Trypanosoma brucei. Our analyses revealed that the predicted histone methyltransferase SET27 and the Chromodomain protein CRD1 are tightly concentrated at RNAPII transcription start regions (TSRs). Here, we report that SET27 and CRD1, together with four previously uncharacterized constituents, form the SET27 promoter-associated regulatory complex (SPARC), which is specifically enriched at TSRs. SET27 loss leads to aberrant RNAPII recruitment to promoter sites, accumulation of polyadenylated transcripts upstream of normal transcription start sites, and conversion of some normally unidirectional promoters to bidirectional promoters. Transcriptome analysis in the absence of SET27 revealed upregulated mRNA expression in the vicinity of SPARC peaks within the main body of chromosomes in addition to derepression of genes encoding variant surface glycoproteins (VSGs) located in subtelomeric regions. These analyses uncover a novel chromatin-associated complex required to establish accurate promoter position and directionality.Kinetoplastids are a highly divergent lineage of eukaryotes with unusual mechanisms for regulating gene expression. We previously surveyed 65 putative chromatin factors in the kinetoplastid  Kinetoplastids are a group of highly divergent eukaryotes that separated from the main lineage early in eukaryotic evolution. Reflecting this, kinetoplastids display many unusual molecular and biochemical features distinct from those of conventional eukaryotes. For example, gene expression regulation in these organisms is very different from conventional model eukaryotes such as yeasts, fruit flies, nematodes, and vertebrates, all of which are members of the Opisthokonta clade .Trypanosoma brucei is a diploid kinetoplastid parasite transmitted to mammals by the tsetse fly in sub-Saharan Africa, where it causes human sleeping sickness and animal nagana. In the mammalian host, bloodstream form (BF) trypanosomes have a surface coat composed of variant surface glycoprotein (VSG). Only one VSG is expressed at a time from a collection of ~2000 distinct VSG genes and gene fragments, many of which are clustered near telomeres , a transition coupled to metabolic reprogramming and replacement of VSG with procyclins on the surface of these parasites proteins . Such trproteins .RNAPII transcription termination regions (TTRs) are marked by the presence of chromatin containing the H3.V and H4.V histone variants and the kinetoplastid-specific DNA modification base J . ApproxiRegulation of trypanosome gene expression is thought to occur predominantly post-transcriptionally via control of pre-mRNA processing, turnover, and translation, suggesting a lack of promoter-mediated modulation. It was therefore surprising that a large number of putative chromatin regulatory factors exhibit enrichment in the vicinity of TSRs . We prevHere, we set out to characterize the Class I factors SET27 and CRD1 which exhibit some of the most prominent peaks at RNAPII TSRs. Our previous affinity purifications and mass spectrometry identified four uncharacterized proteins that interact strongly with both SET27 and CRD1. We show that all six proteins associate with each other and that the four uncharacterized proteins also display sharp peaks at TSRs, coincident with sites where SET27 and CRD1 reside. Thus, these six proteins form the SET27 promoter-associated regulatory complex (SPARC). We further show that SPARC is required for accurate transcription initiation and/or promoter definition.We previously reported that YFP-tagged SET27 (Tb927.9.13470) and CRD1 (Tb927.7.4540) co-immunoprecipitate with each other and with JBP2, a thymidine hydroxylase involved in the synthesis of base J . In addiT. brucei cells. We tagged the proteins on their N termini to preserve 3′ UTR sequences involved in regulating mRNA stability  and the auxiliary subunit JBP2 (jbp2Δ/Δ). However, we were unable to obtain cell lines completely null for any other SPARC component, suggesting these may be essential for viability. To confirm the SET27 and JBP2 gene deletions, we generated RNA-seq libraries using poly(A)-selected RNA. Cells lacking either one (Δ/+) or both alleles (Δ/Δ) showed a partial and complete loss of SET27 and JBP2 mRNA, respectively , though the extent of this defect varied between promoters in terms of both distance from the wild-type start site and RNA amount. Additionally, at 14 out of 54 normally unidirectional promoters, transcription initiated in both directions in set27Δ/Δ cells, effectively converting them to bidirectional promoters (right). These altered transcription patterns were also clearly evident in a metagene analysis of unidirectional promoters  in wild-type, ely lost . Comparipe cells . Metagenpe cells . Thus, wset27Δ/Δ cells prompted us to determine if these changes affect the expression of any mRNAs. Principal component analysis was employed to compare the global transcriptional landscapes of wild-type, set27Δ/+, and set27Δ/Δ cells  in wild-type versus set27Δ/Δ cells revealed widespread transcriptional induction of otherwise silent or weakly expressed genes  were derived from two locations on chromosome 9 near the right telomere. In both cases, these VSG gene transcripts arise downstream of SPARC peaks but are not immediately adjacent to them . In the T. brucei chromosomes as well as genes, particularly VSGs, located further downstream of SPARC sites within subtelomeric regions.These analyses demonstrate that SPARC is required to restrict the expression level of genes in the immediate vicinity of TSRs located within the main body of T. brucei, comprising SET27, CRD1, CSD1, PHD6, PBP1, and PWWP1. Several SPARC components showed homology to known histone mark reader and writer domains, including SET, Chromo, Chromoshadow, PHD, and PWWP, indicating that the association of SPARC with chromatin is mediated through binding to specific histone modification(s). JBP2 also copurified with core SPARC components but it appears to be functionally distinct from them with respect to chromatin binding and impact on transcription initiation. In contrast to a previous report . In contrast, in xpressed  and may Additionally, in cells lacking SET27, we observed derepression of promoter-proximal transcripts derived from retrotransposons. It is possible that a more open chromatin configuration at trypanosome TSRs allows preferential insertion of transposable elements at those locations, similar to what has been observed in other eukaryotes . In wildT. brucei TSRs is reminiscent of the role that the Set1/COMPASS complex plays at Opisthokont RNAPII promoters where it writes the H3K4me mark  and 10% fetal bovine serum (Gibco). PF T. brucei 427 cells were grown at 27°C in SDM-79 medium supplemented with hemin, Penicillin-Streptomycin (Gibco), and 10% FBS (Gibco).BF https://toolkit.tuebingen.mpg.de/tools/hhpred; http://ekhidna2.biocenter.helsinki.fi/dali/; The presence of putative structural/functional domains in the SPARC complex subunits was identified by analyzing: (1) the amino acid sequence composition of individual subunits for remote protein homology using the HHPred web server , offeredT. brucei by electroporation. Cell lines with tagged proteins were verified by anti-GFP or anti-FLAG immunoprecipitation followed by LC-MS/MS.Proteins were tagged N-terminally at their endogenous loci using the pPOTv4 BSR YFP plasmid  and a deSET27 gene deletion was performed in a YFP-RPB1 cell line (WT) via homologous recombination to replace both SET27 alleles with hygromycin (HYG) and G418 drug resistance markers. The JBP2 gene deletion was made using a CRISPR/Cas9 toolkit developed for kinetoplastids  resistance cassette, the SET27 CDS sequence and a fragment from the SET27 3′ UTR were synthesized by Invitrogen. The plasmid was linearized by PsiI (NEB) and transfected by electroporation into oC. Blocked slides were incubated for 45 min at 37oC first with rabbit anti-GFP primary antibody  diluted 1:500 in 2% BSA, and then with Alexa fluor 568 goat anti-rabbit secondary antibody  diluted 1:1000 in 2% BSA. DNA was stained with DAPI. Slides were imaged using a Zeiss Axio Imager microscope.Parasites were harvested by centrifugation, washed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde  for 10 min on ice. Fixation was then stopped by adding glycine. Fixed cells were pipetted onto polylysine slides and permeabilized with 0.1% Triton X-100. Slides were blocked with 2% bovine serum albumin (BSA) for 45 min at 378 cells were harvested by centrifugation and fixed with 0.8% formaldehyde  for 20 min at room temperature. Cells were lysed and chromatin was sheared by sonication in a Bioruptor sonicator (Diagenode) for 30 cycles . Sonication was performed in the presence of 0.2% SDS, after which sonicated samples were centrifuged to pellet cell debris and SDS in the supernatants was diluted to 0.07%. An input  was taken before incubating the rest of the supernatant overnight with Protein G Dynabeads and 10 µg anti-GFP antibody . The following day, the beads were washed first in the presence of 500 mM NaCl, and then in the presence of 250 mM LiCl. The DNA was eluted from the beads using a buffer containing 50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS. The eluted sample was treated with DNase-free RNase (Roche) and Proteinase K, and DNA was purified using a QIAquick PCR Purification Kit (Qiagen). ChIP-seq libraries were prepared as follows. DNA was blunt-ended using a Quick blunting kit (NEB), followed by treatment with Klenow Fragment 3'→5' exo- (NEB) and dATP. NEXTflex barcoded adapters (Bioo Scientific) were ligated to the DNA fragments which were then amplified for 13–18 PCR cycles. DNA fragment purification and size selection were performed using AMPure XP beads (Beckman Coulter). The libraries were sequenced by the Edinburgh Clinical Research Facility on Illumina NextSeq 550 or Illumina NextSeq 2000 instruments. Subsequent ChIP-seq data analysis was based on three replicates for CRD1 (BF), JBP2 (BF), and SET27 (PF), and two replicates for the other YFP-tagged proteins and for the untagged control in BF and PF cells. ChIP-seq data for CRD1 (BF), SET27 (BF), and the untagged control (BF) were taken from 3–5×10ChIP-seq reads were first de-duplicated with pyFastqDuplicateRemover  and then7 parasites were harvested by centrifugation and RNA was extracted from cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA samples were treated with TURBO DNase (Thermo Fisher Scientific) followed by purification with Phenol/Chloroform/Isoamyl alcohol pH 4. RT-qPCR on extracted RNA was performed using Luna Universal One-Step RT-qPCR Kit (NEB). Primers used in the qPCR reactions are listed in jbp2Δ/+, and jbp2Δ/Δ cell lines, and three replicates for the set27Δ/+ and set27Δ/Δ cell lines.1–5×10RNA-seq reads were aligned to the Tb427v9.2 genome  with Bow8 cells were harvested by centrifugation and lysed in a buffer containing 50 mM Tris pH 8, 150 mM KCl, and 0.2% NP-40. Lysates were sonicated in a Bioruptor (Diagenode) sonicator for three cycles . Sonicated samples were centrifuged and the supernatant (soluble fraction) was incubated for 1 hr at 4°C with beads crosslinked to anti-GFP antibody  or to M2 anti-FLAG antibody . The beads were then washed three times with lysis buffer. Proteins were eluted from the beads using RapiGest surfactant (Waters) at 55°C for 15 min. Proteins were digested by filter-aided sample preparation (4×10paration . Brieflyparation  and resuparation . The resparation  and are paration .Plasmids and cell lines made as part of this study are available upon request.https://www.ncbi.nlm.nih.gov/geo/) with accession number GSE208037. The proteomics data generated in this study can be accessed on the Proteomics Identification Database  with accession number PXD036454.The sequencing data generated in this study can be accessed on the NCBI Gene Expression Omnibus  with accession number GSE150253 and on PRIDE (https://www.ebi.ac.uk/pride/) with accession number PXD026743.We have used CRD1 (BF), SET27 (BF), and untagged (BF) ChIP-seq and proteomics data from  Trypanosomes are unusual in the way that they transcribe protein-coding genes. This work defines the role of the SPARC complex in transcription and highlights the role of potential histone readers and writers. This work will be of interest to those in the kinetoplastid community, especially those working on the control of gene expression. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  Reviewer #1 ):In this paper, Staneva et al. describe a novel complex found at RNA PolII promoters that they term the SPARC. The manuscript focuses on defining the core components of the complex and the pivotal role of SET27 in defining its function, and role in PolII transcription. This manuscript is a logical follow on from an initial paper  by the same authors where they systematically analyzed chromatin factors, and their role in both transcription start and termination. What is also very clear, is that this complex is one made of histone readers and writers which suggests its function is to change the chromatin structure around a PolII promoters. The authors show that this complex is necessary for the correct positioning of PolII and directionality of transcription.This was a well-designed study and well written and clear manuscript that provides fascinating insight transcription control in bloodstream form parasites.I have no major comments only a few minor ones.1) Localisation of the different SPARC components appears to be either nuclear or nuclear and cytoplasmic.– Both SET27 and CRD1 show a nuclear and cytoplasmic localisation in the bloodstream form IFA , but only a nuclear localisation procyclic form.Did the authors attempt C terminally tagging SET27, CRD1 to see if this resulted in a change in the pattern?We have not tagged either protein at the C terminus, however SET27 (Tb927.9.13470) has been tagged both N- and C-terminally in procyclic form (PF) cells as part of the TrypTag project (http://tryptag.org). In both cases, SET27 localized to the nucleus, suggesting that the differences in localization we observe for SET27 depend on the life cycle stage, and not on the position of the tag. One caveat is that in the TrypTag project proteins are tagged with mNeonGreen whereas in our study proteins were tagged with YFP. Based on our images, CRD1 appears to be predominantly nuclear in both bloodstream form (BF) and PF parasites. CRD1 (Tb927.7.4540) has been tagged only N-terminally in PF cells as part of the TrypTag project where it has also been classified as mostly nuclear with only 10% of cells showing cytoplasmic localization for CRD1.We are well aware that tags can alter the behaviour of a protein. Absolute confirmation of location will require the generation of antibodies that detect untagged proteins. However, this is a longer-term undertaking. We have added the following statement to the Results section to address the point raised:“We tagged the proteins on their N termini to preserve 3′ UTR sequences involved in regulating mRNA stability . We note, however, that the presence of the YFP tag and/or its position  might affect protein expression and localization patterns”.– The point is made that JBP2 shows a 'distinct cytoplasmic localisation' in PF cells. by this logic, the SET27 localisation in BF is also distinctly cytoplasmic and a nuclear enrichment is not clear.Indeed the reviewer is correct – we have inadvertently over accentuated the significance of this difference in the text. We had emphasized the predominantly cytoplasmic localization of JBP2 in PF trypanosomes as potentially related to its weaker association with other (predominantly nuclear) SPARC components in the mass spectrometry experiments. The presence of SET27 in the nuclei of both BF and PF cells is confirmed by a positive ChIP signal. We have revised the manuscript text by changing “distinct cytoplasmic” to “predominantly cytoplasmic” to describe JBP2 localization in PF cells. We hope that this resolves the issue.– Why would the localisation pattern change between life cycle stages? Surely PolII transcription should remain the same?Although our analysis suggests that there may be some shift in SET27 and JBP2 localization between BF and PF stages, sufficient amounts of these proteins may be present in the nucleus for proper SPARC assembly and RNAPII transcription regulation in both life cycle forms. The proportion of SET27 and JBP2 proteins that localizes to the cytoplasm may have functions unrelated to transcription.2) Several of the images in Supplementary Figure 1B seem to show foci in the nucleus . Do you see foci throughout the cell cycle or just in G1/S phase cells as shown here?We have not systematically investigated protein localization at different cell cycle stages, so we do not have microscopy images for all proteins at all stages of the cell cycle. However, the images we did collect suggest the punctate pattern is preserved for CRD1 in the G2 phase in both BF and PF cells  as we sh3) In Figure 6, what does 'TE' stand for?TE denotes transposable elements. We have added this to the figure legend.4) The authors show this interesting link between SPARC complex and subtelomeric VSG gene silencing.– In the CRD1 ChIP or RBP1 ChIP, are there any other peaks in telomere adjacent regions in the WT cells similar to that seen on chromosome 9A? And does the sequence at this point resemble a PolII promoter?Apart from peaks located on Chromosome 9_3A, there are other CRD1 and RPB1 ChIP peaks in chromosomal regions adjacent to telomeres in WT cells. We observed broadening of RPB1 distribution in these regions upon SET27 deletion, similar to what we show for Chromosome 9_3A. In particular, wider RPB1 distribution on Chromosome 8_5A coincides with upregulation of 10 VSG transcripts. These two loci explain most of the differentially expessed genes (DEGs) detected, but other subtelomeric regions show a similar pattern. We have added the following statement to the Results section to highlight that the phenotype shown for Chromosome 9_3A is not unique:“We also observed a similar phenotype at other subtelomeric regions, such as Chromosome 8_5A where 10 VSGs and a gene encoding a hypothetical protein were upregulated upon SET27 deletion (Supplementary file 3)”.Cordon-Obras et al. (2022) have recently defined key sequence elements present at one RNAPII promoter. We searched for similar sequence motifs but failed to identify them as underlying CRD1 and RPB1 ChIP peaks, highlighting the likely sequence heterogeneity amongst trypanosome RNAPII promoters. To address this point, we have added the following sentence to the Discussion:T. brucei promoter , however, we were unable to identify similar motifs underlying CRD1 or RPB1 ChIP-seq peaks, suggesting that T. brucei promoters are perhaps heterogeneous in composition”.“Sequence-specific elements have recently been found to drive RNAPII transcription from a – In the FLAG-CRD1 IP , the VSG's seen here are not represented (as far as I can tell) in Figure 6B and C. If my reading is correct could, is this a difference in the FC cut off for what is significant in these experiments?set27D/D cells are indeed different from the ones shown in Figure 6 (even after setting the same fold change cutoffs). We have highlighted this by adding the following statement to the Results section: “Gene ontology analysis of the upregulated mRNA set revealed strong enrichment for normally silent VSG genes  which were distinct from the VSG proteins detected in the FLAG-CRD1 immunoprecipitations from set27D/D cells ”.The VSGs detected in the FLAG-CRD1 IP from set27D/D cells, though it is likely that these represent unspecific interactions”.The VSGs in the mass spectrometry experiments likely represent unspecific interactors of FLAG-CRD1. To clarify this, we have added the following statement to the Results section: ”Instead, several VSG proteins were detected as being associated with FLAG-CRD1 in Reviewer #1 (Significance (Required)):Trypanosomes are unusual in the way that they transcribe protein coding genes. Recent advances have defined the chromatin composition at the TSS and TTS, and the recent publication of a PolII promoter sequence(s) further adds to our understanding of how transcription here is regulated. Defining the SPARC complex now add to this understanding and highlights the role of potential histone readers and writers. I think that this will be of interest to the kinetoplastid community especially those working on control of gene expression.T. brucei.Our lab studies gene expression and antigenic variation in Reviewer #2 ):In this manuscript, the authors identify a six-membered chromatin-associated protein complex termed SPARC that localizes to Transcription Start Regions (TSRs) and co-localizes with and (directly or indirectly) interacts with RNA polymerase II subunits. Careful deletion studies of one of its components, SET27, convincingly show the functional importance of this complex for the genomic localization, accuracy, and directionality of transcription initiation. Overall, the experiments are well and logically designed and executed, the results are well presented, and the manuscript is easy to read.There are a few minor points that would benefit from clarification and/or from a more detailed discussion:T. brucei can only occur from one of ~15 subtelomeric bloodstream expression sites, which include the ESAGs. This result implies that VSG genes are also transcribed from "archival VSG sites" in the genome, not only from expression sites. Are there VSGs from the silent BESs among the upregulated VSGs? Is there precedence in the literature for the expression of VSGs from chromosomal regions besides the subtelomeric expression sites?1) The concomitant expression of many VSGs (37) in a SET27 deletion strain is remarkable and has important implications for their normally monoallelic expression. It is well established that VSG expression in wild-type set27D/D cells are annotated as located in BES regions. While it is possible that recombination events have resulted in gene rearrangements between the reference strain and our laboratory’s strain, at least some of the upregulated VSGs are likely to be transcribed from non-BES archival sites. VSG transcript upregulation from non-BES regions was also recently described by López-Escobar et al. (2022).Our analysis of differentially expressed genes (DEGs) revealed that 43 VSG genes (37 of which are subtelomeric) and 2 ESAG genes are upregulated in the absence of SET27. Both ESAGs but none of the upregulated VSGs in set27D/D are still relatively lowly expressed . This is presumably insufficient to coat the surface of T. brucei, and expression from BES sites instead may be required to achieve this. We have revised the manuscript Discussion section to make these points more clear:We note that the upregulated mRNAs in set27D/D cells we detected upregulation of 43 VSG transcripts, none of which were annotated as located in BES regions. Recently, López-Escobar et al. (2022) have also observed VSG mRNA upregulation from non-BES locations, suggesting that VSGs might sometimes be transcribed from other regions of the genome. However, the VSG transcripts we detect as upregulated in set27D/D were relatively lowly expressed  and may not be translated to protein or be translated at low levels compared to a VSG transcribed from a BES site”.“Bloodstream form trypanosomes normally express only a single VSG gene from 1 of ~15 telomere-adjacent bloodstream expression sites (BESs). In contrast, in 2) The role of SPARC in defining transcription initiation is compelling. It's less clear to the reviewer if the observed transcriptional silencing within subtelomeric regions can also ascribed to SPARC. Have the authors considered the possibility that some components of the SPARC may be shared by other chromatin complexes, which could be responsible for the transcriptional activation of silent genes in SET27 deletion mutants?We cannot rule out indirect effects through the participation of some SPARC components in other complexes operating independently of SPARC. Indeed, the transcriptional defect within the main body of chromosomes appears to be somewhat different from that observed at subtelomeric regions, particularly with respect to distance from SPARC. We have added a statement in the Discussion section to highlight the possibility raised by the reviewer:“However, an alternative possibility is that transcriptional repression in subtelomeric regions is mediated by different protein complexes which share some of their subunits with SPARC, or whose activity is influenced by it”.3) The authors mention that the observed interaction of FLAG-CRD1 with VSGs in the immunoprecipitations  is evidence for the actual expression of normally silent VSGs on the protein level. This is true, but it should be spelled out that this interaction is nevertheless likely an artifact, at least the physiological relevance of these interactions is questionable.We agree that these are likely background associations and have added the following statement to the Results section to clarify this point:set27D/D cells, though it is likely that these represent unspecific interactions”.“Instead, several VSG proteins were detected as associated with FLAG-CRD1 in To avoid unnecessary confusion we have also removed the following sentence from the revised Discussion:set27Δ/Δ cells indicate that some of the normally silent VSG genes are also translated into proteins in the absence of SET27”.“The interactions of FLAG-CRD1 with VSGs in the affinity selections from 4) "ophistokont" is misspelled in the introductionThanks for noticing. We have corrected it to “Opisthokonta”.Reviewer #2 (Significance (Required)):Trypanosoma brucei, a highly divergent eukaryotic organism that is renowned for unusual features and mechanisms in gene regulation, metabolism, and other cellular processes. While post-transcriptional regulation is prevalent and relatively well established in T. brucei, much less is known about the mechanism of transcription initiation and transcriptional control, in part due to the general paucity of well-defined conventional promoter regions in this organism (only very few have been identified thus far). In this context, the work by Staneva et al. is highly significant and represents an important contribution to the field of gene regulation and chromatin biology in T. brucei and other related kinetoplastid parasites.The manuscript by Staneva et al. addresses the fundamental regulatory mechanism of gene transcription in the protozoan parasite


Deregulation of transcription factor AP2 alpha (TFAP2A) and RNA polymerase III (Pol III) products is associated with tumorigenesis. However, the mechanism underlying this event is not fully understood and the connection between TFAP2A and Pol III–directed transcription has not been investigated. Here, we report that TFAP2A functions as a positive factor in the regulation of Pol III–directed transcription and cell proliferation. We found TFAP2A is also required for the activation of Pol III transcription induced by the silencing of filamin A, a well-known cytoskeletal protein and an inhibitor in Pol III–dependent transcription identified previously. Using a chromatin immunoprecipitation technique, we showed TFAP2A positively modulates the assembly of Pol III transcription machinery factors at Pol III–transcribed gene loci. We found TFAP2A can activate the expression of Pol III transcription-related factors, including BRF1, GTF3C2, and c-MYC. Furthermore, we demonstrate TFAP2A enhances expression of MDM2, a negative regulator of tumor suppressor p53, and also inhibits p53 expression. Finally, we found MDM2 overexpression can rescue the inhibition of Pol III–directed transcription and cell proliferation caused by TFAP2A silencing. In summary, we identified that TFAP2A can activate Pol III–directed transcription by controlling multiple pathways, including general transcription factors, c-MYC and MDM2/p53. The findings from this study provide novel insights into the regulatory mechanisms of Pol III–dependent transcription and cancer cell proliferation. TFAP2 factors regulate gene expression by binding to the promoters of its target genes containing AP2 binding sites (GCC(N)3/4GGC) , 2, 3, 4and HER2 , 6, 7 buS1 genes , 9. TFAPS1 genes , 11. FurS1 genes , 16, 17.S1 genes , 9, 18 aS1 genes , 21, 22.S1 genes , 24, 25,S1 genes , 27, 28.S1 genes . MiR-204S1 genes . In lungS1 genes . The zinS1 genes . Despitenc886 is a typical gene transcribed by Pol III and its expression is inhibited by CpG methylation (RNA polymerase III (Pol III) products are a subset of abundant noncoding RNA molecules, including 5S rRNA, tRNA, U6 snRNA, and 7SL RNA. These RNAs, accounting for about 15% of transcription activity in eukaryotes, play essential roles in several fundamental cellular activities that include ribosomal assembly, translation, protein transportation, RNA processing, and cell growth , 35, 36.hylation . Deregulhylation , 46, 47.hylation . Howeverhttps://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA726417). Based on the RNA-Seq data, we analyzed differential expression genes between two groups of samples and found that TFAP2A mRNA reads were drastically enhanced by FLNA depletion (A and B). In addition, we found that FLNA knockdown in SaOS2 cells severely affected cell shapes of SaOS2 but did not affect the shapes of SaOS2 nuclei and nucleoli (C and D). To determine whether this result can be reproduced in other cell types, we generated HeLa cell lines stably expressing FLNA shRNA and control shRNA using FLNA-expressing lentiviral particles, and TFAP2A expression was assessed by RT-qPCR and Western blot using the resulting cell lines. As expected, FLNA silencing enhanced TFAP2A expression at both mRNA and protein levels (E and F). This result is in agreement with that obtained from SaOS2 cells, suggesting that FLNA can inhibit TFAP2A expression. To confirm the inhibitory role of FLNA in TFAP2A expression, we generated a HeLa cell line with FLNA overexpression and its control cell line using a lentiviral transduction system. TFAP2A expression was analyzed by RT-qPCR and Western blot. We showed that FLNA overexpression reduced the expression of TFAP2A mRNA and protein in HeLa cells (G and H). Taken together, these results indicate that FLNA may repress TFAP2A expression in SaOS2 and HeLa cells.In our previous work, we found that cytoskeletal FLNA suppresses Pol III–dependent transcription in human transformed cell lines . To undeepletion , A and Bnucleoli , suggestnucleoli , C and Dn levels , E and FLa cells , G and HD and I). Thus, we next focused on the role of TFAP2A in Pol III–directed transcription in HeLa and 293T cell lines. The effect of TFPA2A depletion on the synthesis of Pol III products was first investigated by transfecting TFAP2A siRNA into 293T cells. RT-qPCR and Western blot analysis confirmed TFPA2A downregulation after transient transfection of TFAP2A siRNA (A and B). Interestingly, TFAP2A depletion significantly inhibited the synthesis of Pol III products, including 5S rRNA, U6 RNA, 7SL RNA, and tRNA-Met (C). Meanwhile, we tested the effect of TFAP2A siRNA transfection on the expression of TFAP2A and Pol III products in HeLa cells; and the results are consistent with those obtained in the assays with 293T cells (D–F), indicating that TFAP2A is required for maintaining the normal synthesis of Pol III products. To confirm this result, we generated 293T cell lines that stably express TFAP2A shRNA or control shRNA. As expected, TFAP2A shRNA stable expression dampened TFAP2A expression and the synthesis of Pol III products (J–L). These results suggest that TFAP2A plays a positive role in Pol III–directed transcription. To validate the positive role of TFAP2A in Pol III–dependent transcription, we assessed the effect of TFAP2A overexpression on Pol III products by performing transient transfection with 239T cells. Notably, TFAP2A overexpression stimulated the expression of Pol III products in 293T cells (A and B). Comparable results were obtained when HeLa cells transiently transfected were analyzed (C and D). Next, 293T and HeLa cell lines with HA-TFAP2A stable expression were generated and similar experiments were performed. As expected, stable expression of HA-TFAP2A enhanced the expression of Pol III products in both cell types (E–H). Altogether, we showed that TFAP2A can positively regulate Pol III–directed transcription.Given that FLNA inhibits both Pol III–directed transcription and TFAP2A expression, we asked whether there is a connection between TFAP2A and Pol III–dependent transcription. Before answering this question, we initially investigated TFAP2A expression in several cell types, including SaOS2, 293T, and HeLa cell lines, by immunoblotting. Unexpectedly, TFAP2A expression level in SaOS2 cell line was very low and hard to detect by Western blot, but expression of TFAP2A in 293T and HeLa was quite abundant , D and I2A siRNA , A and BtRNA-Met C. Meanwh3T cells , D–F, inproducts . Consistproducts , J–L. Th3T cells , A and Banalyzed , C and Dll types , E–H. AlA–D). In contrast, TFAP2A overexpression enhanced them in these cell types . Since EdU compound (5-ethynyl-2′-deoxyuridine) is widely applied to proliferation assays, we next investigated the effect of TFAP2A alteration on cell growth by performing EdU assays. EdU assays revealed that TFAP2A downregulation reduced the rate of EdU-positive cells (E–H), whereas TFAP2A upregulation enhanced the rate of EdU-positive cells . These data further confirmed that TFAP2A can promote the proliferative activity of 293T and HeLa cells. To understand whether the enhancement of cell proliferation is caused by the activation of Pol III–dependent transcription, we analyzed proliferative activity and Pol III–dependent transcription in the 293T cell line expressing HA-TFAP2A and its control cell line in the presence or absence of 54 μM ML-60218 (a Pol III transcription inhibitor). As shown in J, TFAP2A overexpression enhanced 293T cell proliferation and expression of Pol III products, which is consistent with the result as described above . Strikingly, the presence of ML60218 reversed the activation of proliferative activity and Pol III–directed transcription induced by TFAP2A overexpression (J). Western blot showed that TFAP2A expression was not significantly affected by the treatment with ML-60218 . Next, we determined whether TFAP2A silencing affects the assembly of Pol III transcription machinery factors at Pol III target loci by ChIP assays. ChIP analysis showed that TFAP2A silencing reduced the occupancies of components of the Pol III transcription machinery at Pol III target loci, including 5S rRNA, 7SL RNA, and tRNA-Met loci (A–D). BRF1 and GTF3C2 showed very low occupancy on the U6 snRNA gene locus. This result is rational because U6 snRNA transcription does not require the involvement of BRF1 and GTF3C2. However, the occupancy of POLR3A showed significant reduction at this locus. These data suggest that TFAP2A can regulate Pol III–dependent transcription by controlling the recruitment of components of the Pol III transcription machinery to Pol III–transcribed gene loci. To understand how TFAP2A expression alteration affects the assembly of the Pol III transcription machinery at the Pol III–transcribed loci, we analyzed the expression of Pol III transcription-related factors using Western blot and cell lines with TFAP2A deletion or overexpression. It is well established that Pol III general transcription factors play essential roles in Pol III transcription initiation (E). Comparable results were obtained when HeLa cells were analyzed (F). In contrast, TFAP2A overexpression activated BRF1 and GTF3C2 expression but did not impact on TBP expression in these cell types (G and H). These results suggest that TFAP2A can regulate the assembly of the Pol III transcription machinery at Pol III target loci by affecting BRF1 and GTF3C2 expression.To understand how TFAP2A regulates Pol III–directed transcription, we initially investigated whether TFAP2A binds to the promoters of Pol III target loci by performing chromatin immunoprecipitation (ChIP) assays. The results showed that TFAP2A did not bind to the Pol III target loci except the ne locus A. Next, Met loci , A–D. BRitiation . Thus, titiation E. Comparanalyzed F. In conll types , G and HJ and A and B). Conversely, TFAP2A overexpression enhanced MDM2 and c-MYC expression in which p53 expression was reduced in the same cell types (K and L and C and D). Rho A is an upstream factor of several signal pathways that are required for Pol III–directed transcription (J and A and C). PTEN, a factor in the AKT/PTEN pathway, has been shown to regulate Pol III–directed transcription (J and A and C). Taken together, these findings suggest that TFAP2A indirectly regulates Pol III–dependent transcription by affecting the expression of multiple factors, including transcription factor subunits BRF1 and GT3C2, oncogenic c-MYC and MDM2, and tumor suppressor p53.Pol III–dependent transcription is also controlled by oncogenic factors, tumor suppressors, and signaling transduction factors , 34. Nexcription . In theson J and , A and Ccription . In theson J and , A and CBrf1 and Gtf3c2 genes at the transcriptional step (A–D). To determine whether TFAP2A can modulate the transcription of Brf1 and Gtf3c2 genes, we searched TFAP2A-binding sites from the Brf1 and Gtf3c2 gene promoters. Surprisingly, the Brf1 promoter 4 (Brf1P4) and the Gtf3c2 promoter 2 (Gtf3c2P2) contain a canonical TFAP2A binding motif (TFAP2A consensus sequence: GCC(N)3/4GGC) (E). Next, we examined whether TFAP2A can bind to these two promoters by performing ChIP assays using HeLa cells. ChIP qPCR revealed that TFAP2A displayed specific occupancy at Brf1P4 and Gtf3c2P2 (F and G). Since TFAP2A binds to Brf1P4 and Gtf3c2P2, we investigated the effect of TFAP2A expression change on the activities of Brf1P4 and Gtf3c2P2 by performing reporter gene assays. 293T or HeLa cell lines with TFAP2A silencing or overexpression and their corresponding control cell lines were transfected with the reporter gene vectors driven by Brf1P4 or Gtf3c2P2, respectively. Reporter assays showed that TFAP2A silencing reduced the activities of Brf1P4 or Gtf3c2P2 (J). In addition, mutations of TFAP2A-binding sites within these promoters inhibited the activities of Brf1P4 or Gtf3c2P2 (K and L). These results indicate that TFAP2A can directly modulate the transcription of Brf1 or Gtf3c2 genes by binding to their promoters. Since the depletion of BRF1 or GTF3C2 has been shown to inhibit Pol III–directed transcription 3/4GGC) E. Next, Gtf3c2P2 , F and GGtf3c2P2 , whereasGtf3c2P2 J. In addGtf3c2P2 , K and Lcription , the resp53 and c-MYC promoters. Unexpectedly, ChIP results showed that TFAP2A did not bind to the promoters of c-MYC and p53 genes (B), suggesting that TFAP2A indirectly regulates the expression of c-MYC and p53. We next focused on the role of MDM2 in Pol III–mediated transcription. First, we examined whether MDM2 overexpression can reverse the inhibition of Pol III–directed transcription caused by TFAP2A downregulation. To this end, both 293T and HeLa cell lines stably expressing TFAP2A shRNA were further infected by lentiviral particles expressing mCherry-MDM2, and the generation of cell lines expressing both TFAP2A shRNA and mCherry-MDM2 were verified by Western blot (A and S7A). We showed that mCherry-TFAP2A expression reversed the inhibition of Pol III–directed transcription caused by TFAP2A silencing in both 293T and HeLa cells (B and S7B). Interestingly, consistent results were obtained when human cervical epithelial cells expressing TFAP2A shRNA and mCherry-MDM2 were generated and used for similar assays . How TFAP2A regulates MDM2 expression remains unclear. To address this question, MDM2 mRNA expression in the cell lines with TFAP2A silencing or overexpression was detected by RT-qPCR. We showed that TFAP2A can activate MDM2 mRNA expression (D and E and F and G), suggesting that TFAP2A regulates MDM2 expression at the transcriptional step. To support this assumption, we searched TFAP2A-binding sites within the Mdm2 gene promoter. Surprisingly, the Mdm2 promoter contains two putative TFAP2A-binding sites upstream of transcription starting site (F). We next examined whether TFAP2A binds to the Mdm2 promoter by performing ChIP assays. ChIP-qPCR results showed that TFAP2A exhibited specific occupancy at the Mdm2 promoter compared with that of control IgG (G), indicating that TFAP2A can bind to the Mdm2 promoter. We next asked whether TFAP2A expression change affects the Mdm2 promoter activity. To answer this question, we prepared the DNA fragments of the wildtype MDM2 promoter and its mutant containing TFAP2A-binding site mutations by PCR and inserted them into the pGL3-basic reporter vector (F). Luciferase assays were performed using the cell lines transiently transfected with the promoter-driven reporter vectors. The data from luciferase activity showed that TFAP2A downregulation inhibited the activity of the Mdm2 promoter activity, whereas TFAP2A overexpression activated its activity (H and I). Intriguingly, mutations of TFAP2A-binding sites reduced the Mdm2P1 activity (J and Mdm2 gene transcription by binding to the Mdm2 promoter. MDM2 was identified to be an inhibitor of p53 (J). Thus, we determined the effect of mCherry-MDM2 expression on p53 expression in 293T and HeLa cell lines with TFAP2A silencing. As expected, mCherry-MDM2 expression inhibited the activation of p53 expression induced by TFAP2A silencing (K and S7K). However, mCherry-MDM2 expression in the TFAP2A shRNA-expressing cells did not rescue the expression of BRF1 and GTF3C2. Similar results were observed when mCherry-MDM2 was expressed in human cervical epithelial cells with TFAP2A silencing (B and C). Collectively, these results indicate that TFAP2A can indirectly regulate Pol III–directed transcription by controlling the MDM2-p53 axis.We have shown that TFAP2A can regulate the expression of MDM2, p53, and c-MYC in the above experiments, and both c-MYC and p53 have been reported to modulate Pol III–directed transcription , 39. To 53 genes B, suggesern blot A and S7ALa cells B and S7Br assays . Since ar assays C and C–Eing site F. We nextrol IgG G, indicar vector F. Lucifer assays C and C–Ety J and . These dr of p53 ; moreover of p53  and J. Tr assays C and C–Eilencing , B and CA and B and A and B). Analysis of Pol III products by RT-qPCR revealed that TFAP2A silencing inhibited the activation of Pol III–directed transcription induced by FLNA depletion (C and S9C), indicating that TFAP2A is required for the activation of Pol III transcription induced by FLNA silencing. These data suggest that FLNA and TFAP2A can play opposite roles in Pol III–dependent transcription. FLNA has been shown to suppress tumor cell proliferation (D and E and D and E), suggesting that TFAP2A is required for the inhibition of cell proliferation mediated by FLNA. This result is in agreement with that obtained in the analysis of Pol III products (C and D and C and D). TFAP2A has been shown to regulate Pol III–dependent transcription by controlling BRF1 and GTF3C2 expression (A and B). Interestingly, TFAP2A depletion inhibited the activation of BRF1 and GTF3C2 induced by FLNA silencing in SaOS2 and HeLa cells (E and F and E and F). This result is consistent with that obtained using cell lines expressing TFAP2A shRNA only (E and F), indicating that TFAP2A modulates FLNA-mediated Pol III transcription by controlling BRF1 and GTF3C2 expression. Taken together, TFAP2A is required for the regulation of Pol III–directed transcription mediated by FLNA.Previous work showed that FLNA downregulation enhances Pol III–directed transcription ; furtherepletion C and S9Cferation . Thus, cpression . Thus, wpression , A and BRNA only , E and FH-①). On the other hand, the activation of oncogenic c-MYC (H-②) and MDM2 (H-③) increases the interaction between c-MYC and TFIIIB and decreases the interaction between p53 and TFIIIB, which subsequently activates Pol III–dependent transcription. These regulatory pathways revealed by this study extend the understanding of the mechanism of Pol III–directed transcription.Based on the data obtained in this study, we proposed a model by which TFAP2A regulates Pol III–dependent transcription. Specifically, TFAP2A expression is inhibited by FLNA; however, TFAP2A upregulation activates the expression of BRF1, GTF3C2, c-MYC, and MDM2. On the one hand, the increase of BRF1 and GTF3C2 expression directly enhances Pol III transcription machinery assembly at target loci to activate the synthesis of Pol III products H-①. On tic c-MYC H-② and Mand MDM2 H-③ increJ), indicating that Pol III–mediated transcription activation contributed to the promotion of cell proliferation by TFPA2A overexpression. These findings provide a novel explanation from about how TFAP2A promotes cancer cell proliferation.TFAP2A has been identified for decades as a transcription factor that regulates RNA polymerase II–directed gene transcription , 6, 7. IBrf1 and Gtf3c2 gene transcription by binding to the promoters of Brf1 and Gtf3c2 genes (H-①). It has been shown that Specificity protein 1 (Sp1) regulates Pol III–directed transcription by controlling transcription of Brf1 and Gtf3C2 genes (Brf1 and Gtf3C2 genes contain Sp1- and TFAP2A-binding sites (H). Moreover, TFAP2A participates in the inhibition of Pol III–directed transcription mediated by FLNA, perhaps, using similar mechanisms. These findings put extra weight on the understanding of the regulatory mechanism of Pol III–dependent transcription mediated by FLNA, where FLNA may modulate Pol III–directed transcription by a complicated regulatory network.General transcription factors TFIIIB and TFIIIC tightly control transcription initiation directed by Pol III , 34. We c2 genes  and 5. Tc2 genes H-①. It hC2 genes . RecentlC2 genes . FurtherC2 genes . Since tng sites , it is png sites , 40. In ng sites  and 6. Mng sites , suggestng sites H. MoreovIn summary, we found that TFAP2A functions as a positive regulator in Pol III–directed transcription and promotes the proliferative activity of 293T and HeLa cells. TFAP2A is also required for the regulation of Pol III transcription mediated by FLNA. Mechanistically, TFAP2A modulates Pol III–directed transcription by controlling the activities of multiple pathways, including the expression of general transcription factor subunits BRF1 and GTF3C2, c-MYC, and MDM2/p53. Our findings provide novel insights into the regulatory mechanisms of Pol III–directed transcription and cell proliferation.The lentiviral expression vectors such as pLV-U6-EGFP-Puro, pLV-U6-mCherry-Puro, and pLV-EF1α-EGFP-Puro were purchased from Inovogen Tech Co. FLNA shRNA lentiviral particles (SC-35374-V) and control shRNA lentiviral particles (SC-108080) were obtained from Santa Cruz Biotech. Human cell lines, including SaOS2, HeLa, and 293T, were derived from American Type Culture Collection (ATCC). SaOS2 cells were cultured in McCoy’s 5A complete medium (HyClone), while HeLa and 293T cells were cultured in Dulbecco’s modified Eagle’s medium complete medium (HyClone). DNA and RNA oligonucleotides were purchased from Sangon Biotech. PCR DNA purification and gel extraction kits were obtained from Axygen Co. Biological reagents such as restriction enzymes and transfection reagents were obtained from Thermo Scientific. All chemicals were obtained from Sinopharm Chemical Reagent Co.Filamin A gene was amplified by PCR using the cDNA synthesized with 1 μg of the total RNA extracted from 293T cells. The HA-Filamin A fusion gene was generated by PCR using the resulting Filamin A gene and inserted into the pcDNA 3.0(+). TFAP2A shRNA-coding DNA fragments were synthesized by Sangon Biotech. After annealing and phosphorylation, DNA fragments were loaded downstream of the U6 promoter at the pLV-U6-mCherry-Puro plasmid. MDM2 cDNA fragments were amplified by PCR and loaded downstream of the mCherry gene at the pLV-EF1α-mCherry-Puro plasmid to form a mCherry-MDM2 fusion gene. A TFAP2A gene was amplified by PCR using the cDNA synthesized as described above; thereafter, the HA-TFAP2A fusion gene was generated by PCR using the resulting TFAP2A gene and loaded downstream of the EF1α promoter at the pLV- EF1α-EGFP-Puro plasmid.A For TFAP2A siRNA transient transfection, 293T and HeLa cells were seeded in 12-well plates. After 24 h, cells in each well were transfected with 6 pmol/L siRNA fragments and 2 μl of transfection reagent. Forty-eight hours post transfection, cell samples were harvested and used to analyze TFAP2A expression with RT-qPCR and Western blot techniques. For the generation of cell lines stably expressing TFAP2A shRNA or HA-TFAP2A protein, 293T cells cultured in six-well plates were transfected with 3 μg of TFAP2A shRNA or HA-TFAP2A-expressing lentiviral vectors and the packaging plasmids pH1 (2 μg)and pH2 (1 μg). After 48 h, the culture medium containing viruses was collected and filtered with a sterile filter (0.45 μm in diameter). Next, 293T or HeLa cells cultured 12-well plates were incubated with the lentivirus-containing medium for 48 h, selected for 72 h at the final concentration of 10 μg/ml puromycin and screened by diluting the drug-resistant cells into 96-well plates. After expansion, individual cell colonies were transferred into 12-well plates and continually cultured in a complete medium containing 10 μg/ml puromycin. Cell lines with stable expression were verified by RT-qPCR and Western blot. SaOS2 cell lines stably expressing FLNA shRNA or control shRNA were achieved by direct transduction with lentiviral particles (Santa Cruz Biotech). SaOS2 and HeLa cell lines stably expressing both FLNA shRNA and TFAP2A shRNA were obtained by incubating the FLNA shRNA-expressing cells with the medium containing TFAP2A shRNA-expressing lentiviral particles. The HeLa cell line expressing both TFAP2A shRNA and mCherry-MDM2 was generated by incubating the TFAP2A-depleted HeLa cell line with the medium containing mCherry-MDM2-expressing lentiviral particles. After selection, cell lines with shRNA or protein stable expression were verified by Western blot using antibodies against FLNA, TFAP2A, HA, and mCherry.2O. Next, 1 μl of cDNA was used for quantitative PCR (qPCR) in which 20 μl of reaction mixture contained 10 μl of 2 × SYBR Green Master Mix (Vazyme Co) and 5 pmoles of the primers detecting Pol III target genes. Quantitative PCR was performed using a Bio-Rad real-time PCR detection system. The resulting data were processed with CFX Manager 3.1 software (Bio-Rad), where an Actin or GADPH gene was used as a reference gene.SaOS2, 293T, and HeLa cell lines expressing FLNA shRNA or TFAP2A shRNA were grown in 12-well plates. At 90% confluence, cells were harvested and total RNA was prepared using an RNA extraction kit (Axygen). Reverse transcription was performed at 42 °C in a 20-μL reaction mixture containing 0.5 μg of RNA and 10 U of reverse transcriptase (New England Lab). After incubating for 1 h, the reaction was terminated by heating the sample for 15 min at 85 °C, followed by diluting it with 80 μl of ddHSaOS2, 293T, and HeLa cell lines were cultured in six-well plates. At 90% confluence, cells were harvested and lyzed using 250 μl of 1 × SDS loading buffer. After heating for 10 min at 100 °C, 20 μl cell lysate was loaded into wells within an SDS-PAGE gel for electrophoresis. After that, immunoblotting was performed using antibodies against the factors, including FLNA , TFAP2A , HA , GAPDH , BRF1 , GTF3C2 , c-MYC , MDM2 , p53 , RhoA , mCherry , TBP , and PTEN .Cell proliferation assays were performed using different methods, including cell counting, MTT, CCK8, and EdU. For cell counting and EdU assays, 293T and HeLa cell lines expressing TFAP2A shRNA or HA-TFAP2A and their control cell lines were cultured in 12-well plates. Proliferation assays were performed as described , 53, 54.2O was used to elute the DNA. One microliter of DNA samples (1/40) from a ChIP assay was used for one qPCR reaction; meanwhile, 1 ng of genomic DNA from input (0.05% input) was used as a positive control during qPCR. The relative enrichment of each Pol III gene locus was obtained by comparing the relative DNA quantity from 1 μl of ChIP sample to that from 1 ng input DNA.HeLa cell lines stably expressing TFAP2A shRNA or control shRNA were cultured in 10-cm dishes. At 90% confluence, cell lines were fixed for 10 min using 10 ml of 1% formaldehyde freshly prepared with 1 × PBS before adding 1 ml of 2.5 M glycine solution into the medium to quench the fixation. After washing twice with 1 × PBS solution, the fixed cells were lysed with 1 ml ChIP lysis buffer , followed by disrupting for 20 min in an ultrasonicator and centrifuging for 10 min at 12,000 rpm. The supernatant was retained and diluted 7 times with RIPA buffer . The rest of the procedures for ChIP assays were performed as described , 54. ChrBrf1 gene promoter 4, Gtf3C2 gene promoter 2, and MDM2 gene promoter 1 were amplified from 293T genomic DNA by PCR. The resulting promoter DNA was inserted into pGL3-basic reporter vector (Promega). 293T and HeLa cells were cultured in 12-well plates for 24 h and were subjected to transient transfection with the Brf1P4 or Gtf3c2P2-driving reporter vectors and the vectors expressing β-galactosidase, where three biological replicates for treatment or control groups were designed. After 48 h, cell samples were harvested and lysed with 100 μl of lysis buffer provided by a dual-light luciferase detection kit (Promega). Five microliters of cell lysate was used to detect the activities of luciferase and β-galactosidase based on the manual provided by the kit. The luciferase activity of each sample was normalized by the activity of β-galactosidase within the same sample. Relative luciferase activity was obtained by comparing the luciferase activity from the treatment samples to that from the control samples, where the activity of the control sample was arbitrarily set as 1. For reporter assays using MDM2P1-driving reporter vectors, luciferase activity for treatment and control was normalized by the protein quantity used in each assay.The DNA fragments for p Values were obtained by a Student’s t test or two-way analysis of variance.The experiments including RT-qPCR, cell proliferation assays, ChIP assays, and reporter assays were performed in three biological replicates. The mean and standard deviation (SD) for the data from gene expression analysis (RT-qPCR), cell proliferation assays, reporter assays, and ChIP assays (qPCR) were calculated using the GraphPad Prism 6.0 software. Each column or point in all digital graphs in this article represents the mean±SD of three biological replicates (n = 3). All data were included in main figures in the article and figures in the supplementary file.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Cynoglossus semilaevis is an economical marine fish with obvious female-biased size dimorphism. So, it is important to explore the molecular mechanism beyond gonadal development for sex control in aquaculture industry. RNA-binding protein Ewing Sarcoma protein-like (ewsr1) gene is important for mouse gonadal development and reproduction, however there are limited studies on this gene in teleost. In this study, two ewsr1 genes were cloned and characterized from C. semilaevis. The ewsr1-w gene, located in W chromosomes, showed female-biased expression during C. semilaevis gonadal development. In addition, knock-down effect and transcriptional regulation of Cs-ewsr1-w further suggested its essential role in ovarian development. This study broadened our understanding on ewsr1 function in teleost, and provided genetic resources for the further development of sex control breeding techniques in C. semilaevis aquaculture.Sexual dimorphism is a phenomenon commonly existing in animals. Chinese tongue sole Cs-ewsr1-w and Cs-ewsr1-z were cloned in Chinese tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of Cs-ewsr1-w was 1,767 bp that encoded 589 amino acids, while Cs-ewsr1-z was 1,794 bp that encoded 598 amino acids. Real-time PCR assays showed that Cs-ewsr1-w exhibited significant female-biased expression and could be hardly detected in male. It has the most abundant expression in ovaries among eight healthy tissues. Its expression in ovary increased gradually from 90 d to 3 y with C. semilaevis ovarian development and reached the peak at 3 y. After Cs-ewsr1-w knockdown with siRNA interference, several genes related to gonadal development including foxl2, sox9b and pou5f1 were down-regulated in ovarian cell line, suggesting the possible participation of Cs-ewsr1-w in C. semilaevis ovarian development. The dual-luciferase reporter assay revealed that the -733/-154 bp Cs-ewsr1-w promoter fragment exhibited strong transcription activity human embryonic kidney (HEK) 293T cell line. The mutation of a MAF BZIP Transcription Factor K (Mafk) binding site located in this fragment suggested that transcription factor Mafk might play an important role in Cs-ewsr1-w basal transcription. Our results will provide clues on the gene expression level, transcriptional regulation and knock-down effect of ewsr1 gene during ovarian development in teleost.Ewsr1 encodes a protein that acts as a multifunctional molecule in a variety of cellular processes. The full-length of  Cynoglossus semilaevis shows typically female-biased size dimorphism . Af. Af31]. ewsr1-w and ewsr1-z from C. semilaevis genome [Taq Mix , 0.5 μL forward/ reverse primers, and 1 μL cDNA template. The PCR program was set as follows: 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and 72 °C for 10 min. PCR products were purified by FastPure Gel DNA Extraction Mini Kit , connected to pEASY-T1 vector, transformed, and sequenced in Beijing Ribioco Biotechnology Co., Ltd. . The 3′ and 5′ untranslated regions (UTR) were amplified by using SMARTer RACE 5′/3′ Kit  with the primers listed in RNA was extracted from each sample by using Trizol Reagent . The quality and quantity of RNA was checked with agarose gel electrophoresis and P100 Series Spectrophotometers . The first strand cDNA was synthesized with 800 ng RNA as the template by using PrimeScript RT Kit with gDNA eraser . Gene specific primers for gene cloning were designed by Primer Premier 5.0  based ons genome . The mixhttp://smart.embl.de/, accessed on 26 March 2022), and NetPhos-3.1  . The BLASTP Program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)  was used for multiple sequence alignment. The MEGA X , amino acid sequence, molecular weight, protein domains, and phosphorylation sites were predicted and analyzed by DNAstar (V7.1.0) , SMART , 2 μL cDNA, 0.4 μL of each sense and anti-sense primers, and 0.4 μL ROX Dye II. The qPCR program was set as default settings followed by the dissociation curve, that is, 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 60 °C for 30 s. The relative mRNA expression of Cs-ewsr1-w and Cs-ewsr1-z were processed by using 2−ΔΔCt method [p < 0.05.The expressions of  primers  via qPCRt method . The datCs-ewsr1-w. The primers were listed in Cs-ewsr1-w-F1~F6 by using TSV-S1 Trelief® SoSoo Cloning Kit .Based on the TFs prediction, six promoter plasmids with luciferase report were constructed by serial-deletion to detect the promoting activity of regulatory elements of 2 at 37 °C. The pGL3-Cs-ewsr1-w~F1~F6 were transfected into HEK293T cells by using Lipo8000TM Transfection Reagent . Meanwhile, pGL3-basic and PGL3-control plasmids were used as the negative control and the positive control, respectively. The pRL-TK plasmid was transfected at the mean time as the internal reference. Dual Luciferase Reporter Gene Assay Kit  was employed to measure the promoter activities. Each experiment was performed in triplicates following the standard protocol provided by the manufacturer. Data obtained from Varioskan Flash spectral scanning multimode reader  were analyzed by LSD (Least-significant difference) in SPSS 25.0, and the significance was regarded at p < 0.05.Human embryonal kidney (HEK) 293T cells were maintained in DME/F-12 containing 10% fetal bovine serum  and 1% bFGF  in 5% COhttp://alggen.lsi.upc.es/, accessed on 2 April 2022)   were synthesized in Sangon Biotech . C. semilaevis ovarian cells were cultured in L-15 medium supplemented with 1% bFGF and 15% FBS at 24 °C. Cs-ewsr1-w siRNA was transfected into the cells by using riboFECTTM CP Transfection Kit  following the protocol described in the previous study [Cs-ewsr1-w-siRNA and negative control (NC) groups. At 48 h post transfection, the cellular status and the florescence of 5cy3-transfected cells would be checked. When it reached 90–95% of cell confluency and the percentage of transfection reached ~80%, it would be a good timing to harvest ovarian cells for the following experiments. After cell collection and RNA extraction, reverse transcription and qPCR assays were performed following the methods mentioned above. The relative expression levels of sex-related genes, such as Forkhead Box L2 (foxl2), SRY-box transcription factor 9b (sox9b), POU Class 5 Homeobox 1 (pou5f1) were measured with the primers listed in The specific siRNA of us study . Three rCs-ewsr1-w (GenBank accession no. 103397238) located in W chromosomes, with the full length of 2297 bp containing the ORF region of 1767 bp encoding 588 amino acids (Cs-ewsr1-z (GenBank accession no. 103398620), located in Z chromosome, was 2230 bp in full length. It contains 1794 bp ORF region encoding 598 amino acids  was cloned by genomic DNA with specific primers Cs-ewsr1-w-P-F/R  after in vitro Cs-ewsr1-w siRNA interference (RNAi), while no significant variation of Cs-ewsr1-z gene was detected. The down-regulation of sex-related genes was detected after Cs-ewsr1-w RNAi (p < 0.05), including foxl2, sox9b and pou5f1. Among that, foxl2 expression significantly dropped by18.8-fold compared with the control group. The expression levels of sox9b and pou5f1 were significantly reduced by half  and zebrafish (Danio rerio) [sox9b was detected in gonads of early-stage C. semilaevis, indicating its potential involvement in gonadal differentiation [Pou5f1  is a key TF regulating embryonic stem cell pluripotency, primordial germ cell formation, early embryonic and gonadal germ cell development [Pou5f1 analogue in teleost plays a post-embryonic role in adult gonad and gametes development [Cs-ewsr1-w knockdown on several sex-related genes, we proposed that it might be a positive regulator in ovarian development of C. semilaevis. In the future, analysis including in vivo trials would be conducted for further investigation on its mechanism.Based on our qPCR results, elopment . Many stelopment ,42,43,44elopment . Sox9b, o rerio) ,47. It fo rerio) ,49,50. Bntiation . Pou5f1 elopment . Pou5f1 elopment ,53,54. BCs-ewsr1-w promoter -733 to -154 bp was the core region that had a great effect on transcription regulation. After site-direct mutagenesis on the TF-binding sites, the activity of TF Mafk-binding site decreased significantly, suggesting potential involvement of Mafk in Cs-ewsr1-w transcription. Mafk, a member of small MAFs family, was essential for mice embryonic development [C. semilaevis, revealing the indispensable involvement of ubiquitination pathway in female differentiation [Cs-ewsr1-w transcription during C. semilaevis ovarian development. The regulation mechanism between them are worthy for further functional studies. In the future, further studies will be performed to get a clearer picture on the regulation of Cs-ewsr1-w by TF Mafk during female differentiation and ovarian development.Promoters contain sequence-specific binding sites for many TFs, and the TFs could recognize and bind target sequences to guide underlying transcription and regulate transcriptional activity ,56,57. Ielopment . It reguelopment . Numerouewsr1 genes were cloned and characterized from Chinese tongue sole (Cs-ewsr1-w and Cs-ewsr1-z). The female-biased gonad expression of Cs-ewsr1-w was observed from 90 d to 3 y, suggesting its potential roles in ovarian development. Its knockdown significantly down-regulated the expressions of foxl2, sox9b and pou5f1. The activity analysis, and the prediction and verification of transcription factors for Cs-ewsr1-w promoter shed some lights on the transcription regulation of this gene. Our findings suggested the potential roles of Cs-ewsr1-w in C. semilaevis ovarian development, providing fundamental information for further exploration on its biological functions in teleost.In this study, two


Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol , on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress–induced transcription of Heat Shock Factor 1–regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1–target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription. Such structures play important roles in regulating various cellular processes, including RNA metabolism, protein synthesis, ribosome biogenesis, DNA damage repair, and cell type-specific gene transcription, thereby enabling cellular differentiation or permitting cells to evade stressful environments , undersronments , 9, 10; ronments ; and theronments , 13.Electrostatic, weak hydrophobic, pi–pi stacking, and pi–cation interactions are critical in the formation of most biomolecular condensates . A commoSaccharomyces cerevisiae, HeatShockProtein (HSP) genes regulated by Heat Shock Factor 1 (Hsf1) are transcriptionally activated and engage in robust physical interactions in response to acute heat shock (HS), coalescing into intranuclear foci  analysis revealed that while activator recruitment was only mildly affected, RNA polymerase occupancy within promoter and gene bodies was abolished. These results demonstrate that at moderately elevated temperatures, aliphatic dialcohols potently affect the recruitment of RNA polymerase and/or prevent its retention on DNA. In parallel, histone H3 occupancy was significantly increased, well beyond the histone density typically seen under transcriptionally quiescent conditions.Here, we have tested the hypothesis that perturbation of an genes , 10. WhiA). In the first, “HD pretreatment,” cells were pretreated with 5% 1,6-HD or 2,5-HD for 2.5 min at 25 °C followed by upshift to 39 °C over the next 7.5 min. In the second, “HD post-treatment,” cells were subjected to a 25 to 39 °C upshift for 7.5 min followed by addition of 5% HD over the final 5 min. In both protocols, transcription was terminated by addition of 20 mM sodium azide, RNA was isolated, and transcript abundance was measured by RT–quantitative PCR (qPCR). The concentration of HD and times of incubation were selected based on previous work with mammalian nuclear condensates , consistent with previous observations . In the post-treatment protocol, when cells were heat shocked for 7.5 min followed by HD treatment for the final 5 min , heightened transcript levels were observed for the three genes, yet transcript abundance was frozen at 7.5 min heat-shocked levels (B and C and S1A), consistent with a normal transcriptional response for the first 7.5 min. Collectively, these results argue that HS-induced transcription is abrogated upon exposure of cells to either 1,6-HD or 2,5-HD. Interestingly, in cells exposed to 2,5-HD and maintained at the non–heat shock (NHS) temperature of 25 °C, a modest increase in transcript abundance was observed in certain cases  cells, transcript levels of the Hsf1-dependent genes rvations . In contOne explanation for the dramatic cessation of transcription is that 5% HD combined with moderately elevated temperature is deleterious to global metabolic activity such that even short-term cell viability is severely compromised. To rule this out, we measured cell viability by trypan blue staining and metabolic status by methylene blue dye exclusion in cells treated (or not) with HD. By these criteria, cells maintained at 25 °C were viable and metabolically active following either 5 min or 10 min treatment with 5% 1,6-HD or 2,5-HD, similar to untreated cells . Therefore, the repressive effect of HDs extends to HS-induced genes that do not prominently coalesce. A similar outcome was observed for the dual Hsf1- and Msn2-regulated HSP12 gene (C) that has been shown to coalesce . Consistent with this, mature transcript levels of these genes were also reduced (data not shown). Thus, at moderately elevated temperature, 1,6-HD and 2,5-HD inhibit not only the transcription of HS-induced genes but also that of housekeeping genes.To address whether HDs affect constitutive Pol II transcription, we designed primers spanning exon–intron junctions to specifically identify nascent pre-mRNA transcripts. Using this strategy, we found that nascent transcript abundance of both protocol , E and FWe next investigated the mechanism by which HD exposure, in concert with elevated temperature, inhibits Pol II transcription. A number of scenarios could be imagined. In one, the GSTF is prevented from gaining access to its cognate binding site (UAS) in chromatin. In another, the GSTF gains access, but recruitment of RNA polymerase is prevented. In a third, both GSTF and Pol II recruitment are permitted, but subsequent steps in the transcriptional cascade—namely, promoter escape and elongation—are prevented.HSP genes. Under NHS conditions, Hsf1 is present in the nucleus in an inactive complex with Hsp70 and the Hsp70 cochaperone Sis1 . At HSP82, 1,6-HD treatment prevented the further increase in Hsf1 binding that occurs in response to HS (A). As this enhanced occupancy has been previously attributed to cooperative binding of Hsf1 to low-affinity HS elements . These observations are therefore consistent with the notion that HD abrogates thermal stress–induced gene transcription whether it is added prior to or during exposure to elevated temperature. Analysis of the constitutively expressed ACT1 and TUB1 genes led to a similar outcome  (B and C and B). This increased nucleosome density, as much as 15-fold over the HD-untreated basal state, offers a potential mechanism to account for the decrease in Pol II occupancy elicited by the HDs. The increase in nucleosome density implied by these ChIP data is all the more remarkable in light of the minimal transcription occurring at these genes under NHS conditions (B and C and S1A)  where histones have already been displaced by prior HS . At genes whose chromatin is less dynamically regulated—PGM2, HSP12, ACT1, and TUB1—both pre-HD and post-HD treatment led to a substantial enhancement of H3 density (twofold to fivefold) throughout regulatory and coding regions (B and C). These results indicate that the combination of elevated temperature and HD stabilize chromatin structure to a remarkable degree, in certain cases exceeding by 10-fold the nucleosome compaction seen in control noninduced cells.To gain further insight into the role played by HDs, we examined H3 occupancy, which serves as a proxy for nucleosome density. As previously observed, acute HS induces gene-wide displacement of histones at Hsf1 target genes and S2B) , 22. PreB , 22. Pand S1A) . The inc regions , B and CSIR-silenced heterochromatic loci. As shown in A and B, quiescent (ARS504 and the PHO5 promoter) and heterochromatic (HMLα and YFR057w) loci responded in a similar fashion to HD/HS treatment as the transcriptionally active ones. Therefore, even at loci that are rarely transcribed, the combination of HD  and HS suppresses Pol II occupancy to near-undetectable levels while dramatically increasing histone H3 density. Second, we asked if HD pretreatment similarly impacted actively transcribing Pol III genes. Indeed, occupancy of Rpc1, the large Pol III subunit, is nearly obviated at two such genes, SCR1 and RPR1; concomitantly, H3 occupancy at each increased ∼10-fold. Thus, the profound effect of HD/HS treatment on RNA polymerase and nucleosome occupancy is not restricted to Pol II transcribed loci. Third, we attempted to demonstrate one aspect of the phenomenon, increased chromatin compaction, using an orthogonal approach. For this analysis, we used an H2A-mCherry–expressing diploid yeast strain and determined chromatin volumes in cells exposed to HD , HS, both HD and HS, or neither one. Consistent with the notion that chromatin compaction increased in cells exposed to the HD/HS treatment, the volume of chromatin decreased significantly (∼25%) in cells exposed to the dual treatment . While exposure to 2,5-HD alone had little effect—consistent with H3 ChIP analysis of NHS cells (A and B). The HS result is consistent with a recent report that yeast cells subjected to mild HS exhibit a diminished nuclear volume , diminished nuclear volume cannot be the entire basis for the substantial increase in nucleosome compaction arising from dual HD/HS exposure.To provide additional evidence for this striking phenomenon, we used three strategies. First, we investigated the effects of the HD pretreatment protocol on histone density and Pol II occupancy at weakly expressed euchromatic loci and HS cells —exposurer volume . Therefoblue bars). Likewise, cells subjected to thermal stress alone (either 7.5 or 12.5 min) were able to fully recover . Cells were able to form colonies following either 5- or 10-min exposure to 5% HD at 25 °C and to a degree similar to that observed for untreated cells  does not have the same effect: 2,5-HD causes a slight increase in Pol II occupancy, whereas it has no effect on H3 density; 1,6-HD has no effect on Pol II occupancy although it does slightly increase H3 abundance at certain genes  is due to their low—but nonzero—level of transcription that might result in H3 displacement and potential unfolding of higher-order chromatin structure. However, the fact that the HD/HS protocol elicits a substantial increase in H3 abundance at genes embedded in silent heterochromatin (B) whose transcription is virtually undetectable . By conIn conclusion, while others have pointed out limitations and caveats to the use of 1,6-HD in studying biomolecular condensates , 45, ourStrains used in this study are listed in To measure total RNA abundance, cells from a 12.5 ml midlog culture were metabolically poisoned by adding 20 mM sodium azide  following HS/HD treatment, harvested at 4 °C, and lysed by vortexing with glass beads in 300 μl 200 mM Tris–HCl (pH 7.5), 500 mM NaCl, 10 mM EDTA for 10 min at 4 °C, twice with a break of 2 min on ice. Nucleic acids were then purified by phenol–chloroform extraction and precipitated through addition of 3 volumes of ice-cold 100% EtOH, 300 mM sodium acetate, and 2 μl of RNA-grade glycogen , incubating on ice for 2 h. RNA was then pelleted by centrifugation, washed in 70% ethanol, air dried, and dissolved in 50 μl of nuclease-free water.SCR1 Pol III transcript was used as a normalization control. Primer sequences are provided in TURBO-DNase kit  was used to remove DNA contamination, and a 2 μg aliquot of purified RNA was used to synthesize complementary DNA (cDNA)  in a Bio-Rad thermal cycler set at 25 °C for 10 min, 37 °C for 2 h, and 85 °C for 5 min in a 60 μl reaction volume. Following this, 2 μl of the resultant cDNA were used for qPCR using Bio-Rad 2× iTaq Universal SYBR Green Supermix and gene-specific primers. Relative cDNA levels were calculated using the standard curve method . To corrTo perform ChIP assays, 50 ml midlog W303-1A cultures were used in either the HD pretreatment or HD post-treatment protocol. In each, HS was terminated by adding formaldehyde to a final effective concentration of 1% (363 mM). Given that HD reacts in a 1:1 stoichiometry with HCHO to form an acetal, to compensate for its presence, 786 mM 2.16%) formaldehyde was added to the +HD samples (5% HD = 423 mM). Following 10 min incubation at either 39 °C (HS samples) or 25 °C (NHS samples), 363 mM glycine was added for another 10 min to quench unreacted HCHO. Cells were harvested at 4 °C, washed, and stored at −80 °C. Cell lysis, chromatin sonication, and immunoprecipitation were conducted essentially as described  were grown to midlog (absorbance at 600 nm = 0.6) in synthetic dextrose complete (SDC) medium supplemented with adenine. Independent cultures were grown for each of three conditions ; a paired NHS control was evaluated in each case. Cells were attached to a smart substrate with reservoir  by coating the substrate with 100 μg/ml concanavalin A (Sigma–Aldrich) prior to cell attachment. Cells were incubated for 20 min at 25 °C; the medium was then replaced with either SDC or SDC supplemented with 5% 2,5-HD for 2.5 min. Cells were then subjected to an instantaneous 7.5 min 25 to 39 °C HS by using the VA-HEAT temperature controller (or kept at 25 °C). Images were acquired using an Olympus CSU W1 Spinning Disk Confocal Microscopy System set at 10% laser power. For each field, 11 z-planes (each imaged for 100 ms) were taken with interplanar distance of 0.5 μm using a 100× objective. To quantify volume occupied by H2A-mCherry, we used the 3D Object Counter plugin in Fiji/ImageJ (Open-Source) . A manuaTo measure the viability and metabolic status of the cells immediately following HS/HD treatment (HS conducted at 42 °C), a 100 μl aliquot of midlog culture of RMY005 (absorbance at 600 nm = 0.5) was diluted 1:10 in 1 ml YPDA (25 °C). The cell suspension was centrifuged and resuspended in 30 μl YPDA to remove most traces of HD. For the viability assay, cells were stained with an equal volume of 0.4% trypan blue  in phosphate-buffered saline for 1 min. To assay metabolic activity, cells were stained with 0.1 mg/ml of methylene blue  in 2% dihydrate sodium citrate solution for 5 min at room temperature. Viable cells  and metabolically active cells (those that excluded dye) were counted by light microscopy using a hemocytometer.To quantify long-term viability, midlog RMY005 cells were diluted 2500-fold with YPDA following the aforementioned HS/HD treatment, and 50 μl (∼200–300 cells) were plated on rich medium. The cells were allowed to grow for 1.5 to 2 days before counting colony-forming units. Controls for this and the aforementioned assays were cells maintained at 25 °C throughout the HD/HS protocol.david.gross@lsuhs.edu).All data described here are contained within the article as either a main figure or a supporting figure. Primary data will be made available upon request. Address all inquiries to David S. Gross (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Chromatin structure is a major regulator of DNA-associated processes, such as transcription, DNA repair, and replication. Histone post-translational modifications, or PTMs, play a key role on chromatin dynamics. PTMs are involved in a wide range of biological processes in eukaryotes, including fungal species. Their deposition/removal and their underlying functions have been extensively investigated in yeasts but much less in other fungi. Nonetheless, the major role of histone PTMs in regulating primary and secondary metabolisms of filamentous fungi, including human and plant pathogens, has been pinpointed. In this review, an overview of major identified PTMs and their respective functions in fungi is provided, with a focus on filamentous fungi when knowledge is available. To date, most of these studies investigated histone acetylations and methylations, but the development of new methodologies and technologies increasingly allows the wider exploration of other PTMs, such as phosphorylation, ubiquitylation, sumoylation, and acylation. Considering the increasing number of known PTMs and the full range of their possible interactions, investigations of the subsequent Histone Code, i.e., the biological consequence of the combinatorial language of all histone PTMs, from a functional point of view, are exponentially complex. Better knowledge about histone PTMs would make it possible to efficiently fight plant or human contamination, avoid the production of toxic secondary metabolites, or optimize the industrial biosynthesis of certain beneficial compounds. The interaction between the highly basic histones, due to numerous arginine and lysine side chains, and the negatively charged phosphate backbone of DNA maintains a charge neutralization that allows nucleosome stabilization [linker histone H1, is not directly incorporated into the nucleosome, but interacts with it to ensure chromatin cohesion as well as contribute to chromatin dynamics [facultative to refer to genomic regions that can dynamically adopt open or compact conformations, or as constitutive when genomic regions with a definitively compact conformation are considered. Literature regarding the biological functions of chromatin structure changes is diverse and abundant, and chromatin dynamics are largely described to be major players in a wide array of events, such as growth, differentiation, adaptation to changing environments, or genome stability and evolution.Eukaryotic genomic DNA (gDNA) is organized in a dynamic structure, known as chromatin. Its fundamental unit is the nucleosome, i.e., ~146 base pairs of gDNA wrapped around a histone octamer containing two copies of each histone, H2A, H2B, H3, and H4 [Histone genes are tightly regulated; they are typically expressed during the replication phase of the cell cycle , causing nausea, vomiting, and immunosuppressive diseases; fumonisins mostly produced by Fusarium verticillioides and Fusarium proliferatum on corn may provoke human oesophageal cancer upon chronic exposure [Studies focusing on yeasts have often pioneered investigations on chromatin structure dynamics. The budding yeast ng genes , with th studies . Fungal  studies . These lexposure . Despitelocus [The production of secondary metabolites is largely dependent on environmental parameters, such as nutrient sources , pH changes, or oxidative stress ,16. Non-locus . This cllocus .The genomic location of these clusters has been identified as a key factor of their regulation . Indeed,S. cerevisiae, and S. pombe to a lesser extent  and severe defects in capsule production [Ustilago maydis responsible for corn smut disease, the loss of UmGcn5 (HAT) leads to a downregulation of genes known as involved in pathogenicity associated with a dramatic loss of virulence in planta [U. maydis morphogenesis [F. graminearum, the loss of H3K14 and H3K4 acetylation in ΔFgSas3 mutant, or H3K9, H3K14, H3K18, and H3K27 in ΔFggcn5 mutant, showed a sharp growth rate reduction, respectively of 40% and 70% [ΔFgsas3 and to the complete loss of asexual reproduction ability in ΔFggcn5, suggesting specific functions for these acetyltransferases. In Beauveria bassiana, an entomopathogenic ascomycete, H3K56ac, deposited by the HAT Rtt109, is also required for normal growth and conidiation [A. flavus, Rtt109, responsible for both H3K9ac and H3K56ac, is also involved in growth, asexual reproduction, and in pathogenicity [N. crassa, H3K14ac deposited by Gcn5 is an active mark for light-induced gene expression [al-3 gene (light inducibility gene) has been observed concomitantly with al-3 mRNA expression induction. In addition, the substitution of H3K14 by glutamine (which cannot be acetylated) leads to the loss of photo-inducibility, proving its function in light response [F. graminearum, FgSAS3 and FgGCN5 exhibited a role in the oxidative and osmotic stress sensitivity [Studies on histone acetylation have highlighted its major role in developmental events and in pathogenicity in fungi. In the yeast, sed host , the deloduction . In the n planta . In addiogenesis . In the  and 70% . In addiidiation . In a sigenicity . In addipression . Followiresponse . In F. gsitivity . AltogetMagnaporte oryzae that infects rice and wheat, H3K18ac deposited by Sas3 [C. albicans, in the ascomycete Cochliobolus carbonus, or in U. maydis [Conversely, proper histone deacetylation is also essential for development. For instance, in the phytopathogenic ascomycete  by Sas3  is deace by Sas3 . This de by Sas3 . The dec by Sas3 . Actuall. maydis ,53,54. AA. nidulans [A. nidulans, the loss of GcnE (homologous to GCN5) leads to an impaired production of sterigmatocystin and terrequinone and to only 3% of penicillin production compared with the WT [A. nidulans, that H3K9ac and H3K14ac deposited by GcnE have been localized by ChIP-seq in the promoter region of the sterigmatocystin biosynthesis positive regulatory gene aflR upon the induction of secondary metabolism production [A. nidulans. In F. graminearum, the loss of acetylated H3K4, H3K9, H3K14, H3K18, and H3K27 by the deletion of FgGcn5 and FgSas3 leads to virtually no DON production concomitantly with a significant downregulation of genes contained in DON cluster [A. parasiticus, similar observations about the positive role of histone acetylation on secondary metabolite production have been made [Aflr to Aflr-binding sites located in promoter regions of the aflatoxin biosynthesis gene cluster, leading to a co-ordinated activation of aflatoxin production. By contrast, three F. graminearum PKS (polyketide synthases (PKS), involved in the biosynthesis of zearalenone (ZEA), fusarielin, and arsellinic acid, respectively, are up regulated in ΔFgSAS3 [The involvement of chromatin structure changes in modulating the production of fungal secondary metabolites was first described in nidulans . Since tnidulans . In A. nh the WT . These roduction . In addioduction ,59,60, e cluster . In A. peen made . In this ΔFgSAS3 . These c ΔFgSAS3 ,63.The role of histone methylation on chromatin structure remains unclear, despite much literature on the subject. From the biochemical point of view, histone methylation allows the tighter wrapping of histones tails around DNA, leading to a restricted access to DNA. In addition, this modification serves as docking sites for other proteins, especially those containing a chromodomain . The majF. fujikuroi, mutants devoid of the KMT Ash1, responsible for the deposition of H3K36me3, were lacking two subtelomeric regions of chromosome I and X, suggesting a role of Ash1-dependant H3K36me3 on chromosome stability [Δset2Δash1, suggesting that subtelomeric deposition of H3K36me3 depends on Ash1 and requires the presence of Set2 [S. pombe. The deposition of H4K20me by SpSet9 is an essential mark associated with H2A phosphorylation for recruiting Crb2 (DNA damage checkpoint protein) to nuclear specific loci of DNA damage [Methylation is involved in various molecular processes, such as DNA damage repair (DDR), genome integrity, chromatin status regulation, and recently the siRNA mechanism. In tability . This ge of Set2 . AnotherA damage . This exN. crassa, in which NcDIM5 (H3K9 methyltransferase) has been identified as responsible for the deposition of H3K9me3 [N. crassa [A. nidulans, in which similar underlying mechanisms for heterochromatin formation are observed [F. graminearum showed that the deletion of FgHp1 leads to the loss of H3K9me3, demonstrating a modification of this sequential mechanism [S. pombe, the small interfering RNA (siRNA) pathway is involved in H3K9me3 positive feedback and in gene silencing inheritance [epe1 partially suppresses the requirement of siRNA for gene silencing. Here, the siRNA pathway and the deposition/removal of H3K9me3 are intertwined mechanisms for gene silencing inheritance.One of the most studied histone methylation marks may be H3K9me3, mostly known as involved in heterochromatin formation. This mechanism has been elucidated in  H3K9me3 . H3K9me3 H3K9me3 , allowin. crassa . This meobserved . Contrasechanism . In S. peritance . Cells lF. graminearum leads to severe growth defects and decreased virulence [Colletotrichum higginsianum, the deletion of CclA required for the deposition of H3K4me3 leads to reduced mycelial growth, asexual sporulation, and spore germination, a decreased production of secondary metabolites production associated with a strongly reduced virulence on plants [N. crassa, H3K4me3 has been identified as playing a role in the normal clock gene expression, notably by regulating the proper expression of frq gene, which is a major gene associated with the regulation of circadian and light-system [N. crassa under temperature fluctuation [N. crassa strains with deficiency in histone methylation showed that H3K36me deposition/removal follows a rhythmic cycle, leading to the cyclic activation of the frq locus [F. fujikuroi strongly affects vegetative growth and conidiation and reduces its pathogenicity in planta [N. crassa, the loss of H3K36 mono-, di-, and tri-methylation leads to slower growth, conidial defect, and female sterility [In filamentous fungi, the role of histone methylation in developmental processes has been extensively studied. Four main histone methylations were mostly studied, H3K4me, H3K9me, H4K27me, and H3K36me for their major role in developmental and virulence regulation. H3K4me is typically described as an active gene mark. The reduction of H3K4me in irulence . In the n plants . In N. ct-system ,81. In actuation . Moreoverq locus ,83. Thesn planta . In N. cterility .N. crassa ΔNcDim5 displays a moderately altered phenotype, showing slow and irregular growth [F. verticilloides ΔFvdim5 mutant exhibited severe defects in conidiation and in perithecium formation [F. graminearum did not exhibit phenotypic differences compared to the wild type [F. graminearum and F. fujikuroi, it has been shown that almost a third of their respective genome is enriched with H3K27me3 deposited by KMT6, suggesting its fundamental role in these fungi [F. graminearum, the lack of KMT6 leads to no more H3K27me3 and strong developmental defects, such as sterility, slow growth, an aberrant fate of germination, and reduced pathogenicity [M. oryzae, the same deletion leads to slower growth [F. fujikuroi is lethal [Regarding the heterochromatic mark H3K9me, roles seem to differ notably depending on the considered species. r growth . Moreoveormation . In contild type . Such diild type . In F. gse fungi ,86. In Fgenicity . In M. or growth . More drA. nidulans three protein arginine methyltransferases (PRMTs) have been identified: RmtA (arginine methyltransferase A) and RmtC involved in H4R3 methylations, and RmtB involved in the arginine methylation of H2A, H3, and H4 [rmtA and ΔrmtC A. nidulans mutants exhibited growth retardation under osmotic stress [RmtC mutants, although less pronounced. These observations are consistent with post-translational modifications of histones allowing a rapid and adapted response to environmental changes.As mentioned earlier, histone arginine can also be modified by methylation, although this PTM is less documented than lysine methylation. In , and H4 . ΔrmtA ac stress . The samF. graminearum and F. verticilloides, H3K4me has been identified mainly as a transcriptional activator of biosynthetic gene clusters. For instance, the loss of Set-1 in F. verticillioides and F. graminearum, involved in the deposition of H3K4me3, leads to a decrease of fumonisins and DON production, respectively, coupled with a downregulation of the expression of their respective biosynthetic gene clusters [Epichloë festucae, in several Aspergillus species and in C. higginsianum leads to the induction of biosynthetic gene clusters [F. fujikuroi leads to an increased production of fusarins and bikaverin [F. graminearum and A. nidulans [The role of histone methylation in the regulation of secondary metabolism in filamentous fungi has been scrutinized in various studies. In clusters ,90,91. Cclusters ,92,93,94nidulans ,95. In tA. nidulans, ChIP-seq (Chromatin-ImmunoPrecipitation-sequencing) experiments demonstrated that H3K9me3 deposited by ClrD is associated with the silenced sterigmatocystin cluster [A. nidulans’ genome leads to a global over-expression of secondary metabolite genes. In the same way, the loss of H3K9me3 in F. graminearum leads to higher levels of production of aurofusarin, involved in its red pigmentation [F. graminearum after the loss of H3K9me3. This decrease, contrasting with other observations in filamentous fungi, may be explained by a de-repression of a DON cluster repressor or a combinatorial effect with an unknown player.H3K9me3 is associated with the repression of the biosynthetic gene cluster. In  cluster . In addientation . In contF. graminearum and F. fujikuroi, secondary metabolite genes expression was de-repressed in KMT6 lacking or knock-down mutants, including aurofusarin gene cluster, nine genes included in the fusarin C cluster, and some genes included in the DON cluster [E. fistucae, involved in the deposition H3K27me3, leads to a de-repression of Lolitrem and ergot alkaloids biosynthesis clusters [Finally, H3K27me3 revealed itself as an important player in repressing the production of secondary metabolites . In F. g cluster ,86. In tclusters .As a whole, these examples  to lysine chains . This adS. cerevisiae, H2BK123ub is tightly linked to gene transcription activation [GAL1 mRNA levels and 40% of SUC2 mRNA levels [S. cerevisiae H2BK123ub is further involved in modulating nucleosome occupancy through the chromatin remodeller Chd1, involved in the maintenance of a high level of H2B monoubiquitylation, notably towards the 3′ ends of genes [S. cerevisiae, the substitution of H2BK123ub and adjacent lysine residues, avoiding the ubiquitylation to others neighbouring sites, showed mitotic and meiotic defects [C. neoformans, decreased levels of H2BK123ub in Δbre1 mutants led to growth defects and reduced self-filamentation correlated with the upregulation of 27 genes involved in filamentation suppression [A. alternata, a plant necrotrophic fungus, was revealed by the deletion of AaBre1 that caused growth retardation, decreased conidiation, and reduced virulence when infecting Chrysanthemum morifolium [One of the most studied histone ubiquitylations in fungal species is H2BK123ub, highly conserved from yeast to human H2BK120ub) ,116. Thetivation . This trtivation ,120,121,ub ,116. tivation . SupportA levels . On the A levels ,125. TheA levels . S. cereof genes . H2BK123of genes . This nuof genes . In addi defects . In C. npression . A functrifolium . As a whS. cerevisiae  and E2 (Ubc9 in yeast) that combine to a handful of known SUMO E3 enzymes  and a few other candidates with, to date, no demonstrated activity  is a 10 kDa ubiquitin-like modifier that, similar to ubiquitin, can covalently bind lysine side chains of numerous proteins, including histones. In er fungi ), there ity  to fluconazole, which showed no antifungal capacity on these species when tested alone [C. glabrata to azoles and of S. cerevisiae to Brefeldin A has been highlighted [Fungi are major producers of secondary metabolites, which are incredibly structurally diverse molecules. Many of them are of considerable interest for pharmaceutical drug development. In contrast, some of these secondary metabolites represent a huge health hazard. In fact, just few fungal human pathogens are responsible for almost 1.5 million deaths per year . Antifun species . This co species  and the  species . For inse arrest . TSA als. oryzae ,177,178.. oryzae ). One waglabrata . It alsoed alone . To datehlighted ,181. TheThe plurality and diversity of histone PTMs and their cross-interaction identification and understanding in filamentous fungi represent a challenging issue to be addressed. Deepening studies about “classical” PTMs, such as acetylation and methylation, coupled with studies about “non-classical” PTMs, such as lysine acylation, would allow a better understanding of these PTMs, their interactions, and their role in filamentous fungi. Recent views of the dynamics of histone PTMs deposition and removal underline a possible role of chromatin to store chemical groups, such as acetate derived from acetyl-CoA, to be released when the cell needs it (discussed in ). For ex