-enriched gene regulatory elements that associated with temporal gene expression. Based on tissue-specific H3K27ac and H3K4me3 marks, we successfully produced the first-ever SCN gene-regulatory map. We found that a large majority of SCN enhancers not only show robust 24-h rhythmic modulation in H3K27ac occupancy, peaking at distinct times of day, but also possess canonical E-box (CACGTG) motifs potentially influencing downstream cycling gene expression. To establish enhancer–gene relationships in the SCN, we conducted directional RNA-seq at six distinct times across the day and night, and studied the association between dynamically changing histone acetylation and gene transcript levels. About 35% of the cycling H3K27ac sites were found adjacent to rhythmic gene transcripts, often preceding the rise in mRNA levels. We also noted that enhancers encompass noncoding, actively transcribing enhancer RNAs (eRNAs) in the SCN, which in turn oscillate, along with cyclic histone acetylation, and correlate with rhythmic gene transcription. Taken together, these findings shed light on genome-wide pretranscriptional regulation operative in the central clock that confers its precise and robust oscillation necessary to orchestrate daily timekeeping in mammals. Most organisms possess intrinsic circadian (∼1-d) clocks that align their molecular, behavioral, and physiological processes to changing daily environmental conditions. In mammals, the phase and amplitude of circadian processes are directed and synchronized by the suprachiasmatic nucleus (SCN) of the hypothalamus, which facilitates robust 24-h oscillations in peripheral tissues . In turnThe TTFL is also active in peripheral tissues and cells but is far less robust than that of the SCN, typically damping after a few cycles, ex vivo, whereas the SCN can continue to oscillate indefinitely in explant culture. This raises the question that apart from the distinctive intra- and inter-cellular coupling, what features of transcriptional architecture render the SCN such a powerful clock. We hypothesize that precise regulation of spatiotemporal gene expression in the SCN is imperative for its powerful daily timekeeping. Daily rhythms of electrical activity, metabolic rate, and intra- and inter-cellular signaling are driven by cascades of transcription, co-ordinated by clock-controlled genes (CCGs), many of which are transcription factors (TFs) sensitive to the TTFL. Such rhythmic gene transcription depends on the sequential co-ordination of multiple layers of epigenetic events, ranging from chromatin accessibility to TF binding . The regRecently, active enhancer sites have also been reported to transcribe enhancer RNA (eRNA) dynamically in response to chemical and electrical stimuli, learning, and behavioral experiences . This clAt the tissue level, the persistence and precision of the SCN as a circadian timekeeper are unique, but the cell-autonomous regulatory factors that confer this uniqueness to the SCN are poorly understood. Genomic mapping of regions with associated histone modifications such as H3K4me3 and H3K27ac has been the cornerstone for identifying active promoter and enhancer sites, respectively . Typicalcis-elements, which presumably are vital to drive the SCN circadian rhythm. Furthermore, we performed directional bulk RNA-seq at six distinct time points to study the relationship between observed rhythmic H3K27ac occupancy and target gene expression. Along with rhythms in the coding mRNA fraction, we also noticed cycling expression in the noncoding bidirectional eRNA. Thereby, our study offers a framework to understand the dynamic pretranscriptional regulation operative in the SCN central clock that potentially contributes to maintaining its uniquely stable circadian timekeeping.Here, we used histone chromatin immunoprecipitation (ChIP) sequencing (histone ChIP-seq) to map genome-wide gene promoter and active enhancer sites in the SCN. Next, with a view to identifying putative SCN-enriched regulatory processes, we compared the identified histone modification peak profiles in the SCN with those found in the cerebral cortex. This revealed considerable enrichment associated with genes highly expressed in the SCN , supportSupplemental Fig. S1; Supplemental Fig. S2A,B; Supplemental Table S1). The SCN-enriched H3K4me3 sites were found adjacent to TSSs of the genes known to be highly expressed in the tissue, as exemplified in Supplemental Figure S2C .To identify DNA regulatory elements influencing gene transcription in the SCN, we focused on SCN-enriched promoter and enhancer sites marked by H3K4me3 and H3K27ac, respectively. C57BL/6J mice were kept in standard 12-h/12-h light–dark conditions, and eight animals per time point (Methods) were used for subsequent brain dissections. The harvested SCN and cerebral cortical brain regions were then used for H3K4me3 and H3K27ac ChIP-seq, and the resulting sequencing reads were mapped to the mouse genome (Methods). Initial principal component analysis (PCA) of aligned reads arising from H3K4me3 and H3K27ac occupancy at ZT3 confirmed high-quality ChIP signal and tissue-specific enrichment. The reads associated with H3K4me3 and H3K27ac, respectively, not only were found to be separate from their corresponding input sample but also showed clear distinction based on the brain region A. In addgure S2C , whereasSupplemental Table S2A). These SCN-enriched H3K27ac sites were dominant at distal intergenic (34.7%) and intronic regions, including first introns (38.9%) (Usp29 (±0.2 Mb) gene at Chr 7 and the Zfhx3 (±1 Mb) gene at Chr 8. It is noteworthy that the SCN TF ZFHX3 has been implicated as an important regulator in setting the robustness and speed of the circadian clock  that differentially marked active enhancer sites between the two brain regions ( (38.9%) C, which  (38.9%)  wherein  (38.9%) D,E SCN-s (38.9%) . Further (38.9%) . Overall (38.9%) , layer 3an clock , and theSupplemental Fig. S3A; Supplemental Table S3A), mostly confined around the TSS of genes involved in learning and synapse organization. In contrast, no changes in H3K4me3 sites were seen within the SCN . This stability of histone trimethylation levels in the SCN is in marked contrast to studies in peripheral clock(s) . This reinforces the potential role of H3K27ac-marked active enhancers, as opposed to H3K4me3 marks, in regulating time-of-day-dependent gene transcription. On further inspection, a significant proportion of identified differentially H3K27ac-marked enhancer sites was found proximal to genes implicated in circadian entrainment, such as Gnas, Rasd1, and Cacna1a , with almost negligible occurrence in the cortex. Moreover, active enhancer sites that showed day–night fluctuation in both the SCN and cortex (n = 31) did not follow the same trend; for example, a differential H3K27ac site mapped to calcium binding gene Calm3 is strongly elevated at ZT3 in the SCN but is elevated at ZT15 in the cortex  was then investigated by comparing the antiphasic (12-h-apart) time points, ZT3 (3 h after light on) and ZT15 (3 h after light off), in both the SCN and cerebral cortex. We observed approximately 551 differential H3K4me3 sites in the cortex at FDR ≤ 0.05 (clock(s) , whereine cortex D, where e cortex . Hence, Supplemental Fig. S3C). Indeed, we found a considerable number of active enhancer sites in the SCN  with differential abundances at distinct times of day. In addition, there was almost negligible overlap between H3K27ac-marked differential sites noticed from distinct antiphasic comparisons; for instance, an enhancer peak differential between ZT3 and ZT15 did not overlap with any found between ZT7 and ZT19 . Using this approach, we clearly identified a unique active enhancer site within the Per2 gene showing increased histone acetylation at ZT11 in comparison with ZT23, coinciding with the peak and trough of host gene expression levels  is concordant with the downstream gene transcription . Thus, variability in histone acetylation levels seems to be clearly associated with changing transcriptional output in the SCN, as noted with the rhythmic expression of the core-clock genes Per2 and Cry1. About 50% of the differing histone acetylation levels perfectly correlated (Pearson's correlation (r) = 1) with the host gene expression . To our knowledge, this method of strategic in-depth examination in the time-dependent occurrence of histone modifications has never been reported previously. We clearly observed the time-dependent differing histone acetylation levels arising from TTFL components to downstream CCGs in the SCN.To understand further the dynamics of H3K27ac occupancy in the context of daily timekeeping in the SCN, we profiled its occurrence every 4 h starting from ZT3 (Methods) and compared the peak profiles between additional antiphasic time points , based on the expectation that rhythmic genes under circadian regulation would have peaks and troughs in expression level ∼12 h apart . TF binding sites for CAAT/enhancer-binding protein (C/EBP) alpha (CEBPA) and RAR-related orphan receptor (ROR), both belonging to the PARbZip family  by both JTK_CYCLE . The canonical E-box (CACGTG) element facilitating circadian transcriptional activation by binding of CLOCK and BMAL1 (P < 0.05), suggesting a pivotal role of cis-regulatory elements in the maintenance of rhythmic transcriptional output in the SCN . For example, RAR-related orphan receptor alpha (Rora) is known to regulate the rhythmic expression of Bmal1 , we also observed uniform phase (peak abundance) distribution for the rhythmic H3K27ac sites at the 24-h period . For that reason, a high proportion of these rhythmic histone acetylation sites were found ± 50 kb from the rhythmic gene TSS. In addition, functional annotation of the rhythmic genes that are under the control of the phase-separated cycling H3K27ac levels in the SCN revealed diverse roles  comparisons. Active enhancer sites that were found to be distinct between these were compiled (n = 1021) and further assessed for circadian oscillation using the Extended Circadian Harmonic Oscillator (ECHO) application . BrieflyTK_CYCLE  and ECHOnd BMAL1  was overnd BMAL1 C. In prind BMAL1 . Thus, tof Bmal1 , and it h period B. Althouse roles E, ranginSupplemental Table S8). Out of these, we subselected IBSs that revealed greater SCN-specific H3K27ac occupancy  and focused on the high-confidence SCN actively transcribing enhancer sites (n = 883), as represented in Supplemental Figure S5, A and B. Based on previous findings, chromatin remodeling and eRNA transcription are shown to precede mRNA expression present at adjacent cis-loci  SCN-specific enhancer sites compared with the cortex . A subsecis-loci . Therefor > 0.5), we noted 7795 high-confidence eRNA–gene pairs from a total of 46,022 identified pairs . As expected, the actively transcribing enhancers positively correlated with highly expressed SCN target genes as shown in the representative examples at the Vipr2 and Cry2 loci . The distance of enhancer from the target gene had no effect on the subsequent strength of transcriptional correlation, consistent with the suggested role of eRNA in regulating both adjacent and distal gene expression  to both a 0.4-Mb distant target gene, prolyl 3-hydroxylase 1 (P3h1), and a 1.5-Mb separated gene, rearranged L-myc fusion (Rlf). Overall, we were clearly able to show the prevalence of noncoding eRNA in the SCN, arising from enriched histone acetylation sites, which are well placed to regulate local tissue-specific gene expression. Our data set pointed out toward the involvement of not just histone modifiers but also succeeding noncoding transcript in regulating the target gene transcription and prompted us to study its role in daily timekeeping.Next, the expression of SCN-specific eRNA was evaluated at six distinct time points across the day starting from ZT3 (Methods). To determine the target gene under the control of each actively transcribing enhancer, we studied the correlation between eRNA and all annotated protein-coding gene expression falling within ∼±2 Mb from the peak center B. This aP < 0.05)  in the SCN, with their relative expressions peaking across different times of the day. Based on their phase of peak expression, circadian SCN eRNAs were divided into 12 groups  . Approximately 66% of circadian eRNAs oscillated with a peak phase between ZT14 and ZT20, whereas 34% oscillated in other phases. This irregular phase distribution of SCN eRNAs agrees with the previous finding wherein circadian eRNAs in liver peaked predominantly between ZT18 and ZT3  B. Circadtervals) B or rhyt and ZT3 . BearingSupplemental Table S11). Subsequently, the expression level of each identified eRNA was examined across various times of the day for a positive correlation (r > 0.5) with the target gene transcription present at ∼±2-Mb distance . Unlike the principal nearest neighboring gene approach, this strategy helped us to identify a repertoire of target genes whose expression is possibly regulated by distantly transcribing enhancers in the SCN . The recognized target genes were seen to be involved in divergent biological processes based on the disparate H3K27ac abundance as shown in Bmal1]) , prokineticin receptor 1 (Prokr1), etc., mediating circadian rhythms, whereas those identified from differential histone acetylation between ZT7 and ZT19 were found to influence genes involved in mRNA processing. Therefore, the noncoding eRNAs driven by temporally fluctuating histone acetylation levels add an important layer of control over the gene expression machinery in the SCN.To explore the link between differing histone acetylation at distinct times of day and eRNA expression levels, we assessed the intergenic class of differential H3K27ac sites (n = 216) for the occurrence of eRNA transcription. To accomplish this, strand-specific RNA abundance was examined for each intergenic site showing differential histone acetylation between antiphasic time intervals. About 53% of these intergenic H3K27ac sites revealed bidirectional transcription, a reliable measure for eRNA abundance (P < 0.05) . Taken together, we uncovered a proportion of intergenic sites with cycling H3K27ac levels , which in turn transcribes rhythmic eRNA to regulate gene transcriptional machinery in the SCN. A characteristic example of this phenomenon is presented , wherein rhythmic H3K27ac abundance and subsequent eRNA transcription  are seen to regulate the circadian expression of the SCN neuropeptide receptor gene Vipr1 , where the relative expression of varying eRNA directly coordinated with the target Vipr1 mRNA transcription during the day. As expected, the eRNA and target Vipr1 mRNA had high expression at ZT15 and low expression at ZT3, showing an in-phase relationship. Herewith, this comprehensive analysis of dynamic histone acetylation in conjunction with enhancer transcription helped us to unfold an important layer in the systematic regulation of circadian gene expression in the SCN.Of note, ∼38% of these eRNAs arising from dynamic histone acetylation sites showed 24-h periodic transcription  E. Thus, ne Vipr1 . This waSupplemental Table S1) and H3K27ac-bound enhancers  around TSSs of the genes reported to be highly expressed in the central clock . This led us to produce the SCN regulatory map, highlighting the active enhancer sites that distinguishes SCN from the other brain region(s). These SCN-enriched active enhancer sites were found to be in close proximity of the genes associated with not just circadian entrainment but also calcium signaling and neuropeptidergic synaptic function , with no difference in the cortex. This strongly suggests SCN-specific cis-elements that confer daily change in chromatin accessibility, potentially to control downstream rhythmic gene transcription. It clearly shows the importance of temporal control in genome-wide histone modifications within distinct brain regions in the context of daily timekeeping. The differential intensity noted in histone modification in the SCN was only observed for H3K27ac, and not for the H3K4me3 mark, which is profoundly enriched around the gene TSS. A plausible explanation for this phenomenon could be the occurrence of H3K4me3 marks around both poised and active gene TSSs  and ZT15 (night) and compared the resulting peak profiles. We clearly observed a significant proportion of varying H3K27ac peak abundance between day and night exclusively in the SCN , and found robust 24-h oscillation  (Per2 gene expression peaks around ZT8 (middle to late day) in the SCN, whereas it is reported to attain highest level around ZT14 (early night) in the liver. Hence, the systematic SCN transcriptomics conducted in the present study reconciled with the prior studies  . For exa studies  and offeFos gene . Moreover, at certain loci, we also observed rhythmic eRNAs arising from differential (not rhythmic) H3K27ac levels that point toward the possibility of histone acetylation being in charge of the chromatin accessibility (open or closed chromatin), whereas the corresponding eRNAs modulate the downstream target gene transcription. Nonetheless, our current finding is an extension to the formerly studied role of noncoding RNA  and Home Office Project License 19/0004. WT C57BL/6J were maintained and provided in-house by MRC Harwell. Animals were group-housed (four to five mice per age) in individually ventilated cages under 12-h/12-h light–dark conditions with food and water available ad libitum.WT C57BL/6J mice  were used for SCN tissue collection as described by Gapdh (GAPDH-TSS) and myoglobin exon 2 (MBex2).ChIP was conducted with Diagenode ChIP-seq/ChIP-qPCR profiling service (Diagenode G02010000). Briefly, the chromatin was prepared using the true MicroChIP kit (Diagenode C01010130). Samples were fixed with 1% FA for 8 min. Chromatin was sheared using a Bioruptor Pico sonication device (Diagenode B01060001) combined with the Bioruptor water cooler for seven cycles, using 30-sec/30-sec on–off settings. Then, shearing was performed in 0.65-mL Bioruptor Pico microtubes (Diagenode C30010011). Twenty-five microliters of this chromatin was used to assess the size of the DNA fragments obtained by a high-sensitivity NGS fragment analysis kit (DNF-474) on a fragment analyzer . ChIP was performed using a IP-Star compact automated system (Diagenode B03000002) following the protocol of the aforementioned kit. Five hundred nanograms of chromatin was immune-precipitated using 1 µg of each of the following Diagenode antibodies: H3K4me3 , H3K27ac , and rabbit IgG . Chromatin corresponding to 1% was set apart as input. The DNA after reverse-cross-linking was quantified using a Qubit dsDNA HS assay kit (Thermo Fisher Scientific Q32854). Moreover, qPCR analysis was made to check ChIP efficiency using KAPA SYBR FAST (Sigma-Aldrich) on a LightCycler 96 system (Roche), and results were expressed as % recovery = 100 × 2^((Ct_input-6.64) − Ct_sample). Primers used were the following: the promoter of H3K4me3 and H3K27ac immune-precipitated genomic DNA along with their corresponding input samples was sent to Oxford Genomics Centre, University of Oxford, for library preparation using ChIP-seq protocol and paired-end sequencing on NovaSeq 6000 platform (Illumina).https://usegalaxy.eu/) using the ChIP-seq pipeline. Paired-end FASTQ files were quality assessed by removing low-quality bases (Phred < 20) and trimmed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic v0.36 (http://www.usadellab.org/cms/index.php?page=trimmomatic), respectively. FASTQ files containing trimmed sequences were then aligned to the mm10 genome assembly to generate binary alignment map (BAM) files with Bowtie 2 v2.3.4.1 (https://samtools.github.io/hts-specs/) (http://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf).Raw sequence data in the form of a pair of fq.gz were processed using tools on the Galaxy EU server (v2.3.4.1 . Aligned-specs/)  and used-specs/)  with the-specs/)  was usedBulk RNA-sequencing (RNA-seq) was performed at Oxford Genomics Centre, University of Oxford. RNA was extracted, DNase-treated, and purified  for four biological replicates per time point. Five hundred nanograms to 1 µg of total RNA underwent quality control (NanoDrop), and libraries were prepared for directional ribodepleted RNA-seq using NEBNext reagents. RNA-seq libraries underwent sequencing  on a NovaSeq 6000 (Illumina) platform.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and Illumina adapters were trimmed with Trim Galore! v0.4.3 . Then the reads were aligned to the mm10 genome assembly using STAR v2.7.8a (http://bioconductor.org/packages/release/bioc/html/limma.html) was adopted to quantify differential gene expression, and normalized logarithmic CPM values were generated for downstream analysis.Paired-end FASTQ files were quality assessed (Phred < 20 removed) with FastQC ( v2.7.8a  with the v2.7.8a  via SAMt v2.7.8a  from thehttps://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) as previously reported .The gene list derived from Bioconductor-based ChIPseeker v1.28.3  was fed Logarithmic CPM values across all time points for H3K27ac occupancy, gene, and eRNA feature were analyzed for significant circadian oscillations using JTK_CYCLE  and ECHOVipr1, eRNA; Chr 9: 123,334,375–123,335,717). 7500 Applied Biosystems Software v2.3  was used to obtain the relative quantification values and examine melt curves. All RT-qPCR data were normalized to the Rpl32 reference control and analyzed using the standard curve method (Vipr1 (TCAACAACGGGGAGACAGAC [forward] and GGCCATGACGCAATACTGGA [reverse]), eRNA (TGGTTAAGAGCGCCTACAGC [forward] and GCTGTCTTCAGACACTCCAGA [reverse]), and Rpl32 (AGGCACCAGTCAGACCGATA [forward] and TGTTGGGCATCAGGATCTGG [reverse]) were designed and screened for target specificity with the National Center for Biotechnology Information's (NCBI) Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/). The primer sets were validated before use .Total RNA was extracted from the SCN tissue as described above at three distinct time points; ZT3, ZT15, and ZT23. For each time point, RNA was extracted from two to four biological replicates, where each biological replicate comprises three to four individual SCN punches. One hundred nanograms RNA per sample replicate was reverse-transcribed . The synthesized cDNA was used for RT_qPCR using an Applied Biosystems 7500 real-time PCR instrument  in triplicates for the genes of interest (e method  The primwww.graphpad.com). The Pearson's correlation between the target mRNA and eRNA across the tested time points was set at alpha = 0.05.Gene expression differences from qPCR were plotted and analyzed with GraphPad Prism v9.5.1 for Windows, GraphPad Software (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE217943.All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


Gryllus bimaculatus. Accompanying spermatid differentiation, the SNBP-based genome is strikingly reorganized as ~25nm-thick fibers orderly coiled along the elongated nucleus axis. This chromatin spool is further found to achieve large-scale helical twisting in the final stages of spermiogenesis, favoring its ultracompaction. We reveal that these dramatic transitions may be recapitulated by a surprisingly simple biophysical principle based on a nucleated rigidification of chromatin linked to the histone-to-SNBP transition within a confined nuclear space. Our work highlights a unique, liquid crystal-like mode of higher-order genome organization in ultracompact cricket sperm, and establishes a multidisciplinary methodological framework to explore the diversity of non-canonical modes of DNA organization.Spermiogenesis is a radical process of differentiation whereby sperm cells acquire a compact and specialized morphology to cope with the constraints of sexual reproduction while preserving their main cargo, an intact copy of the paternal genome. In animals, this often involves the replacement of most histones by sperm-specific nuclear basic proteins (SNBPs). Yet, how the SNBP-structured genome achieves compaction and accommodates shaping remain largely unknown. Here, we exploit confocal, electron and super-resolution microscopy, coupled with polymer modeling to identify the higher-order architecture of sperm chromatin in the needle-shaped nucleus of the emerging model cricket  Orsi et al. identify an elegant solution to DNA packing in cricket sperm nuclei, whereby relatively simple biophysical changes in chromatin properties result in a liquid crystal-like twisted spool organization that favors ultracompaction. Indeed, there is a remarkable diversity in the morphology of sperm nuclei in animals, including. falciform-shaped (mouse), rod-shaped (rooster), drill-bit-shaped (house sparrow), or the needle-shaped sperm nucleus that is common among insects2. Fitting into these complex morphologies, the paternal nucleus typically undergoes a high level of compaction − ~10x to ~40x compared to somatic interphase cell nuclei, depending on the species5. Abnormal compaction and/or morphology are generally associated with reduced fertility8, illustrating the physiological importance of these mechanisms.During spermiogenesis, post-meiotic spermatogenic cells differentiate to fulfill their main function of transporting an intact paternal genome into the egg. This genome must fit into a cell that is morphologically optimized for the specific requirements of sexual reproduction in every species10. Actin filaments and microtubules play a critical role in this remodeling process10. The sperm head is at least in part shaped by a stack of F-actin hoops anchored at the acrosome-nucleus junction  and a transient microtubule-actin basket-like structure known as the manchette, that encircles the nucleus. These structures are thought to act as scaffolding fibers contributing to constrained spermatogenic cell elongation by contractile forces. Additionally, as the manchette forms, nuclear pores are redistributed caudally and are no longer found in fully mature spermatozoa11. Concomitantly, the importance of multiple sperm-specific lamin, lamin-associated and LINC (linker of nucleoskeleton and cytoskeleton) proteins for sperm nuclear apico-basal polarization, shaping and compaction is well-documented13.Differentiating, post-meiotic male germ cells (spermatids) undergo re-shaping accompanied by removal of excess cytoplasm and formation of sperm-specific structures typically including the apical acrosome and the basally anchored sperm flagellum16. In most eukaryotic cells, chromatin is organized as a string of nucleosomes, composed of ~146 bp of DNA wrapped around an octamer of histone proteins. Histone proteins can further undergo post-translational modifications that alter their properties and enable their interaction with effector complexes17. While nucleosomes are essential to organize chromatin and modulate its functions in somatic nuclei, the vast majority of histones are removed from chromatin during sperm differentiation and replaced by Sperm-specific Nuclear Basic Proteins (SNBPs) in many animal species, including mammals and insects16. In mouse and Drosophila, histones first become broadly acetylated20, a modification that entails changes in the biophysical properties of the chromatin, favoring DNA accessibility and increasing short-range rigidity21. Acetylated histones are further recognized by testis-specific bromodomain proteins (BRDTs)23. Via BRDTs, acetylated histones are evicted and replaced on DNA by Transition Proteins (TPs) and Protamines in mammals or Transition protein-like (TPL) and Protamine-like proteins (PLs) in Drosophila. These SNBPs are highly basic, fast-evolving small proteins25. In addition, Protamines and PLs usually feature multiple cysteine residues, which can form intra- and/or inter-molecular covalent disulfide bonds that participate in Protamines and PLs stability on DNA28. Disulfide bond reduction at fertilization is thus required for paternal chromatin decompaction, PL removal, in preparation of nucleosome assembly and paternal genome incorporation into the first zygotic nucleus29. In vitro, mammalian Protamines acting as multivalent cations can organize DNA into ~150nm-wide tori32. Extracted chromatin from vertebrate sperm confirmed the presence of torus- as well as rod-shaped structures33. Yet, the actual in situ configuration of the genome in mature sperm is notoriously difficult to access, in large part due to the extreme density and insolubility of this substrate.In contrast, the link between sperm nuclear morphology establishment and genome tridimensional reorganization has remained unclear. At the chromatin level, spermiogenesis in many animals involves widespread remodeling34. In insects, although sperm chromatin is generally diffuse in spermatocytes (meiotic germ cells) and highly compact in mature sperm, genome organization in intermediate spermatid stages can widely vary between species35. A frequently observed feature is the progressive thickening of chromatin fibers, sometimes further organizing into lamellae, which have been proposed to arise from liquid-liquid phase separation 35. Patterned chromatin condensation has notably been observed during spermiogenesis in several orthopteran insects including the grasshopper Chortophaga viridifasciata36, the cave cricket Ceutophilus nigricans37 or the bush katydid Scudderia38. As a striking case in point, pioneering studies on the two-spotted cricket Gryllus bimaculatus revealed a highly regular configuration of DNA in maturing sperm, consisting of ~30nm-thick chromatin fibers that were bundled, stretched, and coiled along the needle-shaped nucleus axis39. This remarkable organization is arguably among the simplest solutions to higher-order genome folding in any cell type or species, thus providing a unique opportunity to dissect the mechanisms underlying large-scale remodeling of nuclear architecture.Important insights into this issue came from electron microscopy studies of insect spermatogenesis carried out over the last seventy years, which revealed an outstanding diversity in the large-scale re-configuration of the nuclear contentG. bimaculatus male germ cells. By exploiting confocal, electron and single-molecule localization microscopy, we identify the successive chromatin structural and architectural transitions that accompany spermiogenesis. To understand their mechanistic, physical bases, we establish a simple polymer modeling approach to simulate genome dynamics in the restricted sperm nuclear space. By iteratively confronting simulations and observations, we reveal that chromatin spooling can be explained by a nucleated, gradual increase in chromatin rigidity during the histone-to-protamine transition. We further uncover that the alignment of spooled fibers along the nuclear axis does not precede, but rather follows antero-posterior cell polarization and elongation, indicating that chromatin configuration may adapt to cell shape in a liquid crystal-like fashion. Finally, we provide evidence that spooled fibers twist around the nucleus axis during terminal differentiation to achieve an ultracompact configuration. Our results demonstrate how a relatively simple set of changes in the biophysical properties of the chromatin fiber may lead to radical tridimensional reorganization of the genome that allows its fitting into the sperm nucleus.Here, we investigate the underlying principles of such drastic spatial rearrangements of the genome in G. bimaculatus, i.e. the differentiation of haploid post-meiotic germ cells. Based on nuclear morphology, by analogy to well-characterized spermiogenesis stages in Drosophila14, we identified multiple steps in germ cell proliferation and differentiation including spermatocytes, round spermatids, canoe (elongating) spermatids, needle-shaped spermatids .To understand the mechanical underpinnings of this striking transition, we developed a simple polymer model considering the specific features of sperm chromatin (see “Methods” section). We modeled chromatin as a polymer consisting of identical 30 nm monomers, whose kinetics are controlled by the interplay between thermal motion, steric repulsion, and bending rigidity. This long chain was confined within a flexible and discrete nuclear envelope, modeled as a polymerized elastic shell. Positively charged SNBPs are expected to result in a higher stiffness of sperm chromatin compared to that of nucleosomal chromatinFirst, we simulated the re-organization of a polymer upon a progressive, uniform increase in bending rigidity, starting from an isotropic, disordered conformation of an initially flexible chain. In these conditions, multiple segments of the polymer progressively coiled in different directions Fig. , left anTo understand how this inhomogeneously oriented coiled polymer could fill the elongating nucleus during spermiogenesis, we imposed an arbitrary stretching of the envelope through an external radial force applied at two opposite poles Fig. , right. cis along the fiber (see “Methods” section). In particular, we simulated the dynamical reorganization of chromatin when the local bending rigidity was first increased in a progressive fashion at a limited number of nucleation sites, initially located near the center of the nucleus . H4ac accompanies and is required for the histone-to-protamine transition in other species, including ern Fig. A, 4. In 45. Such liquid crystal phases are commonly observed in dense solutions of semiflexible polymers46, and typically arise from the local tendency of stiff polymer segments to spontaneously align along a common axis to optimize packing. In our case, the presence of a spherical envelope interferes with this effect, preventing the establishment of a unique, uniform direction of fiber alignment throughout the entire nuclear interior. Thus, our cylindrically-symmetric spool may be generically explained in terms of the interplay between spherical confinement and orientational, liquid-crystalline order44.The strong local alignment of the spool-like folded arrangement that arises from this rigidification process Fig.  does notThe nucleation model results in a spool that features a distinct axis imposed by the orientation of the spooled chromatin fiber. We thus wondered whether the definition of the antero-posterior axis of the cell preceded or rather was determined by chromatin orientation. Since the flagellum is a clear marker of cell posteriority, we performed immunofluorescent staining on α-Tubulin and observed that flagellar microtubules start forming in early round spermatids Fig. , indicatTo test whether chromatin orientation adapts to cell elongation, we similarly introduced an external stretching force acting on the spherical envelope encapsulating our spooled, stiff polymer Figs.  and 5C. ent Fig. , to \docids Fig. . Transveids Fig. A and 5D.ids Fig. . Togethe47 that readily labeled DNA in situ in whole mount testes. We performed single-molecule localization microscopy (SMLM) detecting this dye and reconstructed high-resolution images of spermatid nuclei  fluorophore coupled to the DNA dye Hoechst33382lei Fig. . At advalei Fig. . We furtlei Fig. . Interes39. At the flagellum-nucleus junction, we noticed a structure reminiscent of a cytoskeletal mesh in physical contact with chromatin  causes reduction of disulfide bonds between Protamines, their eviction from chromatin, and a rapid decompaction of the needle-shaped nucleus29. In our simulations, the unconstrained needle-shaped polymer as a whole shortened through lateral swelling and curling  in sperm55. Accordingly, NUT or Gcn5 mutants display partial failure in histone-protamine replacement, together with aberrant sperm compaction and shaping. In crickets, we further show a histone acetylation pattern consistent with nucleation and spreading of this modification. The spreading of histone modifications has been well described for repressive heterochromatin methylation marks such as lysine 9 and lysine 27 on histone H3. In mice and Drosophila, enzymatic complexes can both establish and recognize the methylation marks, therefore feeding a spreading mechanism on neighboring nucleosomes. These modifications alter the mechanical properties of chromatin and attract additional complexes, which favors the formation of nuclear sub-compartments60 where these processes are further catalyzed61. Histone acetylation can analogously target DNA regulatory elements (notably enhancers and promoters) via DNA sequence-specific transcription factors recruiting HATs and bromodomain proteins63. It was further proposed that a feedforward loop involving NUT, p300, and BRDT helps spreading histone acetylation in mouse sperm23. The targeting mechanisms for HATs acting on mouse or Drosophila sperm is unknown but the rather homogeneous nuclear distribution of acetylation favors a model in which histones are randomly and synchronously acetylated as a nucleus-wide wave, with the notable exception of genomic regions resisting the histone-protamine transition20. Yet, these mechanisms must differ in cricket sperm, where early acetylation displays a sharp spatially defined sub-nuclear pattern. The molecular bases for such nucleation of acetylation are unknown but may be key to account for the strikingly different genome reorganization in crickets compared to mice and Drosophila. Identification of the enzymes responsible for acetylation in crickets, as well as the genomic distribution of histone acetylation and HAT binding during spermiogenesis is needed to further explore these questions.Illustrating the importance of these mechanisms, failure in histone hyper-acetylation correlates with infertility in mice and menDrosophila sperm64, which features a homogeneous histone hyperacetylation distribution. In mice, Hi-C maps of DNA-DNA contacts have yielded contrasting results, but usually revealed relatively modest differences in sperm cells compared to somatic cells67. These surprising observations imply that the massive histone-SNBP replacement does not cause large-scale genome 3D rearrangements in this species. While these studies need further confirmation, developments of our modeling framework could help identify how histone acetylation and replacement patterns may result in this intriguing preservation of genome organization, which must clearly not occur in crickets.The relative simplicity of the biophysical forces we uncover makes it all the more intriguing that, to our knowledge, this specific chromatin configuration has not been reported in any other cell type or species thus far. We propose that the precise spatio-temporal patterning of acetylation and histone-SNBP replacement in sperm cells is critical to determine chromatin organization. Notably, no particular higher-order genome organization is observed in electron micrographs from At later stages of spermiogenesis, the twisting of the chromatin fiber spools may contribute to achieve maximum compaction. While we could not unequivocally identify its mechanistic bases, we suggest that unidirectional rotational movements of the flagellum may be involved. A limited number of turns may be enough to transmit sufficient rotational forces onto chromatin and the nucleus as a whole to explain the observed deformations. This finding would imply a nuclear structuring role for the flagellum, in addition to its motility and sensory roles. Whether flagellar components interact with chromatin and how they could exert such mechanical forces would deserve further experimental interrogation. Importantly, our results also suggest that additional processes may be required towards the final steps of spermiogenesis in order for sperm cells to acquire a robust needle-like morphology despite this persisting torque. While the cricket SNBPs remain to be identified, we speculate that the effect of DTT in mature sperm may be due to reduction of disulfide bonding among these proteins. Future work should thus aim at identifying whether cricket SNBPs bear cysteine residues and whether their intermolecular bonds might serve to stabilize such a nuclear configuration, so that flagellar movement may drive cellular motion without altering cell shape.45. Previous theoretical works have already suggested that the coupling between polymer stiffness and confinement may lead to liquid-crystal-like folding patterns71. For example, a configuration known as the condensed Hopf fibration, similar to our spooled chromatin arrangement  state, characterized by the conjunction of local orientational order with a dynamic, liquid-like mode of molecular motionent Fig. , has beeThe unique combination of order and fluidity provided by this original packing mechanism may in our case play a central functional role in the maturation of cricket sperm cells. Indeed, our results suggest that the strong orientational alignment of the chromatin fiber, associated with its ability to dynamically reorganize following changes in nuclear morphology and the application of torsional forces, may be key to achieve maximal levels of compaction and elongation in mature sperm nuclei. Hence, our findings would support a model in which irreversible intermolecular interactions, potentially induced by disulfide protamine bonds, should be established at the very final stage of spermiogenesis, in order to “freeze” the resulting liquid-crystalline chromatin assembly into a crosslinked state resilient to external perturbations.72. These so-called cholesteric liquid crystal phases are commonly observed in the mature sperm heads of many vertebrates73, and have been more broadly proposed to fulfill a variety of biological purposes and functions74. Such cholesteric interactions could in our case serve to further stabilize the twisted fiber configuration uncovered in mature spermatids, and thus contribute to the establishment and maintenance of optimal packing and elongation in the cricket sperm. Interestingly, similar cholesteric arrangements have also been reported in dinoflagellate chromosomes75, which are characterized by unusually-large genome sizes, and feature a near-total absence of histones and histone-like proteins76. Therefore, our results would more generally suggest the combination of histone replacement and liquid-crystalline organization as a convergent evolutionary process for the ultradense packaging of chromatin within highly-confined nuclear environments.In this context, the potential influence of the detailed fiber structure on the observed chromatin arrangements would warrant further investigations. For instance, the chiral symmetry of many liquid-crystal-forming biopolymers such as DNA is generally known to promote twisted, non-uniform patterns of orientational order, which may in turn give rise to a higher level of hierarchical organizationGryllus bimaculatus white-eyed mutant strain was a gift from C. Extavour and T. Mito. Crickets were reared at constant 29 °C under a 12-h light–dark photoperiod, fed ad-libitum with powdered mouse food, quenched with tap water, and allowed to lay eggs in soaked tissue paper. For imaging experiments and protein extraction, pairs of testes were dissected from sexually active adult males, while mature sperm was isolated from adult female spermatheca. Animals were anesthetized with CO2 prior to dissection.The 77.For immunofluorescence staining, testes were dissected in PBS 0.1%, Triton X-100 (PBS-T) and fixed at room temperature in 4% formaldehyde in PBS for 30 minutes. Testes were washed three times (10 min each) with PBS-T and incubated with anti-H4ac , anti-Histones  or anti-αTubulin  primary antibodies in the same buffer on a wheel overnight at 4 °C. They were then washed three times (10 min each) with PBS-T and secondary antibody incubations using DyLight 550 conjugated goat anti-mouse IgG  and DyLight 488 conjugated goat anti-rabbit IgG  were performed identically. Testes and sperm samples were mounted in DAKO mounting medium containing DAPI (2 µg/ml). Images were acquired on an LSM 800 confocal microscope (Carl Zeiss). Images were processed with Zen imaging software (Carl Zeiss) and FIJI software78 Fiji plugin.Fixed testes or sperm samples were mounted in PBS medium containing 100 nM Hydroxymethyl Silicon-Rhodamine-Hoechst probe We acquired the streams using MetaMorph software (Molecular devices) on a home-built setup consisting in an IX81-ZDC2 microscope equipped with a temperature-controlled incubation cage kept at 27 °C. The intensity and wavelengths of the illumination were controlled through Labview to pilot an acousto-optical tunable filter . Wide-field illumination was achieved by focusing the laser beams to the back focal plane of a 100x 1.49 numerical aperture (NA) oil-immersion apochromatic objective lens (Olympus). Data collection was obtained using an Evolve EMCCD camera (Photometrics) with continuous 642 nm light illumination, at high power. Images were reconstructed using the ThuderSTORMTestes were dissected in water and fixed for 20 h at 4 °C in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate pH 7.35. After four washings of 12 h at 4 °C in 0.15 M sodium cacodylate, samples were postfixed 1 h at RT in 1% OsO4 / 0.15 M sodium cacodylate. They were dehydrated in ethanol solutions (30°/50°/70°/80°/95°/100°) for 30 min each, and in propylene oxide 2 times 15 min. After 6 baths of substitution and 3 baths of impregnation, samples were embedded in epoxy resin in flat silicon molds and polymerized at 56 °C for 48 h. Ultrathin sections were cut with a UC7 Leica ultramicrotome. Ultrathin sections were contrasted in uranyl-acetate and lead citrate solutions. Sections were observed with a Philips CM120 transmission electron microscope at 120Kv .Testes or sperm samples were homogenized by roto-douncing in 5% SDS, 50 mM TEAB, 50 mM DTT. The homogenates were incubated overnight at 37 °C, pelleted and clear lysates were collected and quantified. 1 or 2 µg of total protein were separated in 4–12% acrylamide gels, transferred to PVDF membranes and stained with Pierce Reversible Protein Stain Kit for total protein visualization. Stain was washed away, membranes were blocked in 5% non-fat milk and stained with an anti-H3 antibody  in TBS-T with 1% non-fat milk. Membranes were washed in TBS-T, stained with HRP-coupled secondary antibody  for 2 h at room temperature, and washed again with TBS-T. Proteins were revealed by chemiluminescence using Pierce SuperSignal West Pico substrate and imaged in a ChemiDoc imaging system.Sperm samples were isolated from female spermatheca, spread in PBS onto microscopy slides. Excess liquid was removed and sperm was mounted in PBS-Triton 0.3% containing 5 µg/ml of Hoechst with 25 mM DTT for immediate visualization.Each electron micrographs shown is a representative example from at least 10 similar observations from 3 independent experiments. Confocal microscopy images were chosen as representative from at least 10 similar observations in at least 2 independent experiments. STORM images shown are representative of an ensemble of >20 images collected from 4 independent experiments. Western blots were performed 3 independent times with similar results.4479, and was kept fixed throughout the simulations to mimic the static cross-links of the nuclear envelope lamina network80. Self-avoidance was achieved through the use of a truncated and shifted Lennard-Jones potential with exclusion radius The nuclear envelope was described as a cross-linked biopolymeric shell consisting of a fixed number 81.The chromatin fiber was modeled as a single linear polymer comprised of mentclass2pt{minimf ~ 10% of the total chromatin content, as estimated by electron microscopy imaging of canoe-stage spermatids (see main text). Accordingly, the initial envelope radius was set to The effective diameter of the chromatin fiber was set to 83. A standard Langevin thermostat was used to thermalize the system at 84. The model unit of energy was set to 85. A vanishing bending rigidity Molecular dynamics (MD) simulations were run on multiple graphics processing units via the HOOMD-blue software packageThe chromatin chain was initialized as a dense, self-avoiding random walk encapsulated within the bounds of the initial spherical envelope conformation. Relaxation runs of age Fig. . \documecis-spreading of acetylation marks along the chain. The above protocol was then repeated over In the case of nucleated stiffening simulations, all loci located within a distance entclass1pt{minima85, over To simulate the directional elongation of the nuclear envelope, two antipodal vertices were identified along an arbitrary stretching axis To model the effects of geometric twist on spermatid morphology, a uniform torsional field of axis ids Fig. . The systin Fig. . For dec44r  Q-tensor,Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Trypanosoma brucei, pulse labelling of nascent RNAs with 5-ethynyl uridine (5-EU) was performed with cells depleted of TbMex67 and complemented with a dominant-negative mutant (TbMex67-DN). RNA polymerase (Pol) II transcription was unaffected, but the procyclin loci, which encode mRNAs transcribed by Pol I from internal sites on chromosomes 6 and 10, showed increased levels of 5-EU incorporation. This was due to Pol I readthrough transcription, which proceeded beyond the procyclin and procyclin-associated genes up to the Pol II transcription start site on the opposite strand. Complementation by TbMex67-DN also increased Pol I-dependent formation of R-loops and γ-histone 2A foci. The DN mutant exhibited reduced nuclear localisation and binding to chromatin compared to wild-type TbMex67. Together with its interaction with chromatin remodelling factor TbRRM1 and Pol II, and transcription-dependent association of Pol II with nucleoporins, our findings support a role for TbMex67 in connecting transcription and export in T. brucei. In addition, TbMex67 stalls readthrough by Pol I in specific contexts, thereby limiting R-loop formation and replication stress.TbMex67 is the major mRNA export factor known to date in trypanosomes, forming part of the docking platform within the nuclear pore. To explore its role in co-transcriptional mRNA export, recently reported in  Trypanosoma brucei is an early branching unicellular eukaryote and the parasite responsible for human and animal trypanosomiasis in sub-Saharan Africa. This pathogen goes through a very complex life cycle between its insect (tsetse fly) and mammalian hosts, accompanied by unusual features in terms of gene expression. With few exceptions, protein-coding genes are organised in polycistronic transcription units (PTUs) preceded by a single transcription start site (art site . Polycisart site . Steady art site . In contart site . These eart site .T. brucei are transcribed by RNA polymerase II (Pol II), but some developmentally regulated genes are transcribed by RNA polymerase I (Pol I). This is the case for the procyclins and variant surface glycoproteins (VSG), the surface proteins expressed by procyclic forms and bloodstream forms in their insect and mammalian hosts, respectively (Most protein-coding genes in ectively . Althougectively ). The acectively . This loectively  and is aectively ,24. By cectively ,26. In cectively .T. brucei are unregulated. However, there are specific chromatin marks at the boundaries of the PTUs. Modified histones H3K4me3 and H4K10ac, and histone variants H2AZ and H2BV map to open chromatin at the transcription start sites, while H3V and H4V map to compact chromatin at transcription termination sites   . The nucnscripts . In genenscripts .T. brucei, designated Mex67b (Tb927.11.2340) and Mex67L (Tb927.10.2060) are Mex67 paralogs, based on their interaction with TbMtr2 and the presence of LRR and NTF2-like domains (https://www.biorxiv.org/content/10.1101/2022.06.27.497849v1). The genes encoding them were too divergent to be identified as relatives of TbMex67 by BLASTN searches. Mex67L was described as having a non-canonical function as RNAi did not cause retention of polyA RNA in the nucleus. RNAi against Mex67b did cause polyA retention, but had no effect on growth of procyclic forms, suggesting that it might affect a non-essential class of RNAs. This contrasts with depletion of TbMex67 which results in both polyA RNA retention and a growth phenotype -seq revealed that this was the result of Pol I readthrough and concomitant nucleosome depletion. Furthermore, both Pol I subnuclear localisation and nucleolar organisation were affected. Together, these phenomena suggested a role for TbMex67 in connecting the transcription and export machineries. Consistent with this, we verified that TbMex67 binds chromatin at actively transcribed genes, assisted by its zinc finger motif, and that the truncated form loses its chromatin-binding properties and nuclear localisation.T. brucei brucei Lister 29–13 , cul, cul46))6 ml−1 were pulsed with 200 μm 5-ethynyl uridine (5-EU) for 10 min. Nuclear RNA isolation and copper-dependent click-it chemistry were performed as described (Procyclic forms at a density of 5 × 10escribed . cDNA li−1 tRNA). Cy3-labelled oligo-d(T)20 (2 ng μl−1 in hybridisation solution) was incubated overnight at room temperature. Cells were then washed with decreasing concentrations of SSC (from 4 × to 1×), and nuclei were stained with Hoechst 33342. Images were captured with a Leica DM 5500 B microscope and analysed using LAS AF software (Leica) and Fiji.RNA FISH was performed according to Cassola and colleagues : cells w4Cl and permeabilised with Triton X-100 (0.1% in PBS). After blocking in PBS/3% BSA, cells were incubated with primary antibody (1:100 for anti-Pol I (https://cellprofiler.org). All antibodies employed in this study are listed in For immunofluorescence, cells were harvested by centrifugation, washed once in PBS, fixed in 4% paraformaldehyde in PBS followed by 10 min incubation with 50 mM NHti-Pol I , L1C6  in PBS for 8 min at room temperature to cross‐link protein complexes. The reaction was stopped by adding glycine to a final concentration of 125 mM and incubating for 5 min. Cells were washed in PBS and resuspended in lysis buffer  containing protease inhibitor cocktail . Cells were lysed by sonication using a Branson Digital Sonifier three times 10 s, 30 s interval on ice at 10% power. Two percent of the soluble supernatant was reserved as the input fraction and the remainder was used for immunoprecipitation using anti-HA magnetic beads (Pierce™) or IgG-coated Sepharose beads (Roche). In all cases, an untagged culture was used in parallel as a negative control. Protein cross-linking was reversed by incubation of the sample in Laemmli Buffer for 20 min at 95°C. Immunoprecipitated proteins were identified by western blot analysis.HA-RRM1 and TbMex67-PTP protein complexes were isolated as previously described ,51. Brie6 cell equivalents per lane) were separated on 12% SDS-polyacrylamide gels and transferred to Immobilon-P membrane (Millipore). Membranes were incubated overnight with primary antibodies: anti-TbMex67 , anti-RPB1 , anti-Pol I , anti-histone H3  anti-HA 3F10 , anti-RRM1  and anti-EIF1α . After washing, membranes were incubated with IRDye® 800CW or 680RD (LI-COR Biosciences) secondary antibodies and detected using an Odyssey imaging system (LI-COR Biosciences). All antibodies used in this study are listed in For Western blot analysis, protein samples  by a 2-hour incubation with antibody diluted in lysis buffer see . Eluates8 parasites were washed once in PBS and fixed with 1% paraformaldehyde (w/v) in PBS for 8 min at room temperature; the reaction was stopped by adding glycine to a final concentration of 125 mM for 5 min. Cells were then washed once in PBS and resuspended in lysis buffer ). Subsequently, the lysate was sonicated with a Bioruptor (Diagenode) using the following settings: Power ‘L’, ON/OFF (Sec) 30/90 for 7 min, to produce fragments <1 kb. The soluble chromatin was diluted 1:100 in RIPA buffer  and used for immunoprecipitation. Anti-histone H3 , anti-RPA1  and anti-RPB1  antibodies immobilized on Dynabeads (Thermo Fisher) were used for immunoprecipitation (ChIP experiments were performed as described previously  with minpitation . TbMex67pitation . IPs werpitation . DNA wasT. b. brucei TREU927 genome downloaded from TritrypDB (http://tritrypdb.org/tritrypdb/). The Bowtie2 tool with default parameters was launched in single-end mode with respect to the replicate sequencing libraries. The output was sorted and indexed using SAMtools. Peak calling was performed using MACS2 software using the replicates for each run. Reads from duplicate controls (DNA extracted from the same chromatin samples used for ChIP) were used as input.Illumina reads were mapped and analysed using the Galaxy platform, as described ,53,54. Chttp://www.ebi.ac.uk/ena as study PRJEB57072.Pairwise Spearman correlations were computed using the R package ‘ggplot2’. To generate the profiles heatmaps, alignment coverage was normalised to input coverage using deepTools with the option bamCompare. The normalised alignment as well as the peak enriched intervals (narrow peaks outputs) were used as input files computeMatrix and plotHeatmap. Raw read files are deposited at the European Nucleotide Archives (ENA) ® 800CW or 680RD (LI-COR Biosciences) secondary antibodies; protease inhibitor cocktail ; Stratalinker UV Crosslinker (Stratagene), Bioruptor (Diagenode); Qubit dsDNA HS Assay kit (Invitrogen); Odyssey imaging system (LI-COR Biosciences). Secondary antibodies (Thermo Fisher).Phusion polymerase (NEB M0530S); Bam HI (NEB R0136S), Dpn I (NEB R0176S) and Hind III (NEB R0104S); Ampicilin (Sigma-Aldrich), Blasticidin (Sigma-Aldrich), Nourseothricin (Jena Biosciences), Puromycin (Sigma-Aldrich); Immobilon-P membrane (Millipore); Dynabeads (Thermo Fisher); anti-histone H3 (Abcam ab1791); S9.6 (Kerafast); anti-HA 3F10 (Sigma-Aldrich); IRDyeT. brucei brucei Lister 427 29.13, E. coli TG1 competent cells, pLEW100 and pLEW101 vectors .https://cellprofiler.org/) after developing a suitable pipeline and statistical analysis with Mann-Whitney U-test by Prism v.6 (https://www.graphpad.com/scientific-software/prism/).Imaging quantification was performed with Cell profiler (T. b. brucei TREU927 genome (http://tritrypdb.org/tritrypdb/). LeishGEdit (http://www.leishgedit.net/). Galaxy platform . Cell profiler (https://cellprofiler.org/). Prism v.6 (https://www.graphpad.com/scientific-software/prism/).TritrypDB, for et al. (2013). Upon addition of tetracycline (Tet), this cell line down-regulates TbMex67 by RNAi and overexpresses a dominant negative form lacking the N-terminal CCCH zinc finger domain. Growth curves showed that Tet-induced and -uninduced cultures grew at the same rate for the first two days  or a hemagglutinin (HA)-tagged version of this protein . In bothSince these data suggested that chromatin remodeling was occurring, we also performed histone H3 ChIP-seq with the TbMex67 RNAi/DN-OE cell line. In trypanosomes, nucleosomes are known to be partially depleted from regions actively transcribed by Pol I such as the procyclin and ribosomal loci ,56. ConsPol I protein levels were also not affected when uninduced and induced TbMex67 RNAi/DN-OE were compared Figure . HoweverTo explain the stronger impact of TbMex67 RNAi/DN-OE in comparison to the knockdown alone on transcription, RNA export and cell growth, we took a closer look at the protein's modular architecture Figure . It tranThese results, together with the impact on nascent transcription, stimulated us to evaluate the subcellular localisation of TbMex67 in the presence of the transcriptional inhibitor actinomycin D, which binds DNA and prevents initiation and elongation . We founTo analyse TbMex67 proximity to chromatin, we performed ChIP-seq with anti-TbMex67 antiserum on uninduced and induced TbMex67 RNAi/DN-OE cells. In uninduced cells, the endogenous protein showed a pattern of occupancy that was reduced at the beginning and end of each gene, known sites of pre-mRNA processing and nucleosome depletion Figure . ConsistWhen browsing genomic regions transcribed by the different polymerases, TbMex67 ChIP-seq showed occupancy in the procyclin and rDNA loci as well as actively transcribed Pol II genes, such as tubulins and histones, and Pol III genes such as tRNAs Figure . OccupanAs shown above, expression of the DN isoform of TbMex67 led to histone depletion downstream of the procyclin loci. When looking at the general distribution of TbMex67 across the genome, we observed a similar pattern to occupancy by H3 and TbRRM1, a modulator of chromatin structure Figure . To inve−1alpha-amanitin (a concentration which inhibits Pol II and Pol III). This suggests that the S9.6 foci observed by microscopy are mostly products of Pol I transcription. Immunoprecipitation with S9.6 followed by high throughput sequencing (DRIP-Seq) confirmed the R-loop distribution already reported for bloodstream form trypanosomes  that could interfere with Pol II elongation and eventually cause DNA damage and genome instability ,61. Furtanosomes  at sitesanosomes . Enrichmanosomes .Taking these results into account, we monitored the presence of R-loops by S9.6 staining following induction of TbMex67RNAi/DN-OE. S9.6-positive foci increased in intensity and partially co-localised with γH2A foci Figure . The latTrypanosoma brucei, we sequenced nascent transcripts from cells in which the general RNA export factor TbMex67 was depleted by RNAi. Even though we did not find many differences between induced and uninduced cells, two regions on chromosomes 6 and 10 showed striking increases in reads downstream of the procyclin genes when TbMex67 was depleted and complemented with a dominant-negative isoform lacking the N-terminal domain. Labelling of nascent transcripts and ChIP-seq experiments revealed that this was the result of Pol I readthrough into the adjacent Pol II PTU, coincident with diminished H3 occupancy in the induced cells. One consequence of this extensive overlapping transcription might be to trigger RNAi and degradation of the mRNAs from the Pol II PTUs. This did not occur, however, suggesting that the transcripts from both strands either failed to form double-stranded RNA or were not accessible to the RNAi machinery.To analyse the relevance of co-transcriptional export in To the best of our knowledge, the procyclin loci are unique in being chromosome-internal protein coding genes that are transcribed by RNA Pol I and this may have required trypanosomes to develop specialised processes to regulate them. Although the mechanism by which TbMex67 normally prevents readthrough remains unknown, we showed that it is bound to DNA at the procyclin loci and that this binding decreases in the presence of the dominant-negative isoform. Our laboratory has previously shown that the 5′ UTR of PAG1 contains a region that impedes Pol I transcription, and that deleting it allows transcription to extend further downstream . This suThe dominant-negative mutant lacks the CCCH domain, which is only present in trypanosomatid Mex67 and has been proposed to fulfil tasks that are performed by additional adaptor proteins in other eukaryotes, particularly given that TbMex67 lacks an RRM domain. We have shown that the CCCH domain is important for nuclear localisation of TbMex67 and its recruitment to chromatin, as analysed by ChIP. However, it is also possible that this domain binds RNA, which could account for TbMex67’s proximity to transcription sites. Alternatively, this role could be fulfilled through its interaction with TbRRM1, which is also known to bind RNA , or otheThe finding that readthrough does not occur from the rDNA loci implies that, in contrast to the procyclin transcription units, these are insulated by factors that are independent of TbMex67. As far as Pol II transcription is concerned, meta-analyses of nascent transcription and Pol II occupancy of divergent and head-to-tail transcription initiation sites did not show major changes in initiation or termination upon depletion of TbMex67 and overexpression of the dominant negative mutant. This suggests that TbMex67 is also not required to insulate transcription between Pol II PTUs, and that its main or sole function in this context is to facilitate co-transcriptional export. The proximity of Pol II to the nuclear pore is confirmed by our finding that its largest subunit is strongly associated with nucleoporins, and that blocking transcription disrupts this association. It is also consistent with data showing that TbMex67 and TbNup158 not only interact with each other  and withet al. showed yeast Mex67 interacted with Pol II upon heat stress, probably aided by the transcription factor HSF1  and that, at sites of pre-mRNA processing, the whole newly assembled ribonucleoprotein (RNP) complex is targeted to and through the nuclear pore. TbMex67’s interactions with nucleoporins and Ran proteins support this hypothesis . Similartor HSF1 . Our stutor HSF1 . Also litor HSF1 ,70.All the aforementioned interactions place TbMex67 at multiple layers of RNA biogenesis and lead us to speculate that it could also aid genome integrity, as was reported for other export factors in mammalian cells . Indeed,In summary Figure , our reshttp://www.ebi.ac.uk/ena as study PRJEB57072. Proteomics data have been deposited in the Proteomics Identification Database (PRIDE), under accession code PXD039878.European Nucleotide Archives (ENA) gkad251_Supplemental_FilesClick here for additional data file.


Plasmodium falciparum, which catalyzes symmetric dimethylation of histone H3 at R2 (H3R2me2s) and R8, and histone H4 at R3 in vitro. PfPRMT5 disruption results in asexual stage growth defects primarily due to lower invasion efficiency of the merozoites. Transcriptomic analysis reveals down-regulation of many transcripts related to invasion upon PfPRMT5 disruption, in agreement with H3R2me2s being an active chromatin mark. Genome-wide chromatin profiling detects extensive H3R2me2s marking of genes of different cellular processes, including invasion-related genes in wildtype parasites and PfPRMT5 disruption leads to the depletion of H3R2me2s. Interactome studies identify the association of PfPRMT5 with invasion-related transcriptional regulators such as AP2-I, BDP1, and GCN5. Furthermore, PfPRMT5 is associated with the RNA splicing machinery, and PfPRMT5 disruption caused substantial anomalies in RNA splicing events, including those for invasion-related genes. In summary, PfPRMT5 is critical for regulating parasite invasion and RNA splicing in this early-branching eukaryote.Protein arginine methyltransferases (PRMTs) regulate many important cellular processes, such as transcription and RNA processing in model organisms but their functions in human malaria parasites are not elucidated. Here, we characterize PfPRMT5 in  Plasmodium falciparum.PfPRMT5, symmetrically dimethylating histones H3 and H4 in vitro, and responsible for dimethylation of histone H3 at R2 (H3R2me2s) in vivo, is necessary for the regulation of parasite invasion and RNA splicing in  Plasmodium falciparum is a unicellular parasite that causes the most virulent human malaria, responsible for about half a million deaths annually1. In the human host, the parasite’s cyclic multiplication and destruction of the red blood cells (RBCs) are responsible for the clinical manifestations and morbidity of malaria. The intraerythrocytic development cycle (IDC), beginning from the merozoite’s invasion of an RBC and progressing through the morphologically distinct ring, trophozoite, and schizont stages, completes in about 48 h. The IDC is orchestrated by a tightly regulated transcription program dictating waves of stage-specific gene expression3. The transcription program is multi-layered, including specific transcription factors such as the AP2-domain proteins and epigenetic mechanisms5. Epigenetic regulation is accomplished through remodeling the chromatin structure between the active and silent state by mechanisms such as nucleosome positioning, incorporation of variant histones, and reversible modifications of histones. P. falciparum histones carry a myriad of post-translational modifications (PTMs)11, consistent with the large compendium of histone modification enzymes and histone PTM-binding proteins encoded by the parasite genome4. As an example of multifactorial coordination, genes involved in merozoite invasion are regulated by specific AP2 domain-containing transcription factors AP2-I12, PfGCN513, and PfBDP114. Genome-wide profiling of PTMs and functional studies of proteins mediating the deposition, removal, and binding of PTMs provide an ever-improving understanding of epigenetic mechanisms in numerous aspects of Plasmodium biology, laying the necessary foundation for developing “epi-drugs” for malaria chemoprevention.16. In recent years, arginine methylation has attracted increasing attention as a key PTM, mainly due to the discovery of its link to human diseases such as cancer and the recognition of its value in therapeutic development17. A family of protein arginine methyltransferases (PRMTs) catalyzes the deposition of active or repressive histone marks, regulating gene expression19. In addition, they also deposit arginine methylation on non-histone substrates, regulating essential cellular processes such as transcription, cell signaling, mRNA translation, and pre-mRNA splicing20. Because of the availability of two-terminal nitrogens for methylation, arginine can be methylated once (monomethylarginine – MMA) and twice . Depending on the types of modification they catalyze, PRMTs are classified into three subgroups: Type I , Type II , and Type III 21. The human genome encodes nine PRMTs, of which PRMT5 is the predominant Type II methyltransferase. Intriguingly, PRMT5 can be a corepressor and a coactivator, depending on the Arg residues it methylates. PRMT5 deposits the symmetric dimethylation marks on histone H3R8 and the R3 motifs present at H2A and H4, which are associated with transcriptional repression22. Conversely, PRMT5 also symmetrically methylates H3R2, which recruits coactivator complexes and is highly correlated with the active mark H3K4me3 at active promoters24. Moreover, PRMT5 interacts with various partners to specifically methylate non-histone substrates, such as the components in the RNA splicing pathway. Thus, PRMT5 deletion has pleiotropic effects, resulting in defects in RNA splicing, cell differentiation, and development27.Protein PTMs, such as acetylation, methylation, phosphorylation, and ubiquitylation, are central to epigenetics28, the significance of PRMTs in the biology of protozoan parasites has received increasing attention30. The enzymatic activities, substrates, and functions of five Trypanosoma brucei PRMTs, including the Type I PRMTs (TbPRMT1 and 6), Type II PRMT5, and Type III PRMT7, have been investigated, and the knockdown of TbPRMT1 and TbPRMT5 by RNAi resulted in growth defects33. TbPRMT5 was identified as a divergent type II PRMT5 since it exclusively localizes in the cytoplasm and was associated with several proteins involved in a methylation-related function34. Toxoplasma gondii PRMT1 and CARM1 catalyzed histone H4R3 and H3R17 methylation in vitro and attributed to cell cycle regulation and gene activation, respectively36. Three putative PRMTs  have been identified in P. falciparum38, but their functions remain uncharacterized. PfPRMT1 is a Type I PRMT located in the nucleus and cytoplasm, with activities toward histones and non-histone substrates37. PfPRMT5 was found to be associated with the spliceosome core protein PfSmD139, suggesting that PfPRMT5 may regulate splicing in malaria parasites.With the recognition of PRMTs as important therapeutic targets for human diseasesP. falciparum. We provide solid evidence to establish PfPRMT5 as a critical regulator of invasion. We also demonstrate that the PfPRMT5 mediates symmetric dimethylation of histone H3R2 (H3R2me2s), and its genome-wide distribution is consistent with H3R2me2s serving as an active chromatin mark required for efficient transcription of invasion-related genes. In addition, PfPRMT5 disruption caused considerable changes in RNA splicing in the parasite, suggesting the role of PfPRMT5 in regulating splicing.In this study, we characterized the Type II PRMT, PfPRMT5, in P. falciparum genome37, PfPRMT5 (PF3D7_1361000) is an intron-less gene of 2175 bp, encoding a protein of 724 amino acids (aa) with a predicted molecular mass of 85.6 kDa. The conserved catalytic core domain, located in the C-terminus (447–668 aa), bears all signature motifs for SAM-dependent methyltransferases, including the SAM-binding domain , a double-E loop critical for substrate recognition and methylation, and the THW loop , S. pombe (Skb1), and T. brucei (TbPRMT5), respectively  identified in the PfPRMT5 transcript has been identified in transcriptomic studies, which showed a peak level in the early stages of the IDC44. Real-time RT-PCR result was compatible with the findings from these studies, showing peak PfPRMT5 mRNA levels in the ring and early trophozoite stages  identified by RNA-seq45. All four clones from the 3’-RACE analysis detected a single polyadenylation site at 183 bp downstream of the stop codon, the same as determined by amplification-free RNA-seq46. These analyses predicted the PfPRMT5 mRNA to be ~2.5 kb  to determine the subcellular localization of PfPRMT5. Analysis of PfPRMT5 by cell fractionation followed by Western blots with antibodies for the nuclear (H3) and cytoplasmic (PfHLP) compartments detected the presence of PfPRMT5 in both fractions Fig. . In addi22. To determine the enzymatic activity of PfPRMT5 on histones, we purified the endogenous PTP-tagged PfPRMT5 using a tandem affinity purification (TAP) procedure and performed in vitro methylation assays. Although the purified PfPRMT5-PTP did not methylate bovine core histones or recombinant P. falciparum core histones, it displayed methylase activity towards histones in the human mononucleosomes  resulting from two transfection experiments were used to determine the effects of PfPRMT5 disruption on asexual parasite development. In vitro growths of the wild-type (WT) and ΔPfPRMT5 lines were compared over three asexual development cycles. Starting at a 0.1% parasitemia, both ΔPfPRMT5 lines grew significantly more slowly than the WT 3D7 . On day 7, the two ΔPfPRMT5 lines only reached ~3% parasitemia compared to ~11% in WT 3D7 parasites. To determine whether the slower parasite proliferation resulted from an altered developmental pattern during the IDC, we monitored the cell cycle progression of highly synchronized parasites every 2 h by differentiating parasite stages in Giemsa-stained blood smears  and 15.96 ± 0.67 (n = 239) merozoites per segmenter, respectively, which were significantly fewer than 18.01 ± 0.82 (n = 238) merozoites per segmenter produced in 3D7 . In addition, we found that the invasion rate of the merozoites released from the ΔPfPRMT5−1 schizonts was approximately three times lower than that from the WT 3D7 in an in vitro merozoite invasion assay . Altogether, these data indicated that the slower in vitro growth of the ΔPfPRMT5 lines was mostly attributed to the lower numbers of merozoites formed and their reduced invasion rates.To elucidate the function of PfPRMT5 in development, we disrupted the ars Fig. . HoweverPfPRMT5 disruption on histone arginine methylation, we purified parasite histones at the schizont stage and determined methylation at H3R2, H3R8, and H4R3 using methylation-specific antibodies. Compared to the WT parasites, the H3R2me2s level was substantially reduced in the disruptant lines, whereas H3R8me2s and H4R3me2s did not show noticeable changes in abundance, indicating that PfPRMT5 disruption specifically affected symmetric methylation of H3R2 . Results from three biological replicates showed that the overall transcriptomes between the WT 3D7 and ΔPfPRMT5 were similar, with linear correlation ranging from 0.97 to 0.99  and PfPKG56, were among the down-regulated transcripts encoding enzymes at 36 and 46 hpi in ΔPfPRMT5, respectively  associated genes  of peaks were called from three replicates. No real peaks of H3R2me2s were identified in the ΔPfPRMT5 based on the criteria of the presence in at least two of three replicates with at least 50% overlap of the peaks. The complete depletion of H3R2me2s in the ΔPfPRMT5 parasite was observed at the sites of the H3R2me2s peaks in the WT  in the PTP tag. After mapping and peak calling, we identified 334 PfPRMT5 peaks using the same criteria for peak detection , DNA helicase PSH3, heat shock protein 70 (HSP70x), and acyl-CoA binding protein (isoform 2), and PfCDPK1. Among them, PfCDPK1 and DPAP3 were found significantly downregulated after PfPRMT5 disruption in our transcriptomic study , indicating that CUT&Tag-seq of PfPRMT5 could only identify the locations with high PfPRMT5 occupancy, which potentially creates higher H3R2me2s signals in the chromatin. These results are consistent with the published studies which showed that histone modifiers are notoriously difficult to be studied by ChIP-seq because of their highly dynamic nature and relatively weak binding to the chromatin, such as GCN559. Previous studies on PRMT5 and its histone marks in eukaryotic cells were conducted by ChIP-PCR of PRMT5 along with ChIP-seq of its histone marks, suggesting that ChIP-seq of PRMT5 was difficult in those studies61. Recent reports showed two separate ChIP-seq studies identified ~3000 genes whose promoters or gene bodies were enriched with PRMT5 reads in pre-B leukemia and Hela cell lines63. In contrast, ChIP-seq in chicken and pro-B cells revealed several folds more genes enriched with H3R2me2s peaks58. Taken together, there are intrinsic difficulties for PRMT5 ChIP-ing than its histone marks.Compared to 1147 H3R2me2s peaks at 5’ UTR regions of 783 genes, PfPRMT5 peaks at 5’UTRs in 27 of 28 genes, including AMA1, EBA175, and MSP1) at the late schizont stage (active stage) compared to the ring stage (silence stage). The transcription of these genes was reduced (significantly for AMA1 and EBA175) after PfPRMT5 disruption  did not show enrichment in both stages  or two replicates (AMA1 and EBA175) of CUT&Tag-seq, respectively  and 75% (186/247) of the identified proteins from cytosolic and nuclear IPs were consistent with the localization in the cytosol and nucleus, respectively  using the PfPRMT5::PTP parasite line to identify the PfPRMT5 interactomes in both the cytosolic and nuclear fractions. A single-step IP with the IgG Sepharose beads was performed to capture proteins transiently or weakly associated with PfPRMT5. The bound proteins were released by TEV proteinase digestion and identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The same IP procedure was performed with the WT 3D7 as the control, and the IP data were subjected to Significance Analysis of INTeractome using stringent criteria 24. The PfPRMT5 interactomes include several AP2 transcription factors, PfGCN5, PfADA2, and a few WD domain-containing proteins, suggesting the participation of PfPRMT5 in regulating specific genes and chromatin states , and disruption of the ing Fig. , promptiely Fig. . Similare27 Fig. . These c 42 Fig. . InteresMT5 Fig. . Given tPfPRMT5 disruption specifically reduced H3R2me2s and overall sDMA. This activity toward histones H3 and H4 is similar to the mammalian PRMT522, which also reflects the conservation of the N-termini of H3 and H4 between P. falciparum and humans6. In mammalian cells, the dimerization of PRMT5 and association with other components, such as Methylosome protein 50 (MEP50), plCln, kinase RioK1, and Grg4, are required for its catalytic activity and substrate specificity48. Human PRMT5/MEP50 methylated free H3, H4 in the H3/H4 tetramers, H3 in the mono- or oligo-nucleosome from Hela cells, in contrast, it failed to methylate H3 in the recombinant nucleosome47. Human PRMT5/Grg4 methylated both H3 and H4 in the mononucleosome whereas it only methylated H3 in the core histones48. We found that only PfPRMT5 purified from parasites possessed methylase activity, and it required mononucleosomes, not individual histones, as the substrates, suggesting that PfPRMT5’s activity and substrate specificity on histone may also need interactions with partners. In contrast, the TbPRMT5 purified from bacteria and parasites showed efficient methyltransferase activities to free histones34. While our interactome studies identified potential partners of PfPRMT5, further studies are needed to determine the critical partners required for its activity.We comprehensively examined the functions of PfPRMT5 by genetic disruption, transcriptome, interactome, chromatin landscape, and RNA splicing analyses and identified the conserved role of PfPRMT5 in RNA splicing and a parasite-unique role in regulating the invasion process. Consistent with the prediction based on PfPRMT5 sequences, PfPRMT5 displayed type II PRMT activity to symmetrically di-methylate histones H3 at R2 and R8, and H4 at R3 in vitro, whereas 71. In contrast, PRMT5 is also a coactivator since H3R2me2s recruits coactivator complexes and is highly correlated with the active mark H3K4me3 at active promoters72. In our study, although we found that PfPRMT5 could deposit the putative repressive (H3R8me2s and H4R3me2s) and the active (H3R2me2s) histone marks in vitro, we only found evidence of a substantial decrease of the H3R2me2s mark after PfPRMT5 disruption. In agreement, PfPRMT5 disruption predominantly led to the down-regulation of gene expression, suggesting PfPRMT5 mainly serves as a coactivator during the P. falciparum IDC. In comparison, disruption of PRMT5 in model organisms normally causes a higher number of up-regulated transcripts than down-regulated ones27, perhaps due to the de-repression of PRMT5-regulated transcriptions. Furthermore, our analysis of the chromatin landscape by CUT&Tag-seq further confirmed that H3R2me2s is an active mark, overlapping significantly with other euchromatin marks H3K9ac, H3K4me3, and H2A.Z. This active mark is enriched in genes involved in invasion, translation, stress response, cell cycle, cell adhesion, signal transduction, DNA repair, and telomere organization, suggesting PfPRMT5’s participation in transcriptional regulation of a multitude of cellular processes, which deserves further in-depth elucidation.The mammalian PRMT5 has a dual role in depositing both repressive and active histone marks, depending on what complexes PRMT5 resides. H3R8me2s and H4R3me2s are repression marks for transcription in model organisms, and PRMT5 is associated with numerous transcription factors and repressor complexesvar genes suggest their potential involvement in regulating these genes in the subtelomeric regions. Although we noticed that var genes were substantially changed from our microarray, RNA-seq, and CUT&Tag-seq data, these data cannot be used for further in-depth analysis because our parasites are mixed clones expressing different var genes due to long-term culture. To further study the function of PfPRMT5 and H3R2me2s in the regulation of these important genes, additional cloning steps are needed.Although CUT&Tag-seq only revealed that PfPRMT5 occupied a fraction of H3R2me2s regulated genes due to the intrinsic difficulties, our ChIP-qPCR analysis demonstrated the coordinated role of PfPRMT5 and H3R2me2s in regulating invasion-related genes. Higher enrichments of PRMT5 and H3R2mes2 in the 5’ UTRs of certain PfPRMT5 only resulted in the substantial decrease of H3R2me2s but no noticeable changes on H3R8me2s and H4R3me2s are probably because that silence marks (H3R8me2s and H4R3me2s) and active mark (H3R2me2s) are deposited by different PRMTs. Our Western blots showed that silence marks (H3R8me2s and H4R3me2s) consistently appeared during asexual development, in contrast, H3R2me3 only appeared at a high level in the schizont stage, consistent with the expression of PfPRMT5  for gene activation23. In agreement with the above findings, H3R2me2s signals in the ΔPfPRMT5 were completely depleted in the chromatin 77.Unlike human PRMT5 and TbPRMT5, which localize almost exclusively to the cytoplasmtes Fig. . These p70. However, PfPRMT5 may function differently in the splicing process because Plasmodium core spliceosomal Sm proteins, which were associated with PfPRMT5, lack the RG motifs that are normally methylated by mammalian PRMT578, suggesting that PfPRMT5 may methylate Sm protein differently. Probably this different methylation pattern specifies the splicing process to the transcripts related to Plasmodium-specific pathways such as invasion.This study demonstrated that PfPRMT5 is involved in RNA splicing processes like PRMT5 in mammalian cellsP. falciparum. The active chromatin mark H3R2me2s deposited by PfPRMT5 shares extensive overlaps with other euchromatin marks, demonstrating a high level of crosstalk among these epigenetic factors. Its specific regulation of invasion-related genes may be dictated by multiple layers of components including the chromatin structure affecting transcription, specific recruitment by TFs, and transcript stability and translation influenced by RNA splicing. The extensive interactomes of PfPRMT5 imply its involvement in multiple cellular pathways, which awaits future investigations. A better understanding of arginine methylation will enable the design of malaria therapeutics targeting this family of enzymes.Here we provided multiple lines of evidence showing that the type II enzyme PfPRMT5 plays critical roles in regulating general and parasite-specific biology in 79, based on the crystal structure of HsPRMT5 (PDB code 4GQB)80.Four known PRMT5 proteins from Homo sapiens (HsPRMT5/JBP1), Saccharomyces pombe (SpSKB1), Caenorhabditis elegans (CePRMT5), and Trypanosoma brucei (TbPRMT5) were retrieved from GenBank  and aligned with the putative PfPRMT5 (Pf3D7_1361000) sequence by Clustal Omega. The structure of PfPRMT5 was predicted by using the multiple threading alignments in I-TASSERP. falciparum was cultured by the established methods with some modifications81. Synchronization of asexual stages was performed by two rounds of sorbitol treatment at the ring stage. For time-course studies, schizonts were purified by Percoll gradient centrifugation and mixed with fresh RBCs82, and parasites were harvested at 12, 24, 36, and 46 h later to represent ring, early trophozoite, late trophozoite, and schizont stages, respectively.The 3D7 clone of PfPRMT5 mRNA were determined by using the FirstChoice RLM-RACE (RNA ligase-mediated rapid amplification of cDNA ends) kit (Ambion) with PfPRMT5-specific primers 5RACE-R1, 5RACE-R2, 3RACE-F1, and 3RACE-F2 (Supplementary Data 83. PCR products were cloned in TOPO® cloning vector (Invitrogen) and sequenced.Total RNA was isolated from the parasites using Trizol (Invitrogen) and treated with ezDNase enzyme (Invitrogen) to remove contaminating genomic DNA. The 5’ and 3’ UTRs of the PfPRMT5 were studied by quantitative reverse transcriptase-PRC (qRT-PCR) analysis at four stages of the IDC. HotStart-IT SYBR Green One-Step qRT-PCR Master Mix Kit (USB) was used for cDNA synthesis and PCR amplification with primers PfPRMT1-F3 and PfPRMT1-R3 (Supplementary Data seryl-tRNA synthetase gene (PF3D7_0717700) was used as the internal reference. The expression was analyzed as described previously6.The relative transcription levels of -ΔΔCt method with the PF3D7_ 0713400 (an unchanged region) as the internal reference.84.To validate the RNA splicing events, qRT-PCR analysis was performed with eight pairs of primers to measure the levels of transcripts from different splicing events in eight selected genes  1138-2172] was amplified using primers F1 and R1 .To tag the C-terminus of PfPRMT5 with the PTP tag89. The selection was performed with 2.5 nM of WR99210 for approximately four weeks until resistant parasites appeared90. Single clones of parasites were obtained by limiting dilution91. Correct integration of pHD22Y/PfPRMT5-PTP at the PfPRMT5 locus was screened by integration-specific PCR with primers IntF and IntR  at 4 °C for 2 h. The beads were equilibrated with the TEV buffer. 2 ml of TEV buffer and 150 U of TEV protease were added to the beads, and the column was rotated overnight at 4 °C. The eluate was collected and incubated with anti-protein C beads for 2 h at 4 °C. The beads were washed, and binding proteins were eluted with an elution buffer. The final eluate was collected for in vitro methylation assay.TAP of PfPRMT5 was performed using the PfPRMT5::PTP parasite line by established methods92. The digests were analyzed by LC/MS/MS using a Waters NanoAcquity HPLC system interfaced with a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). Peptides were loaded on a trapping column and eluted over a 75 μm analytical column at 350 nL/min. MS and MS/MS were performed at 70,000 FWHM and 17,500 FWHM resolutions, respectively. The fifteen most abundant ions were selected for MS/MS. Parasite proteins were identified by searching the UniProt P. falciparum protein database (v01/2014). Data were filtered at 0.7–1% protein and 0.2% peptide FDR, and at least two unique peptides per protein. Mascot DAT files were parsed into the Scaffold software for validation and filtering to create a non-redundant list per sample.The first affinity purification of TAP was used to capture proteins transiently or weakly associated with PfPRMT5. After incubating the cytosolic or nuclear lysate with the IgG Sepharose beads followed by digestion with TEV protease, the eluate was separated in a 10% Bis-Tris SDS-PAGE gel for 10 min. Proteins in gel were excised, and in-gel digestion was done as described8 schizonts of the PfPRMT5::PTP parasite line. In vitro PfPRMT5 activity was determined using a methylation assay as described37. Briefly, 2 μg of bovine core histones  (Sigma), or purified P. falciparum core histones 94, or human mononucleosome assembled from recombinant human histones without any PTM (EpiCypher #SKU: 16-0009) were incubated at 30 °C for 2 h within 20 μl of methylation assay buffer containing 1 μCi of [3H] S-adenosylmethionine87. The reactions were resolved by 15% SDS/PAGE for fluorography using an auto-radiographic enhancer (Perkin Elmer).PfPRMT5-PTP was purified from 1 × 10P. falciparum histone H3 peptide (250 ng of each peptide) were spotted on a PVDF membrane followed by blocking with 1% casein in Tris-buffered saline (TBS) and incubating with H3R2me2s and H3 antibodies at 1:2000 dilutions. After washing with TBS, the blot was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody diluted at 1:5000 in 1% casein-TBS. Densitometry was used to quantify the degree to which the antibodies detected each peptide. To exclude the cross-reaction of H3R2me2a antibodies to histone H3 peptide with or without H2R2me2s modification, the same dot blots were performed with anti-H3R2me2a.Dot blot was used to verify the specificity of the H3R2me2s antibodies  at each developmental stage were separated by 10% SDS/PAGE and transferred to nitrocellulose membranes. Western blotting was performed using a standard procedure with rabbit anti-protein C antibodies (1 μg/ml) as the primary and HRP-conjugated goat anti-rabbit IgG (diluted 1:3000) as the secondary antibodies. Anti-PfHSP70 antibodies were used as the loading control. The results were visualized with the ECL detection system . The grey values of the bands detected by Western blot were quantified using the ImageJ software.2, 10 mM KCl, 0.5 mM DTT, 0.5 mM EDTA and 1% (v/v) protease-inhibitor cocktail (Roche)] and incubated on ice for 10 min. The parasites were mechanically lysed by 40 strokes in a Dounce homogenizer with a loose pestle and then centrifuged at 700 g for 20 min at 4 °C. The supernatant was centrifuged at 10,000 g for 10 min to obtain the cytoplasmic extract. The pellet was resuspended in three volumes of buffer B  and homogenized with a tight pestle for 40 strokes. The homogenate was centrifuged at 10,000 g for 10 min to obtain the nuclear extract. Equal ratios of the protein extracts (20 μl) were resolved on a 15% SDS-PAGE gel and detected by immunoblotting using the rabbit anti-protein C antibodies (1 μg/ml). For cytoplasmic and nuclear controls, a rabbit antiserum against the recombinant His-tagged P. falciparum histone-like protein (PF3D7_0904700) and anti-histone H3 antibodies  were used, respectively37.To estimate the distribution of PfPRMT5 in the cytoplasmic and nuclear compartments of the parasite, ∼100 μl of parasite pellet was resuspended in 300 μl of a hypotonic buffer A  and late schizont stage  were harvested and crosslinked with 1% paraformaldehyde and then neutralized by glycine (0.125 M). The fixed iRBCs were lysed with saponin  and parasites were treated with a lysis buffer  and then were gently homogenized using a douncer to free the nuclei. Pelleted nuclei were sonicated in a shearing buffer . using a rod bioruptor  at high power for 20 cycles of 30 s ON/30 s OFF, resulting in sheared chromatin of approximately 100–1000 bps. Fifty μl of input samples were set aside, and the remaining chromatin was diluted in an incubation buffer . The chromatin (75 μl/400 ng) was incubated with rabbit anti-H3R2me2s antibodies (Epigentek #A-3705-050) followed by the addition of 20 μl of agarose beads for WT 3D7 and IgG Sepharose matrix (Cytiva 17-0969-01) for PfPRMT5::PTP. Beads were then washed with the following: buffer 1 ; buffer 2 , buffer 3  and finally twice with buffer 4 . The immunoprecipitated (IPed) chromatin was eluted with the elution buffer  at room temperature. The eluted chromatin and input samples were reverse crosslinked and purified by the phenol:chloroform method. For qPCR, 10 ng per well in triplicate was used with the FastStart Universal SYBR Green Master [Rox] . Primer pairs targeting 5’ UTRs of the selected genes were designed to amplify fragments less than 300 bp  − (Input Ct-target–Input Ct-stRNA)] for each primer set targeting specific promoter regions.ChIP-qPCR was performed as described with some modificationsp < 0.05 as established by ANOVA. To analyze the schizont numbers containing different numbers of merozoites, a χ2 goodness of fit test was first used to evaluate if the number of schizonts containing a certain number of merozoites was independent of the parasite lines. Then the proportions of schizonts with a certain number of merozoites were compared among these cell lines based on ANOVA for each merozoite number. The ChIP-qPCR data were analyzed by Mann–Whitney U test. The comparison of expression between H3R2me2s enriched genes and the other genes was analyzed by the Wilcoxon test. To make sure the reproducibility of the experiments, three biologically independent experiments were conducted for growth phenotype , RT-PCR, and ChIP-qPCR.For most experiments, three independent biological replicates were performed. The results are presented as mean ± standard deviation (SD). Results are regarded as significant if Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Reporting Summary


The multisubunit NuA4/TIP60 complex is a lysine acetyltransferase, chromatin modifying factor and gene co-activator involved in diverse biological processes. The past decade has seen a growing appreciation for its role as a metabolic effector and modulator. However, molecular insights are scarce and often contradictory, underscoring the need for further mechanistic investigation. A particularly exciting route emerged with the recent identification of a novel subunit, JAZF1, which has been extensively linked to metabolic homeostasis. This review summarizes the major findings implicating NuA4/TIP60 in metabolism, especially in light of JAZF1 as part of the complex. Lysine acetylation is a global regulatory post-translational modification catalyzed by lysine acetyltransferases (KATs). KATs are best characterized as modifiers of chromatin that hold special significance for cell metabolism. Chromatin, comprising genomic DNA and associated histones, is subject to a multitude of regulatory processes which allow for dynamic adjustment of transcriptional outputs in response to environmental cues. Histone acetylation is a mark of active genes that positively correlates with transcriptional activity. KATs utilize acetyl-CoA as a mandatory co-factor to acetylate their histone and non-histone substrates. Among other sources, glucose and lipid catabolism are the major contributors to cellular acetyl-CoA pools. Acetyl-CoA levels are therefore sensitive to the cellular metabolic context and can be limiting to KAT activity. It has thus been proposed that KATs function at the interface of crosstalk between metabolism and chromatin dynamics . Though Saccharomyces cerevisiae, of which 12 have homologs in the 18-subunit human complex, including the catalytic subunit Tip60/Esa1 . A gene variant defined by a single nucleotide polymorphism (SNP) mapping to intron 1 (rs864745-T) is the most widely reported to associate with T2D and was first identified as a significant T2D risk allele in a meta-analysis of genome-wide association studies (GWAS) conducted in individuals of European descent  but worlas.org; , implyinlas.org; . Since Jlas.org; , it woullas.org; , or if J