Trametes hirsuta LE-BIN 072 ligninolytic system to the presence of various monolignol-related phenolic compounds  in culture media was monitored at the level of gene transcription and protein secretion. By showing which isozymes contribute to the overall functioning of the ligninolytic system of the T. hirsuta LE-BIN 072, the data obtained in this study will greatly contribute to the possible application of this fungus and its ligninolytic enzymes in lignin depolymerization processes.Being an abundant renewable source of aromatic compounds, lignin is an important component of future bio-based economy. Currently, biotechnological processing of lignin through low molecular weight compounds is one of the conceptually promising ways for its valorization. To obtain lignin fragments suitable for further inclusion into microbial metabolism, it is proposed to use a ligninolytic system of white-rot fungi, which mainly comprises laccases and peroxidases. However, laccase and peroxidase genes are almost always represented by many non-allelic copies that form multigene families within the genome of white-rot fungi, and the contributions of exact family members to the overall process of lignin degradation has not yet been determined. In this article, the response of the  Lignin valorization has been a recurring theme in chemistry and biotechnology for over a century. As an essential part of plant cell walls, lignin is the second most abundant biopolymer on Earth after cellulose . The pulTo date, the depolymerization of lignin into low molecular weight products is one of the promising methods for its valorization. Some products of lignin depolymerization can be valuable chemicals in their own right. For example, 15% of the world’s vanillin is currently produced by the oxidation of lignosulfonate or kraft-lignin . AlternaTrametes spp., Pycnoporus spp. and Polyporus spp.—are the best known [Since all current commercial-scale lignin depolymerization methods  require high energy input and harsh conditions, the search for an alternative cost-efficient lignin depolymerization process is an active area of research . One of st known ,15.2O2 as an electron acceptor for the oxidation of lignin and lignin-like compounds [Currently, two groups of enzymes responsible for the oxidative lignin depolymerization by white-rot fungi were identified: laccases and ligninolytic peroxidases . Laccaseompounds . The perompounds ,22. Lignompounds . Manganeompounds ,25. Versompounds .In the genomes of white-rot fungi, laccase and peroxidase genes are almost always represented by many non-allelic copies that form multigene families ,27,28,29Trametes hirsuta (Wulfen) Lloyd 1924 is a typical representative of white-rot fungi. The genome of T. hirsuta contains seven laccase and eighteen peroxidase  encoding genes [T. hirsuta LE-BIN 072 ligninolytic system to the presence of various MRPCs in culture media was monitored at the level of gene transcription and protein secretion. In addition, previous exoproteomic studies of T. hirsuta cultivated on various media were summarized.Belonging to the Core Polyporoid clade, ng genes ,37,38. IT. hirsuta LE-BIN 072 (hereinafter referred to as T. hirsuta 072) on the control GP medium and GP media supplemented with four MRPCs—veratryl alcohol (VA), p-coumaric acid (pCA), vanillic acid (VnA), or syringic acid (SA)—are presented in The dynamics of biomass accumulation and changes in the overall oxidative activity of the cultural broth during cultivation of T. hirsuta 072 showed the same dynamics of biomass accumulation on all the studied media: the logarithmic growth phase was observed between the first and sixth days of cultivation; the stationary phase was observed between the sixth and ninth days of cultivation; and slow biomass decline was observed after the ninth day of cultivation. For all cultivations, the profile of the overall oxidative activity has a pronounced maximum. For the VA- and pCA-containing media, the maximal activity was observed at the sixth day of cultivation, and for the VnA- and SA-containing media—at the ninth day of cultivation.During cultivation, p-hydroxyphenyl lignin units demonstrated higher antifungal properties than compounds based on syringyl units [Existing data on the cultivation of various wood-degrading fungi in the presence of aromatic compounds show that these compounds can have a growth-inhibiting effect even at low concentrations. In particular, it was shown that some MRPCs can exhibit strong antifungal properties depending on the length of the CH chain attached to the benzene ring and the presence of conjugated double bonds, carbonyl or carboxyl functional groups in this chain ,43. Geneyl units . In addiyl units .Dichomitus squalens, an efficient wood-degrading white-rot fungus, did not show any growth inhibition when cultivated with 10 different MRPCs [From published data, it also can be hypothesized that among wood-degrading fungi, those with a more developed ligninolytic system are more resistant to growth inhibition by MRPCs. Accordingly, brown-rot fungi were reported to be more sensitive to the growth-inhibition compared to white-rot fungi ,45,46,47nt MRPCs .T. hirsuta 072  and nine isozymes of ligninolytic peroxidases—one versatile peroxidase (VP2), six manganese peroxidases , and two lignin peroxidases (LiP2 and LiP9). Additionally, T. hirsuta 072 secreted four glycoside hydrolases , five serine proteases (from S09X and S53 families), one glyoxal oxidase, one nucleotidase, and four proteins with unknown function. All secreted proteins had proper signal peptides and were therefore secreted by the classical secretory pathway.The majority of enzymes secreted by lacA of T. hirsuta 072 is a gene that is expressed by default  [T. hirsuta 072 was cultivated on the media containing CuSO4, kraft lignin, synthetic dye , lignocellulose and sawdust of various wood , LacA isozyme was always present in the fungal exoproteome  . As in tproteome  [38,49,5ncreased , on the g medium .T. hirsuta 072 are the most abundant laccase isozymes secreted under various conditions by fungi from Trametes and, closely related to it, Pycnoporus genera. For example, LacA orthologues were found in exoproteomes of three Pycnoporus spp. cultivated on control maltose-containing medium as well as on media supplemented with cellulose, wheat straw, aspen chips, and grass biomass [Trametes versicolor A1-ATF during its cultivation on aspen wafers [T. versicolor FP-101664 exoproteomic response to MRPCs, no laccase isozymes were detected in exoproteomes during cultivations with either syringic or 4-hydroxybenzoic acids [Using previously obtained data about orthologous groups of laccases and peroxidases from different fungi ,53, it c biomass ,55. In an wafers . Howeveric acids .T. hirsuta 072, MRCPs induce secretion of broader spectrum of peroxidase isozymes than lignin itself. In the current study, T. hirsuta 072 secreted from six to nine peroxidase isozymes on all media supplemented with MRCPs, while in the previous studies lignin induced secretion from two to six peroxidase isozymes at different stages of cultivation  were not transcribed on any medium. No correlation was found between gene transcription patterns and their phylogenetic relationships. In addition, genes located in close proximity to each other  did not show similar transcriptional patterns.For both laccase and peroxidase genes, changes in the level of transcription on media supplemented with MRCPs did not differ from those on the control GP medium by more than 10-fold. Transcription of all laccase genes and 15 peroxidase genes was detected on all studied media; three peroxidase genes  from that on the control GP medium. For SA medium after the third day of cultivation, it was reported [lacC and lacB was induced by approximately six-fold, transcription of lacF was induced by 36-fold, and transcription of lacG was undetectable. In the current study transcription of lacB, lacF was also increased by approximately four-fold and transcription of lacG was suppressed by five-fold. Additionally, transcription of lacA and lacD was increased by 4 ± 1- and 11 ± 3-fold, respectively. For VA and VnA, the transcriptional patterns found in this study were completely different from those previously observed after cultivation of T. hirsuta 072 on this media for three days [In general, the transcription patterns of laccase genes on onounced . For pCAreported  that trareported  that traree days .T. hirsuta 072 cultivated in the presence of bromocresol green and kraft lignin [In the case of peroxidase genes, the observed transcription pattern was different from that previously reported for t lignin . WhereaspCA and SA, where the transcription patterns on days 6 and 9 generally coincided with that earlier reported on day 5 [D. squalens [Pycnoporus spp.  [Comparing the results of this study with those obtained for other polypore fungi, it can be assumed that there is a significant time lag between the increase in laccase and peroxidase gene transcription and secretion of the corresponding isozymes into the medium. While it was shown that in principle transcription levels of laccase and peroxidase genes can vary by 2–3 orders of magnitude ,61, the on day 5 , but thesqualens ,64 and Pbarinus) , where oT. hirsuta LE-BIN 072 was obtained from the Komarov Botanical Institute Basidiomycetes Culture Collection . After receiving from the collection, the strain was stored at a temperature of +4 °C on slant wort agar . The fungal culture was stored without reseeding from six months to one year.The strain of the white-rot fungus To obtain starting inoculum, fungal mycelium was subcultured from slant wort agar onto Petri dishes (d = 90 mm) containing wort agar of the same composition. Petri dishes were cultivated in the dark at 28 °C until the surface of the dish was completely covered with mycelium. Then, pieces of mycelium from the Petri dish (~1/4 of the dish) were added to 750 mL conical flasks containing ceramic beads and 200 mL of glucose peptone (GP) medium. The seeded flasks were incubated in the dark at 26–28 °C for 7 days. Before further inoculation on the studied media, the mycelium of the fungus was crushed with ceramic beads at 250 RPM for 15 min. Inoculation was carried out with 20 mL of the resulting suspension.−1): glucose—10.0; peptone—3.0; KH2PO4—0.6; ZnSO4 × 7H2O—0.001; K2HPO4—0.4; FeSO4 × 7H2O—0.0005; MnSO4—0.05; MgSO4 × 7H2O—0.5; CaCl2—0.25. The medium was autoclaved at 120 °C and 1 atm for 30 min.The composition of the GP medium was as follows  at a temperature of 25 °C in a dark aerated chamber of an orbital shaker Innova 44 .For cultivation of To measure the dry weight, samples of the mycelium were dried at 105 °C to a constant weight, and air-dry biomass was determined by the gravimetric method.436 = 29,500 M−1 cm−1). The enzymatic reaction was carried out in 2 mL of 0.1 M sodium acetate buffer (pH 4.5), 1 mM ABTS, and the required amount of culture liquids to reliably determine the initial reaction rates. One unit of enzymatic activity was expressed as μM of product formed per min.The overall oxidative activity was measured using ABTS as a substrate by monitoring the increase in absorbance at 436 nm for 3 min. The substance detected in the reaction was the oxidation product of ABTS , MALDI-TOF/TOF MS/MS analysis, and data processing were performed as previously described in Moiseenko et al. .The RNA extraction, reverse transcription, and qPCR analysis for laccase genes were performed as described in Moiseenko et al. . For theF-test. When a significant (p < 0.05) value of F-statistics was found, differences between means were evaluated using Tukey’s HSD (honestly significant difference) multiple comparison test (p < 0.05).All experiments were performed in three biological replicates, and data are presented as mean ± standard deviation when appropriate. All statistical comparisons were firstly performed using a one-way ANOVA omnibus T. hirsuta 072 can grow not only in presence of lignin but in the presence of monolignol-related phenolic compounds (MRPCs), which can be toxic in general for other fungi. It was demonstrated that for T. hirsuta 072, MRCPs induce secretion of broader spectrum of ligninolytic peroxidase isozymes than lignin. The MRPCs can trigger expression of such peroxidase isozymes as MnP1, MnP2, MnP3, MnP6, LiP2, and LiP9, among which the secretion of LiP2 was never observed in previous studies. In addition, it was shown that such peroxidase isozymes as MnP5, MnP7, and VP2 were always secreted by T. hirsuta 072 regardless of the presence of MRPCs in the medium. It has been suggested that the simultaneous secretion of many peroxidase isozymes is a hallmark of T. hirsuta as a species. In addition, it was shown that it is unusual for fungi closely related to T. hirsuta  to secrete orthologues of the MnP5 isozymes that is always secreted by T. hirsuta 072 by default. Surprisingly, MRPCs did not induce secretion of laccase isozymes other than LacA, which T. hirsuta 072 almost always expresses by default. Additionally, it was again highlighted that in white-rot fungi, gene transcription, protein secretion, and activity observed in cultural broth do not strictly correlate with each other. This important observation must be always accounted for in further investigations of white-rot fungi in which measurements are taken at only one of the mentioned biological levels.In summary, the current investigation highlighted that as primary colonizing white-rot fungus,


Chromatin plays a central role in the conversion of energy in cells: alteration of chromatin structure to make DNA accessible consumes energy, and compaction of chromatin preserves energy. Alteration of chromatin structure uses energy sources derived from carbon metabolism such as ATP and acetyl-CoA; conversely, chromatin compaction and epigenetic modification feedback to metabolism and energy homeostasis by controlling gene expression and storing metabolites. Coordination of these dual chromatin events must be flexibly modulated in response to environmental changes such as during development and exposure to stress. Aging also alters chromatin structure and the coordination of metabolism, chromatin dynamics, and other cell processes. Noncoding RNAs and other RNA species that associate directly with chromatin or with chromatin modifiers contribute to spatiotemporal control of transcription and energy conversion. The time required for generating the large amounts of RNAs and chromatin modifiers observed in super-enhancers may be critical for regulation of transcription and may be impacted by aging. Here, taking into account these factors, we review alterations of chromatin that are fundamental to cell responses to metabolic changes due to stress and aging to maintain redox and energy homeostasis. We discuss the relationship between spatiotemporal control of energy and chromatin function, as this emerging concept must be considered to understand how cell homeostasis is maintained. Maintaining cell homeostasis is fundamental to life. Metabolism controls the balance of acquisition and consumption of energy and is pivotal in maintaining cell homeostasis and cell responses to environmental changes during development, exposure to stress, and aging. Cell respiration and ATP synthesis are regulated by ATP utilization, and macromolecule biosynthesis is highly sensitive to the supply of energy . Althoug2 concentrations, is hypothesized to be a phase separation process [In the study of chromatin’s role in cell homeostasis, a new concept has emerged from the observation that macromolecules including chromatin, RNA, and transcription activators are organized into membraneless subcellular compartments where they participate in biochemical reactions that are under spatiotemporal control , 19. It  process , 24. The process . The yie process –27. RNA  process . HoweverHere, we review the relationship between metabolism and chromatin dynamics while considering changes in metabolism and chromatin structure during aging. We also discuss emerging concepts regarding the regulation of chromatin modification and function by metabolic factors, noncoding RNAs, and phase separation.Drosophila melanogaster), CENPA, macro-H2A, and H1.0) are polyadenylated and are replication independent (expressed throughout the cell cycle) [The basic subunit of chromatin is the nucleosome core particle. Nucleosomes contain a histone octamer wrapped with 147 base pairs (bp) of double-stranded DNA , 29. Thel cycle) . An excel cycle) .Fig. 1ReThe reversible nature of chromatin status enables epigenetic reprogramming, but chromatin structure is also modified by changes in metabolism during aging Fig. . MicrocoD. melanogaster, clusters of histones H2A, H2B, and H2Av (the human H2A.Z homologue) have been found in histone locus bodies (HLBs) adjacent to Cajal bodies in nuclei [D. melanogaster [Histone storage, as opposed to suppressed synthesis, can regulate histone abundance. In n nuclei . Cajal bn nuclei . Cajal bn nuclei . Excess nogaster  Fig. 1)D. melanonogaster , increasnogaster . The nucnogaster . Polyubinogaster . Polyaminogaster , assist nogaster  and may nogaster . Lipid snogaster , 52. Mornogaster , and SAMHistone chaperones help select histones in the assembly or disassembly of nucleosomes, as each chaperone binds a different histone or combination of histones Table . For exaE. coli the number of ATPases of RecQ helicase and the number of hydrolyzed ATP molecules used for the enzyme reaction are limited, as this ATPase loses its structure and function after hydrolyzing a certain number of ATP molecules [ISW2, which encodes ISW2 ATPase in the ISW1 chromatin remodeling complex in yeast, derepresses genotoxic stress response genes upon calorie restriction and extends the replicative lifespan [ISW2 increases stress resistance during aging.ATP-dependent chromatin remodelers catalyze histone deposition and removal. Histone H2A.Z is deposited into chromatin by the SWR-C/SWR1 chromatin remodeling complex , and is removed by the INO80 complexes in yeast –67. Acetolecules . This reolecules  may incrlifespan , suggestAsf1 H3-H4 chaperone activity has been found to stimulate Set2 methyltransferase occupancy of the coding region of a highly transcribed gene in yeast . Set2-caGlucose is the source of central precursor metabolites such as pyruvate, acetyl-Coenzyme A (acetyl-CoA), ATP, and 2-oxoglutarate in prokaryotes and eukaryotes . These mS. cerevisiae, which lacks ATP-citrate lyase (ACLY) [+ -dependent histone deacetylation to synthesize acetyl-CoA for acetylation of histones H3K9, H3K27, and H3K56, which activates genes that synthesize lipids in human [S. cerevisiae, the SAGA histone acetylase complex activates transcription of growth-promoting gene by H3K9 acetylation upon glucose repletion after starvation; however, upon glucose starvation the Rpd3 histone deacetylase complex deacetylates growth-promoting gene, leading to the release of acetate, which may participate to the SAGA-acetylation of genes that promote gluconeogenesis and lipid synthesis [Acetyl-CoA is derived from catabolism of glucose, fatty acids, and amino acids. Acetyl-CoA is produced within the cytosol and mitochondria and pools in the cytosol and nucleus . Cellulae (ACLY) . In mamme (ACLY) , 82. Unde (ACLY) . In thesin human , 84. In ynthesis . Thus, hynthesis . It is uShort-chain acyl metabolic intermediates are substrates of histone lysine acylation including lysine propionylation, butyrylation, crotonylation, and β-hydroxybutyrylation . These hS. cerevisiae, methylation of phosphatidylethanolamine (PE), a class of phospholipids present in membranes, is a major consumer of SAMe and promotes transsulfuration, which transfers sulfur from homocysteine to cysteine, leading to synthesis of cysteine, taurine, and glutathione (GSH) [Histones also function as a methyl sink. The methionine derivative SAMe is the major methyl donor for methylation in the cell. In ne (GSH) , 92. GSHne (GSH) , 92. Whene (GSH) . This hine (GSH) . Interesne (GSH) . Loss ofne (GSH) . In mammne (GSH) . Increasne (GSH) . Misregune (GSH) –97. HencMYC and CCND1 by PKM2 is required for H3K9ac and subsequent expression of cyclin D1 and c-Myc [Histones can be phosphorylated at serine, threonine, or tyrosine residues. Histone phosphorylation plays important roles in DNA damage repair, transcription, and chromatin compaction . Most hind c-Myc . Proteinnd c-Myc . It will2) [90–95% of oxygen consumed by the body is utilized in mitochondria to generate ATP . Oxygen 2) . These r2) , 110. Th2) . Thus, t2)  may be a+ in reactions with nucleophiles [+ is involved in five reactions to capture a released electron and is further reduced to NADH [+ biosynthesis [+ precursor that is converted to nicotinamide mononucleotide, elevates NAD+ levels and promotes Sir2-dependent repression of recombination and gene silencing, leading to extension of replicative lifespan regardless of calorie restriction in yeast [+ availability controls Sir2 activity, and Sir2 feeds back to NAD+ biogenesis via its gene regulation.In humans, the sirtuin (SIRT) family consists of seven class III histone deacetylases (HDACs) . SIRTs aeophiles . For exa to NADH , 113. Inynthesis , 115. Hoin yeast . Thus, NDrosophila, the Ada Two-A-containing (ATAC) acetyltransferase complex activates JNK signaling and transcription of JNK target genes upon osmotic stress [There are direct connections between chromatin modifying complexes and the mRNA translation machinery in response to metabolic changes. The double-stranded RNA-dependent protein kinase (PKR) was initially found to be activated by its binding to viral double-stranded RNA and regulates the innate immune response . In mammc stress . Howeverc stress , 121. Thc stress , 122. tRc stress . Downregc stress . Dysreguc stress , 124. Thdihydrofolate reductase (DHFH) suppress transcription from the downstream major DHFR promoter [SPHK1 promotor, by probing with an in vitro triplex capture assay and EMSA [KHPS1 is transcribed antisense to SPHK1 and binds the SPHK1 promoter. This triplex further recruits p300/CBP, leading to increases in H3K27ac and H3K9ac and in the accessibility of the transcription factor E2F1, resulting in activation of SPHK1 transcription [fragile X mental retardation 1 (FMR1) gene is silenced by hybridization of a transcribed CGG-repeat tract of the 5’ UTR FMR1 mRNA to the complementary CGG-repeat of the FMR1 gene [FMR1 promoter is observed; however, it is unknown whether FMR1 mRNA recruits chromatin modifiers that directly change H3K9me2 levels, or whether this is a consequence of gene silencing by RNA:DNA duplex formation alone. Additional studies are needed to elucidate whether ncRNA alone regulates transcription or whether the association of ncRNAs with chromatin modifiers underlies the transcription regulation (discussed below).Noncoding RNAs function as transcriptional regulators. Long noncoding RNAs (lncRNAs) transcribed from a minor promoter upstream of human promoter . The sugand EMSA . The lnccription . RNA:DNAMR1 gene . H3K9me2Xist and HOTAIR—provides examples of gene silencing by lncRNA. Histone H2AK119 mono ubiquitination (H2AK119ub1) catalyzed by PRC1 initiates polycomb-mediated transcriptional repression and stabilizes H3K27me3 deposition by PRC2 in embryonic stem cells (ESCs) [Xist RNA, and non-canonical PRC1 (vPRC1), PCGF3/5/RING1 complex, leading to Xist-dependent H2AK119ub1 and Xist-mediated gene silencing [Xist and PCGF3/5/RING1 is required for Xist-mediated gene silencing [Xist RNA mediates polycomb interactions with super-enhancers in these structures. The lncRNA HOTAIR is transcribed from the HOXC locus and suppresses HOXD by deposition of PRC2-mediated H3K27me3. A 5’ domain of HOTAIR RNA binds to PRC2 whereas a 3’ domain of HOTAIR binds the LSD1/CoREST/REST complex [HOTAIR prevents gluteal adipocyte development by the interaction of HOTAIR RNA with PRC2 which silences genes that are involved in adipocyte lineage in iliofemoral adipose tissue [HOTAIR to bind chromatin [HOTAIR-mediated transcriptional repression in PRC2-depleted breast cancer cells was also observed [HOTAIR may play a role in the restriction of the PRC2-dependent H3K27me3 loci.The association of silencing factors and lncRNA—such as the association of polycomb-repressive complex 1 (PRC1) and PRC2 with lncRNA s (ESCs) , 129. hnilencing . Synthetilencing . Therefoilencing . It has ilencing   at H3K4me1-marked enhancers bi-directionally transcribes enhancer RNAs (eRNAs) which are synthesized at adjacent genes and positively correlate with the mRNA levels of these genes upon activation of calcium-dependent signaling . This obAn emerging concept is that liquid–liquid phase separation (LLPS) participates in genome organization and remodeling , 138. PhAcetylation on lysines has been thought to neutralize their positive charge and reduces interaction of histones with negatively- charged DNA , 148, prDrosophila and mice [23KRA was not shown. Importantly, Cbx223KRA/23KRA mice as well as Cbx2−/− mice display defects in patterning of the body axis [Highly positively charged LCDR/IDR  of the chromobox 2 (CBX2/M33) subunit of PRC1 causes chromatin compaction of PRC1 in and mice . Inductiand mice . H2AK119ody axis . Multivaody axis . For exaody axis . N-termiDepending on the requirements of cell signaling and metabolism, chromatin utilizes canonical histones or different histone subtypes which are synthesized at different periods. Histone chaperones select canonical histones and/or histone subtypes in response to various stimuli or for different biological processes. However, how histone chaperones sense metabolic status is still unclear. The dual functions of histones for metabolite storage and in chromatin functions make histones and chromatin themselves look like a metabolic pathway. There are still questions about the fate of metabolite-storage in histones and these metabolites. If intergenic regions of histone H4 acetylation function as acetate storage, it must be re-considered whether modifications of nucleosomes are direct regulators of nucleosome functions including transcription. Importantly, the interplay between chromatin modifiers and metabolism increases with the availability of oxygen, which provides cell energy via oxidation, and the goal of this interplay is prevention of the generation of excess reactive oxygen species.RNA molecules directly participate in chromatin functions and transcription through their association with chromatin or chromatin modifiers. The effects of metabolism and aging on the quality of those RNA species  must be elucidated. Accumulated observations of high yields of enhancer RNAs at super-enhancer regions raise questions about how nucleotides are produced for transcripts and enhancer RNAs upon a rapid stimulus. More studies of the mechanisms connecting transcription and translation are needed.+, Na+, and Cl−, which in turn control cell volume [Recent concepts of phase separation in the regulation of super-enhancers recalls the activation of stress-activated kinases (SAPKs), such as the activation of MAP kinase (MAPK) and JNK pathways , 154. Stl volume . Ste20-kl volume . Consequl volume . Changin


Enhancer-mediated gene activation generally requires physical proximity between enhancers and their target gene promoters. However, the molecular mechanisms by which interactions between enhancers and promoters are formed are not well understood. Here, we investigate the function of the Mediator complex in the regulation of enhancer-promoter interactions, by combining rapid protein depletion and high-resolution MNase-based chromosome conformation capture approaches. We show that depletion of Mediator leads to reduced enhancer-promoter interaction frequencies, which are associated with a strong decrease in gene expression. In addition, we find increased interactions between CTCF-binding sites upon Mediator depletion. These changes in chromatin architecture are associated with a redistribution of the Cohesin complex on chromatin and a reduction in Cohesin occupancy at enhancers. Together, our results indicate that the Mediator and Cohesin complexes contribute to enhancer-promoter interactions and provide insights into the molecular mechanisms by which communication between enhancers and promoters is regulated. Here, the authors map chromatin conformation at high resolution after rapid Mediator depletion to show that its loss reduces the frequency of enhancer-promoter interactions and associated gene expression, with a corresponding redistribution of Cohesin. In mammals, enhancers can be located far upstream or downstream of the genes they control. To activate genes, enhancers interact with promoters in dynamic three-dimensional (3D) chromatin structures2. Enhancer-mediated gene activation is therefore closely related to the three-dimensional organization of the genome3. However, the molecular mechanisms by which enhancer-promoter interactions are formed and enhancers drive gene expression remain incompletely understood.Precise spatial and temporal patterns of gene expression in metazoans are regulated by enhancers, which are short non-coding DNA sequences that drive expression of their cognate gene promoters4. In this process, ring-shaped Cohesin complexes translocate along chromatin and extrude progressively larger loops, until they are halted at CTCF-binding elements located at the boundaries of TADs5. Interacting enhancers and promoters are usually located in the same TAD6. Moreover, perturbations of TAD boundaries can cause ectopic enhancer-promoter interactions7. These observations suggest that loop extrusion could be involved in the regulation of enhancer-promoter communication and gene expression. Although it has been shown that depletion of components of the Cohesin complex does not lead to widespread mis-regulation of gene expression10, Cohesin and its associated factors have been reported to be important for the regulation of cell-type-specific genes13. In addition, it has recently been shown that depletion of Cohesin can cause weakening of enhancer-promoter interactions15. These observations suggest that Cohesin-mediated loop extrusion contributes to the formation of enhancer-promoter interactions16. However, the molecular mechanism remains unclear. Furthermore, depletion of Cohesin causes a relatively subtle reduction in enhancer-promoter interaction strength14. This suggests that these interactions are not solely dependent on loop extrusion and that other mechanisms are involved in their formation.Mammalian genomes are organized into compartments and topologically associating domains (TADs). Compartments reflect separation of euchromatin and heterochromatin, whereas TADs represent relatively insulated regions of the genome, formed by loop extrusion19, it has been proposed that Mediator acts as a bridge between enhancers and promoters (reviewed in refs. 23). Initial studies based on knockdown of Mediator subunits over the course of several days provided evidence for this hypothesis26. However, since the Mediator complex has a central function in RNA polymerase II (Pol II)-mediated transcription, its long-term perturbation causes secondary, confounding effects, which complicate the interpretation of these early studies.Active enhancers and promoters are bound by transcription factors and coactivators, including the Mediator complex. Because the tail module of the Mediator complex interacts with the activation domains of transcription factors bound at enhancers and the head and middle modules interact with the pre-initiation complex (PIC) at gene promoters28. These studies did not detect changes in chromatin architecture and enhancer-promoter interactions upon Mediator depletion, despite strongly reduced expression levels of enhancer-dependent genes. On the basis of these findings, it has been concluded that Mediator is dispensable for enhancer-promoter interactions and acts as a functional rather than an architectural bridge between enhancers and promoters28.To overcome these limitations, more recent studies have used rapid protein-depletion strategies to investigate the function of the Mediator complex in gene regulation and genome organization29. It is therefore possible that changes in fine-scale genome architecture, including enhancer-promoter interactions, could not be reliably identified. For a better understanding of the function of the Mediator complex in genome regulation, it is important to examine the impact of acute Mediator perturbations on chromatin architecture with high resolution and sensitivity.A caveat of current studies of the role of Mediator in genome architecture is that enhancer-promoter interactions have been assessed with chromosome conformation capture (3C) methods at relatively low resolutionHere, we overcome limitations of current studies and investigate the function of the Mediator complex by combining rapid protein depletion and high-resolution analysis of genome architecture using both conventional and MNase-based 3C approaches. We find that depletion of Mediator leads to a significant reduction of enhancer-promoter interactions. Interestingly, we also find that Mediator depletion causes increased interactions between CTCF-binding elements. We show that these changes in interaction patterns are associated with a redistribution of the Cohesin complex on chromatin and a loss of Cohesin occupancy at enhancers upon Mediator depletion. These results suggest that enhancer-promoter interactions are dependent on both Mediator and Cohesin and provide support for a model in which the Cohesin complex bridges and stabilizes interactions between enhancers and promoters bound by Mediator.19, its degradation disrupts the integrity of the Mediator complex28. We have therefore used an HCT-116 MED14-dTAG cell line28 to study the function of the Mediator complex in genome regulation. Using immunoblotting enhancers upon Mediator depletion29. However, these studies relied on genome-wide 3C approaches, such as Hi-C and Hi-ChIP, with relatively low resolution (4–5 kb). It is therefore possible that small-scale changes in enhancer-promoter interactions could not be reliably detected.Previous work has shown that Mediator depletion leads to strong downregulation of cell-type-specific genes that are associated with super-enhancers32, a targeted 3C method based on DpnII digestion, to evaluate changes in chromatin interactions with the promoters of these genes. Capture-C interaction profiles display interaction frequencies with selected viewpoints per DpnII restriction fragment and therefore have an average resolution of ~250 bp. By comparing Capture-C data generated in HCT-116 MED14-dTAG cells treated with DMSO or dTAG ligand, we find that Mediator depletion leads to subtle changes in the interaction patterns of the selected gene promoters  experimentsoci Fig. .Fig. 2DeWe find that depletion of Mediator leads to a decrease in the frequency of enhancer-promoter interactions in the 20 regions that we focused on  in DMSO- and dTAG-treated HCT-116 MED14-dTAG cells 43, we wondered whether Mediator and BET proteins might have (partly) redundant roles in enhancer-promoter interactions. We therefore investigated the impact of combined Mediator depletion and chemical BET inhibition on enhancer-promoter interactions with Capture-C , re-initiation, elongation and transcriptional bursting46. Moreover, it is thought that the function of the Mediator complex in gene regulation is (partly) dependent on the formation of nuclear condensates51. In agreement with this model, it has been shown that MED14 depletion leads to dissolved Pol II clusters28. It is possible that the reduced interactions between enhancers and promoters after Mediator loss are not sufficient to establish the required concentrations of transcription factors, coactivators and Pol II for the formation of nuclear condensates in which transcription can be efficiently initiated. Finally, it is important to note that enhancer-promoter interactions are thought to be transient and vary from cell to cell2. It has been shown that enhancer-promoter proximity does not necessarily co-occur with transcriptional burst52; the precise mechanisms by which interactions between enhancers and their target gene promoter relate to transcriptional activation therefore require further investigation.In the context of Mediator depletion, there are several possible explanations for these observations. We have focused our analyses on genes regulated by super-enhancers, which are composed of many individual elements. For example, the 25, the functional relationship between these complexes has thus far been unclear. Our data show that Cohesin levels at enhancers are reduced when Mediator is depleted. A possible explanation for this observation is that Mediator stabilizes Cohesin on chromatin. Although further investigation of the interaction between Mediator and Cohesin is required, this suggests that Cohesin and Mediator cooperate in the formation of enhancer-promoter interactions and provides support for a model in which extruding Cohesin molecules are stalled at Mediator-bound enhancers and promoters and thereby bridge interactions between these elements. These findings indicate that Cohesin extrusion trajectories are dependent on multiple regulatory proteins and that these factors cooperate in the formation of specific 3D chromatin structures in which gene expression is regulated53.Our data indicate that Mediator’s role in enhancer-promoter interactions is (partly) dependent on Cohesin. Although it has previously been shown that Mediator co-localizes with Cohesin14. This suggests that Cohesin has a role in facilitating longer-range enhancer-promoter interactions and that Mediator can function independently on smaller scales. At the level of nano-scale genome organization, we also detect changes in chromatin structure at CTCF-binding sites. Since we only observe these changes at CTCF-binding sites within gene bodies and not at intergenic CTCF-binding sites, we think that these changes are related to the reduced transcription levels following Mediator depletion. This implies that specific higher-order nucleosome structures within genes can form only in the absence of high transcriptional activity, which is consistent with experiments in yeast that have shown that the transcriptional machinery disrupts regular nucleosome spacing54.The high resolution of our data has enabled us to visualize the effects of Mediator depletion on nano-scale genome organization. We find that interactions between the individual elements within super-enhancers and interactions between enhancers and promoters across very small distances are dependent on Mediator. Of note, we have previously shown that Cohesin depletion leads to a reduction of enhancer-promoter interactions across medium and large genomic distances (>~10 kb), but that Cohesin is not involved in regulating short-range enhancer-promoter interactions or interactions within enhancer clusters56. This indicates that enhancer-promoter interactions depend on the Mediator complex and not on the process of transcription. However, it is of interest that we observe increased interactions with CTCF-binding sites following transcription inhibition. This suggests that the increased CTCF-mediated interactions that we detect after Mediator depletion could be related to the reduced levels of transcription that are associated with loss of Mediator. A possible explanation for these observations is that transcribing Pol II molecules form an obstacle to extruding Cohesin molecules; CTCF loops might therefore form more efficiently when transcription levels are reduced. This model fits with previous work that has shown that the distribution of Cohesin is dependent on transcription58 and with two recent reports indicating that Pol II can form barriers to loop extrusion60.Although the changes in chromatin structure at intragenic CTCF-binding sites are likely related to lower transcription levels that result from Mediator depletion, it is important to note that we do not observe a reduction in enhancer-promoter interactions following treatment with triptolide to chemically inhibit transcription. These observations are consistent with several recent reports in which the impact of acute inhibition of transcription was analyzed with high-resolution Micro-C approaches29. On the basis of knockout of the Mediator-CDK module, it has recently been suggested that the Mediator complex is involved in the regulation of heterochromatin domains and genome compartmentalization61. We do not observe clear changes in compartmentalization after 2 h of Mediator depletion, but it is likely that changes in compartmentalization would require more time to manifest64.With the exception of a subtle increase in the strength of TAD and sub-TAD boundaries, we do not observe large-scale changes in genome architecture upon Mediator depletion. This is consistent with previous reports, in which the impact of Mediator depletion has been investigated with lower resolution approaches, such as Hi-C and Hi-ChIP67 and more widely expressed transcription factors, such as LDB1 (refs. 71) and YY1 (refs. 73), have been implicated in enhancer-promoter interactions. It is likely that the regulation of enhancer-promoter interactions is dependent on a complex interplay between multiple regulatory proteins, which might act in a (partly) redundant manner to ensure the formation of robust enhancer-promoter interactions. In line with biochemical and structural evidence21, our data show that the Mediator complex is one of the factors with an important role in regulating enhancer-promoter communication and gene expression, by acting as both a functional and an architectural bridge between enhancers and promoters.Although our data clearly show that enhancer-promoter interactions are dependent on Mediator, we do not observe a complete loss of interactions when Mediator is depleted. This suggests that additional proteins and mechanisms play a role in mediating enhancer-promoter interactions. We find that the interactions that remain following depletion of Mediator are not dependent on BET proteins. However, many other regulatory factors, such as tissue-specific transcription factors28 were cultured in RPMI 1640 medium  supplemented with 10% FBS  and 1× penicillin–streptomycin  at 37 °C and 5% CO2. Cells were passaged every 2–3 d by trypsinization  upon reaching ~70–80% confluency. For MED14 depletion, dTAG stock was prepared by dissolving the dTAGv-1 ligand  in DMSO. HCT-116 MED14-dTAG cells were seeded in culture flasks and grown to ~70% confluency. On the day of depletion, the cells were washed once with PBS, replenished with fresh culture medium containing either DMSO only or dTAG ligand at a final concentration of 0.5 μM, and treated for 2 h. For transcription inhibition, triptolide  stock was prepared by dissolving the drug in DMSO, and HCT-116 MED14-dTAG cells were treated with a final concentration of 1 μM for 45 min, as described previously56. For co-inhibition of BET proteins, treatment of HCT-116 MED14-dTAG cells with dTAG ligand, as described above, was combined with I-BET 151 dihydrochloride  treatment at 1 μM final concentration for 90 min.Wild-type and MED14-dTAG human colorectal carcinoma HCT-116 cells–1 benzonase  and protease inhibitor cocktail containing leupeptin , PMSF , pepstatin A  and benzamide hydrochloride  for 1 h at 4 °C on a rotator. Lysates were cleared by centrifugation at maximum speed for 15 min at 4 °C. Protein concentration was measured using Bio-Rad protein assay kit . For each sample, 20 μg of protein lysate was mixed with 4X LDS sample buffer  supplemented with 50 mM DTT  and denatured for 5 min at 95 °C. Proteins were separated on a NuPAGE 4–12% Bis-Tris gel  and blotted to a PVDF membrane. The membrane was blocked with 5% milk  in 1× PBS containing 0.05% Tween-20 (PBST) for 1 h at room temperature and was cut into two parts to detect higher- and lower-molecular-weight target proteins separately. Cut membranes were incubated with primary antibodies  antibody, Cell Signaling Technology, 3724; GAPDH: 1:2,000, mouse anti-GAPDH antibody (6C5), Abcam, ab8245) at 4 °C overnight. The next day, the membranes were washed three times with PBST and incubated with horseradish peroxidase (HRP)-labeled secondary antibodies , Abcam, ab205718; GAPDH: 1:3,000, goat anti-mouse IgG H&L (HRP), Abcam, ab205719) for 1 h at room temperature. The membranes were washed three times with PBST again and were developed and imaged using INTAS ChemoCam Imager HR.To confirm efficient Mediator depletion, we performed immunoblotting experiments of whole-cell lysates of HCT-116 MED14-dTAG cells treated with dTAG ligand for 0.5, 1, 2, 4, 6 or 8 h. Following treatment, the cells were trypsinized and pelleted. The cell pellets were washed once with PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis and extraction buffer  supplemented with 250 U mL74. After treatment, the cells were trypsinized and pelleted. Cell pellets were resuspended in cell lysis buffer  and incubated on ice for 5 min. The resulting cell lysates were gently transferred to fresh protein LoBind tubes containing 2.5 volumes of cold sucrose buffer . After centrifugation, the supernatants were collected and stored as cytoplasmic fractions. The resulting nuclei pellets were resuspended in glycerol buffer , to which nuclear lysis buffer  was added. After incubation on ice for 2 min, the lysates were centrifuged to precipitate the chromatin–RNA complex. The supernatants were collected and stored as nucleoplasmic fractions. The resulting chromatin pellets were briefly washed once with MNase buffer  and resuspended in pre-warmed chromatin digest buffer , followed by incubation at 37 °C and 1,400 r.p.m. for 3 min. The digestion reactions were quenched by the addition of 25 mM EGTA and centrifuged, and the supernatants were collected and stored as chromatin fractions. The fractions were analyzed by immunoblotting, as described above. The following primary antibodies were used: MED-HA: 1:1,000, rabbit anti-HA-Tag (C29F4) antibody ; GAPDH: 1:2,000, mouse anti-GAPDH (6C5) antibody ; and histone H3: 1:5,000, rabbit HRP anti-histone H3 antibody . All immunoblotting experiments were performed independently for at least three times, with similar results.To further evaluate the efficiency of Mediator depletion, we performed immunoblotting experiments of subcellular fractions  of HCT-116 MED14-dTAG cells treated with dTAG ligand for 2 h75, with some modifications for three biological replicates per experimental condition. Fresh protease  and phosphatase inhibitors  were added to all buffers. Briefly, 6 × 107 cells were crosslinked with 1% formaldehyde for 8 min at room temperature, followed by quenching with 125 mM glycine for 5 min. The fixed cells were scraped from the plates, washed twice with ice-cold PBS and centrifuged. The cell pellets were resuspended in Farnham lysis buffer  and incubated on ice for 10 min. After centrifugation, the nuclei pellets were resuspended in 1% SDS lysis buffer . Following incubation at room temperature for 10 min, IP buffer  supplemented with 5 mM CaCl2 was added to quench the reaction and to further dilute the SDS . The samples were then digested with 20,000 U of MNase  at 37 °C for 20 min, followed by the addition of 20 mM EDTA and 10 mM EGTA to quench the MNase digestion. The digested samples were sonicated, and the chromatin supernatants were collected afterwards. For each IP, 45 μg of sample chromatin and 200 ng of Drosophila S2 MNase-digested chromatin were used. The samples were pre-cleared with Dynabeads Protein G  for 30 min at 4 °C. Pre-cleared samples were incubated with 1.32 µg of rabbit anti-HA-Tag (C29F4) antibody  and 1 μg of Drosophila spike-in antibody  and incubated overnight with gentle rotation. Following incubation, inputs were collected and stored for each sample. The samples were further incubated with Dynabeads Protein G at 4 °C for 3 h. Bead washes were performed at 4 °C for 5 min in the following order: 1× with Buffer 1 , 4× with Buffer 2 , 1× with Buffer 3 , and 3× with TE buffer . The beads were subsequently eluted in elution buffer . The samples were de-crosslinked, and DNA extraction was performed. Library preparations were performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina  with a modified thermocycler program for the End Prep reaction . The amplified libraries were size selected with double-sided (1.0-1.2x) SPRI bead purification. The final libraries were assessed on a fragment analyzer and sequenced using the NextSeq550 Illumina platform (43-bp paired-end reads). Paired-end reads were processed for adapter removal and mapped to the hg38 reference genome using Bowtie276. Duplicates were filtered and removed using SAMtools77. Spike-ins from Drosophila chromatin were used for normalization. Normalized bigwig files were generated using Deeptools78. Peak calling was performed with MACS2 (ref. 79) in DMSO samples using input files for thresholding. Box plots were generated with R using default settings.Calibrated MNase ChIP–seq was performed as described previously81 for three biological replicates per experimental condition. Briefly, 10 × 106 cells per biological replicate were crosslinked, followed by cell lysis. 3 C libraries were generated by DpnII digestion and subsequent proximity ligation. After decrosslinking and DNA extraction, the resulting 3 C libraries were sonicated to a fragment size of ~200 bp and indexed with Illumina sequencing adapters, using Herculase II polymerase  for library amplification. To boost library complexity, indexing was performed in two parallel reactions for each sample. Biotinylated oligonucleotides (70 nt) were designed using a python-based oligo tool82 (https://oligo.readthedocs.io/en/latest/) and used for enrichment of the libraries in two consecutive rounds of hybridization, biotin-streptavidin bead pulldown , bead washes and PCR amplification . The final libraries were assessed on a fragment analyzer and sequenced using the NextSeq550 Illumina platform (75-bp paired-end reads). Data analysis was performed using the CapCruncher pipeline80 (https://github.com/sims-lab/CapCruncher).Capture-C was performed as described previously33 for three biological replicates per experimental condition. Briefly, multiple aliquots of 10 × 106 cells per biological replicate were crosslinked and permeabilized with 0.005% digitonin . For each replicate, the permeabilized cells were pelleted, resuspended in nuclease-free water, and split into three digestion reactions. MCC libraries were generated by digesting the chromatin in low Ca2+ MNase buffer  for 1 h at 37 °C with MNase  added in varied concentrations (17–32 Kunitz U). The reactions were quenched by the addition of 5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)  and pelleted afterwards. The pellets were resuspended in PBS containing 5 mM EGTA, and an aliquot of 200 mL per reaction was tested for digestion efficiency as a control. The reactions were pelleted again and resuspended in DNA ligase buffer  supplemented with dNTP mix  at 0.4 mM final concentration and 2.5 mM EGTA. Subsequently, 200 U mL–1 T4 polynucleotide Kinase , 100 U mL–1 DNA polymerase I large (Klenow) fragment  and 300 U mL–1 T4 DNA ligase  were added. The reactions were incubated at 37 °C and 20 °C for 1–2 h and overnight, respectively. Following chromatin decrosslinking, DNA extraction was performed using DNeasy blood and tissue kit . The size-selected MCC libraries were sonicated, indexed and enriched with a double-capture procedure, as described in ‘Capture-C.’ Biotinylated oligonucleotides (120 nucleotides) were designed using a python-based oligonucleotide tool82 (https://oligo.readthedocs.io/en/latest/). The final libraries were assessed on a fragment analyzer and were sequenced using the NextSeq550 Illumina platform (150-bp paired-end reads). Data analysis was performed using the MCC pipeline33.Micro-Capture-C (MCC) was performed as described previously14, using the Twist Hybridization and Wash Kit . Briefly, indexed MCC libraries were pooled and dried completely in a vacuum concentrator at 45 °C. Dried DNA was resuspended in blocker solution and pooled with the hybridization solution containing a custom panel of biotinylated oligonucleotides  and incubated at 70 °C overnight. Streptavidin bead pulldown and bead washes were performed with Twist Wash Buffers according to the manufacturer’s instructions (Twist Target Enrichment Protocol). Subsequently, post-hybridization PCR was performed with 11 cycles of amplification. PCR-amplified libraries were purified using pre-equilibrated Twist DNA Purification Beads. The final libraries were assessed on a fragment analyzer and sequenced using the NextSeq550 Illumina platform (150-bp paired-end reads). Data analysis was performed using the MCC pipeline33 (https://github.com/jojdavies/Micro-Capture-C) and HiC-Pro pipeline83 (https://github.com/nservant/HiC-Pro) as described previously14. All contact matrices were balanced using ICE-normalization84. The large-scale contact matrices have a resolution of 500 bp – 2 kb (depending on the size of the region); the resolution of the nano-scale matrices is indicated in the figures.Tiled-MCC was performed using the generated MCC libraries, following a tiled enrichment procedure as described previously85 with some modifications. Three biological replicates per experimental condition were used for the experiment. Briefly, 1.5 × 105 washed cells were split over two tubes, followed by centrifugation. Cell pellets were resuspended in fresh cold lysis buffer  and incubated on ice for 3 min. The lysates were washed once with cold PBS, and the resulting nuclear pellets were resuspended in the tagmentation mix . The tagmentation reactions were performed at 37 °C and 1,000 r.p.m. for 30 min, followed by DNA purification using MinElute PCR purification kit . The indexed samples were amplified using Nextera indexing primers and NEBNext High-Fidelity PCR Master Mix , with an initial 5-min extension step at 72 °C. A real-time PCR library amplification kit  was used to calculate the required number of PCR cycles (11 cycles) in order to minimize library amplification bias. Size selection was performed with double-sided SPRI bead purification to remove primer dimers and larger fragments (>700 bp). The final libraries were assessed on a fragment analyzer and sequenced using the NextSeq550 Illumina platform (75-bp paired-end reads). The data from each replicate were down-sampled to the library with the lowest read depth and analyzed using the NGseqBasic pipeline86.Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was performed as described previously40 was performed for three biological replicates  per experimental condition using the CUT&Tag-IT Assay Kit (Anti-Rabbit) , according to the manufacturer’s instructions with some modifications. Briefly, 0.5 × 106 cells were mildly crosslinked with 0.3% paraformaldehyde , followed by quenching with 125 mM cold glycine. Meanwhile, concanavalin A beads were prepared, following the manufacturer’s instructions. The fixed cells were washed, resuspended in wash buffer and incubated with concanavalin A beads for 10 min on a rotator at room temperature. The samples were placed on a magnetic stand to clear the liquid, and the samples were resuspended with ice-cold antibody buffer supplemented with protease inhibitor cocktail and digitonin. Then, 1 μg rabbit anti-SMC1A antibody  or 1 μg rabbit IgG isotype control antibody  was added to each sample, and the samples were incubated overnight at 4 °C on a rotator in 0.2-mL PCR tubes. The next day, the samples were incubated with guinea pig anti-rabbit secondary antibody  for 1 h at room temperature on a rotator, followed by washes with dig-wash buffer. The samples were placed on a magnetic stand to clear the liquid, and the beads were resuspended with CUT&Tag-IT Assembled pA-Tn5 Transposons. The reactions were subsequently incubated at room temperature on a rotator, followed by washes with Dig-300 buffer. After clearing the liquid on a magnetic stand, the beads were resuspended with tagmentation buffer. The tagmentation reactions were subsequently incubated at 37 °C for 60 min. The samples were de-crosslinked, and DNA extraction was performed according to the manufacturer’s instructions. The libraries were amplified by PCR, and size selection was performed with two rounds of SPRI bead purification to remove primer dimers. The final libraries were assessed on a fragment analyzer and sequenced using the NextSeq550 Illumina platform (75-bp paired-end reads). The data were analyzed using the NGseqBasic pipeline86. Peak calling was performed with MACS2 (ref. 79) using IgG controls for thresholding. Normalized bigwig files and meta peak profiles were generated using Deeptools78 and LOESS regression was applied for smoothening of the data. Box plots were generated with R using default settings. Differential binding analysis was performed in R using the DiffBind package. An adjusted P value of 0.05 (Benjamini–Hochberg method) was used to identify differentially bound SMC1A peaks after Mediator depletion (Supplementary Table CUT&Tag87 (ENCSR000ENM) and ChIP–Seq data for CTCF87 (ENCSR000BSE) and MED26 (ref. 27) in HCT-116 cells were analyzed using the NGseqBasic pipeline86. TT-seq data files for HCT-116 MED14-dTAG cells28 were shared by the authors, and differential expression analysis was performed in R using the DESeq2 package88.DNase-I hypersensitivity dataFurther information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-023-01027-2.Reporting SummaryPeer Review FileSupplementary Table 1P value < 0.05) in dTAG-treated cells  compared with DMSO-treated cells  are listed. P values were calculated in R using the DiffBind package and corrected for multiple comparisons.Peaks that are significantly changed (adjusted


Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency (frq). FRQ interacts with FRH (FRQ-interacting RNA helicase) and CKI, forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss of brd-8 reduces H4 acetylation and RNA polymerase (Pol) II occupancy at frq and other known circadian genes, and leads to a long circadian period, delayed phase, and defective overt circadian output at some temperatures. In addition to strongly associating with the NuA4 histone acetyltransferase complex, BRD-8 is also found complexed with the transcription elongation regulator BYE-1. Expression of brd-8, bye-1, histone h2a.z, and several NuA4 subunits is controlled by the circadian clock, indicating that the molecular clock both regulates the basic chromatin status and is regulated by changes in chromatin. Taken together, our data identify auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamic frq expression and thereby a normal and persistent circadian rhythm.In the  A screen for clock mutants uncovered a conserved novel auxiliary NuA4 histone acetylase complex, containing orthologs of BRD-8 and BYE-1, needed for maintaining the timely Negative Element expression required for sustaining a normal circadian rhythm. Neurospora, the White Collar Complex (WCC) formed by WC-1 and WC-2 transcriptionally activates the pacemaker gene, frequency (frq), by binding to two promoter elements: Clock box (C-box) in the dark and Proximal Light-Response Element (pLRE) in the light3. FRQ, the frq gene product, associates with FRH (FRQ-interacting RNA helicase)5 and CKI (casein kinase I)7 to form the FFC complex, promoting WCC phosphorylation at a group of residues9 to repress its activity and thereby close the feedback loop.Circadian clocks control a wide variety of physiological, biochemical, and behavioral processes in most eukaryotes and certain prokaryotes. At the molecular level, circadian systems are composed of positive and negative arms and the latter repress their own expression through inhibiting the positive arm. In frq is strongly activated in the light and is rhythmically transcribed and translated in the dark10, events that are highly regulated and whose progress impacts timekeeping; frq lies in a region of highly accessible chromatin associated with rapid nucleosome turnover when active11. In chromatin, nucleosomes impede transcription initiation by Pol II by inhibiting assembly of the preinitiation complex (PIC) at the promoter12. The positive charges on lysine residues of histones can be neutralized by acetylation, which weakens histone-DNA interactions and reduces accessibility to other proteins relevant to gene expression13. Acetylation also provides recognition sites for chromatin remodeling complexes bearing bromodomains14. Nucleosomes can inhibit transcription elongation by Pol II, and their displacement at coding sequences directly correlates with the transcription pace of Pol II15. The major histone acetyltransferase for histones H4, H2A, and H2A.Z19 in yeast is the thirteen subunit 1.3 MDa NuA4 (Nucleosome acetyltransferase of histone H4) complex, and its catalytic subunit is Esa1. NuA4 is recruited both to gene promoters and coding regions and stimulates the passage of elongating Pol II in transcription initiation and elongation by loosening DNA-histone contacts20. Evidence suggests that most histone acetylation at promoter regions precedes and is independent of transcription. In humans, the orthologous Tip60 complex that bears HAT activity is a multiprotein complex with at least 16 subunits22, and may have arisen as a fusion form of two yeast HAT complexes, NuA4 and SWR, the multi-subunit complex that incorporates histone variant H2A.Z into chromatin21. In addition to histones, TIP60 acetylates BMAL1 in the mammalian clock, providing a docking site that brings the BRD4-P-TEFb complex to DNA-bound BMAL1, promoting release of Pol II and elongation of circadian transcripts23. The BYpass of Ess1 (BYE1) protein is a Saccharomyces cerevisiae transcription elongation factor25 whose human homologs are PHF3 and DIDO26. BYE-1 interacts with Pol II and can positively or negatively regulate transcription elongation26. Histone H4 acetylation determines chromatin higher-order structure and functional interactions between remodeling enzymes and the chromatin fibers27; disruption of Tip60 or NuA4 and impairment of histone H4 acetylation have been implicated in DNA damage repair, plant developmental processes, behavioral variability, neurodegenerative diseases, cell cycle progression, cancer, and ageing44.frq and is required for normal frq expression, period determination, and persistence of rhythmicity. BRD-8 also complexes with an ortholog of Saccharomyces BYE-1, suggesting a connection to transcription elongation. In the absence of brd-8, both histone acetylation and Pol II levels at frq are sharply reduced, consistent with reduced expression of frq. Interestingly, expression of brd-8, bye-1, and several NuA4 subunits is controlled by the circadian clock, forming a nested feedback loop surrounding the core transcription translation feedback loop. These and prior data are consistent with an important role for robust and timely transcription of the negative arm proteins in the circadian feedback loop and suggest a link between the circadian clock and the basic transcriptional machinery.In this study, we have identified a conserved bromodomain-containing NuA4-associated subunit, BRD-8, which is required for the NuA4 HAT activity on histone H4 at multiple loci including Neurospora45 were screened on race-tube medium bearing menadione which facilitates visualization of the circadian phenotypes46. Two deletion strains lacking the same gene, ncu09482 , displayed an arrhythmic overt conidiation phenotype on race tubes  or low (0.01%) percentage of glucose  is even significantly higher in ∆brd-8 than in WT, consistent with differing roles for BRD-8 depending on the promoter element (C-box vs. pLRE) used.In addition to its role as the major transcription factor for brd-8, and consistent with the long period of ∆brd-8, new FRQ in ∆brd-8 appears later than that in WT and is slightly reduced. However, the level of WC-1 in ∆brd-8 is significantly reduced. Active WC-1 is unstable and FRQ, by inactivating WCC, stabilizes WC-16, so to test whether the low WC-1 level is caused by a defective negative arm, ∆brd-8 was backcrossed to the loss of function allele frq9; WC-1 levels are further reduced in the frq9; ∆brd-8 double mutant , the wc-1 and WC-1 levels are raised to those in WT  and purified from protein extracts; three distinct protein bands from the stained gel removed and analyzed by tandem mass spectrometry proved to be proteins encoded by NCU09482 , NCU06787 (ortholog of the NuA4 subunit EAF-3), and NCU05943 (ortholog of the regulator of transcription elongation BYE-124),  was C-terminally tagged with V5 or VHF, while BYE-1 and most NuA4 subunits including VID-21, EAF-6, EAF-3, YAF-9, EAF-7, ESA-1 etc.  best homology with BRD8, in keeping with prior nomenclature precedents (e.g.50), it is designated as BRD-8 (bromodomain protein-8).To understand how loss of tc. Fig. , were intc. Fig. . NGF-1, tc. Fig. , indicateaf-3 shows an impaired long-period core clock by a luciferase assay , indicating that it is an essential gene as it is in yeast16. To confirm its requirement for normal rhythmicity, a construct bearing a hairpin structure specific for esa-1 and driven by the qa−2 promoter (inducible by QA) was transformed into an ESA-1V5 strain, targeting the csr locus to create a ds esa-1 strain affording regulatable knock-down of esa-1; circadian rhythms of this strain were monitored using a luciferase assay in the presence or absence of 10−2 M QA. In the absence of QA, the ds esa-1 strain exhibited a robust rhythm similar to that seen in WT 53 was knocked into a strain bearing frq-promoter-driven luciferase at the his-3 locus. By adding QA, the period in qa-2-driven esa-1E395Q is extended by ~2 hrs compared with that cultured in the QA-free medium as a control  in cultures from the late subjective night. Interestingly, histone H4 densities in ∆brd-8 increase modestly, while the level of acetylated H4 decreases significantly at frq, frh, wc-1, wc-2, and other clock-and light-response-associated genes in ∆brd-8 . Consistent with results from ChIP-sequencing  as well as the coding region of frq  at its native locus. In the presence of QA, the controlled gene is constitutively induced by the qa−2 promoter. Interestingly, the level of brd-8 positively correlated with shortening of the period of the circadian conidiation rhythm within a certain range of QA concentration (10−5 to 10−3 M)  Fig. ; howeverned Fig. , which mned Fig. . The BRDned Fig. , consistned Fig. , suggestNeurospora globally regulates gene expression, we investigated whether the expression of BRD-8, BYE-1, and several additional NuA4 subunits are also under circadian control. Promoters of brd-8, bye-1, eaf-3, vid-21, and esa-1, along with histoneh2a.z, and histone h4-1 were fused with the codon-optimized LUC gene respectively and transformed to the WT strain at the csr locus. The promoters of brd-8, bye-1, eaf-3, vid-21, and histone h2a.z genes were shown to drive rhythms in LUC expression , and the strain bearing BRD-8-LUC showed an overt rhythm with a WT period  is required for Pol II elongation processivity, and CDK-9 (SGV1 in Saccharomyces) is the kinase phosphorylating Ser2. To test whether inhibition of CDK-9 causes a period effect on ∆ent Fig. . CDK-9 wd-8 Fig. , ∆brd-8,hrs Fig. . The datfrequency gene encodes the central scaffold protein that organizes the negative arm repressive complex in the Neurospora clock, playing the same role in Neurospora as that ascribed to the PER proteins in the mammalian clock; as a result, its expression is carefully regulated. frq is rhythmically transcribed and translated in the dark10, a result of chromatin remodelers and modifiers that co-execute their effects at the frq locus, and most research on its transcriptional regulation has focused on transcription initiation at the C-box rather than on steps in transcription elongation. During daylight the frq gene lies in a region largely free of nucleosomes and showing rapid nucleosome turnover11, and in the dark several distinct chromatin remodeling complexes have been identified in controlling frq expression, including CSW (Clockswitch)60, CHD1 (chromodomain helicase DNA–binding)61, SWI/SNF (SWItch/Sucrose NonFermentable)62, CLOCK ATPase (CATP)63, Centromere Binding Factor 1 (CBF-1)64, and IEC-1-INO80 complex65. Histone modifications can also lead to changes in gene expression, especially methylation and acetylation of N-terminal tails of histones which are known to influence condensation as well as serving to call down modifiers69; indeed one study has reported that the lysine methyltransferase SET1 is required for methylation of lysine 4 of histone H3 and normal circadian expression of frq70. Here, by a circuitous route beginning with an unbiased genetic screen, we have identified a phylogenetically conserved auxiliary subunit of the canonical NuA4 histone acetyltransferase complex and shown that it also complexes with elements involved in transcription elongation, and that in the aggregate mutations in these elements impact frq expression and the progress of the circadian clock. In the process, we also discovered that there are multiple NuA4 complexes, presumably with altered activities, and that some include an ortholog of the regulator of transcription elongation, BYE-1, first described in Saccharomyces24 but with orthologs in humans26. Genetic and pharmacological data are consistent with a model in which the NuA4 complex that includes BRD-8 and BYE-1, but not the canonical complex, is involved in controlling frq expression in the service of the clock  and can be reliably measured with precision of +/− 10%. The BRD-8-containing NuA4 complex however, like the other chromatin-regulating and clock-affecting complexes, would not be considered a core clock component because its role in rhythmicity is solely conferred by its action on frq expression whose rhythmicity is driven through WCC, and the rhythm persists in the absence of BRD-8; instead these are all clock-affecting. This said, the data do show that not all genes are bound by nor their expression influenced by complexes that contain BRD-8 and BYE-1. Instead action of these NuA4-associated factors is restricted, although they do appear to be quite important when present: Data shown in Figs. brd-8 resulted in ~50% reduction of histone H4 acetylation at the frq locus, suggesting a higher activity of the BRD-8-containing NuA4 complex.This work follows the lines of Belden, He, Liu, and othersNeurospora, however, no gene having homology to eaf-5 exists, and indeed orthologs of EAF5 are found only in Saccharomyces and closely related species , perhaps to prevent loss of histones during polymerase passage through chromatin26 but also slowing transcription. Although in Saccharomyces BYE1 works as a free protein, its action(s) in Neurospora in conjunction with NuA4 may be similar to those seen in yeast, acting on elongation through regulating Pol II CTD or just enhancing ESA-1 activity on chromatin, an activity not reported in Saccharomyces. In either case, a seeming paradox in the circadian data related to loss of BRD-8 and BYE-1 arises from the facts that (1) loss of BRD-8 which results in increased nucleosome density is expected to slow transcription but (2) loss of BYE-1, a negative regulator of transcription elongation26, would be expected to increase transcription elongation; yet both result in period lengthening. One clue to this lies in the data from Fig. BYE1 was originally identified in Saccharomyces through a mutation that bypassed the requirement for the essential prolyl isomerase ESS1Neurospora BRD-8 that likewise has a single bromodomain. While as noted above, BRD8 is associated with multiple mammalian complexes including NuA4/TIP60, the association of a bromodomain-containing protein in a fungal NuA4 complex has been debated (reviewed in refs. 71). Bdf1 in the Saccharomyces cerevisiae SWR1 complex, has a single bromodomain as does Bdc1, a component of the NuA4 histone acetyltransferase complex In Saccharomyces pombe (reviewed in refs. 71). However, the overall amino acid identity of Neurospora BRD-8 with Bdf1 and Bdc1 is relatively low , Bdf1 and Bdc1 are a third of the size of BRD-8, and perhaps most significantly, neither Bdf1 nor Bdc1 are BLASTP hits with BRD-8. Bdf1 is, however, a component of the yeast SWR complex which contains additional components orthologous to mammalian NuA4/TIP60 components, prompting the observation that the human NuA4 complex corresponds to a near-perfect fusion of the two distinct complexes in yeast, namely NuA4 and SWR1 . This prompted us to re-examine the mass spectrometry data  and it is similar in size to Neurospora WC-1), providing a docking site that brings the BRD4-P-TEFb complex to DNA-bound BMAL1, promoting release of Pol II and elongation of circadian transcripts and revealing a role for control of transcriptional elongation in the mammalian clock23. At first glimpse there are clear parallels to the BRD-8 story here, in particular because WC-1 has been reported to be acetylated72; however, the similarities weaken when examined more closely. Neurospora does have a Brd4 ortholog (BDP-3 encoded by NCU08423) and, unlike BRD-8, it is essential as is Brd4 in humans. The acetylation of WC-1 appears only to be important for light-regulation72, and perhaps most importantly, the structure/function data from Fig. https://thebiogrid.org/116108), is ubiquitously expressed, and has roles in a host of processes; in both systems it seems plausible that not all of these roles would require the bromodomain. In the present case, BRD-8 uses its unstructured region to interact with BYE-1, and deleting its single bromodomain still results in a normal period, while removal of two disordered domains individually phenocopies the whole gene knockout of brd-8  and be independent of the BRD-8/BYE-1 association, and if so the period lengthening seen from loss of either BRD-8 or BYE-1 alone should be additive in the double mutant ∆brd-8, ∆bye-1; however, this is not the case  does not appear in the mass-spectrometry interactome data ; however, the reverse has rarely been confirmed (reviewed in79). Here we have shown that expression of histone h2a.z, as well as a number of NuA4 subunits including brd-8 gene and protein are circadianly controlled  for evidence of circadian regulation of mammalian NuA4/TIP60 components. Interestingly, strong albeit sometimes low amplitude rhythms were seen for gene expression of brd8 as well as Trrap, RUVBL1, and DMAP1. This suggests that in mammals, as in Neurospora, the events surrounding histone acetylation and transcription elongation describe a feedback loop surrounding the core clock that serves to amplify and stabilize the rhythm, contributing to persistence. These data also provide evidence for a mechanism through which the circadian clock can control gene expression through an indirect mechanism not directly mediated by the WCC or CLOCK/BMAL1 complex, the principal means of circadian output control.A wealth of research in recent years has established mechanisms through which epigenetic regulation plays an important role in controlling circadian systems  was used as a clock-WT strain in the race tube analyses and 661-4a , WT in the luciferase assay; this contains the frq C-box fused to codon-optimized firefly luciferase  at the his-3 locus. Neurospora transformation was performed as previously reported62. To test the rhythmicity of gene expression, promoter-driven luciferase (LUC) reporter constructs were generated by transforming four PCR products  with digested plasmid (pRS426) into yeast80. Race tube medium contains 1 × Vogel’s salts, 0.17% arginine, 1.5% bacto-agar, and 50 ng/mL biotin with glucose at 0.1%, and liquid culture medium (LCM) contains 1 × Vogel’s, 0.5% arginine, 50 ng/mL biotin, and 2% glucose. Quinic acid (QA) was added into race tube medium with certain concentrations as indicated. Unless otherwise specified, race tubes were cultured in constant light for 16–24 h at 25 °C and then transferred to the dark at 25 °C81. BRD-8, BYE-1, and NuA4 deletion strains generated by the Neurospora genome project were obtained from Fungal Genetics Stock Center (FGSC).328-4 83, WC-2 84, FRQ 10, and FRH 5. V5 antibody (Thermo Pierce), FLAG antibody (Sigma-Aldrich), and HA  were diluted at 1:5,000 for use as the primary antibody. All uncropped and unprocessed whole blots used for figure preparations were deposited into the Source Data file except for the ones for Supplementary Figure Procedures for preparation of protein lysates and Western blots (WB) were followed as described in refs. 62. 30 g of Neurospora tissue (brd-8VHF) was used for purification of BRD-8. To exclude the possibility that associations with chromatin impacted identification of BRD-8-interactors in purifications, we (1) vortexed lysate at the top speed to mechanically disrupt chromatin: 10 s on/10 s off (put on ice) for a total of 2 min; (2) extensively sonicated protein lysate; (3) included benzonase nuclease in the protein lysis buffer; benzonase is widely used for the removal of nucleic acids (both DNAs and RNAs) from protein samples88. To recover potential weak interactors, 5 g of tissue from brd-8V5 were purified with V5 antibody (Thermo Pierce)-conjugated Dynabeads (Life Technologies). A single protein band or the whole lane89 was cut off from the gel as indicated in the figure and used for mass spectrometry analysis.VHF-tagged BRD-8 were purified with the same method applied for isolation of C-terminal VHF-tagged WC-1V5 . Six samples were analyzed by mass spectrometry.Affinity purification of BRD-8’s interactors using VHF-tagged BRD-8 , desalted, and dried. Peptides were analyzed on a Fusion Orbitrap mass spectrometer (Thermo Scientific) equipped with an Easy-nLC 1000 (Thermo Scientific).Peptides were resuspended in 5% methanol / 1% formic acid and loaded on to an analytical resolving column  pulled in-house  with a 45-min gradient of 5–34% LC-MS buffer B . The Fusion Orbitrap was set to perform an Orbitrap MS1 scan  from 350 to 1,500 m/z, followed by HCD MS2 spectra detected by Orbitrap scanning  for 2 sec before repeating this cycle. Precursor ions were isolated for HCD by quadrupole isolation at width = 0.8 m/z and HCD fragmentation at 29 normalized collision energy (NCE). Charge state 2, 3, and 4 ions were selected for MS2. Precursor ions were added to a dynamic exclusion list ± 10 m ppm for 15 s.90 (release version 2014.01) in high resolution mode against a target-decoy (reversed)91 version of the Neurospora proteome sequence database  with a precursor mass tolerance of +/− 1 Da and a fragment ion mass tolerance of 0.02 Da, and requiring fully tryptic peptides  with up to three mis-cleavages. Static modifications included carbamidomethylcysteine and variable modifications included: oxidized methionine. Searches were filtered using orthogonal measures including mass measurement accuracy (+/− 3 ppm), Xcorr for charges from +2 through +4, and dCn targeting a < 1% FDR at the peptide level.Raw data were searched using COMETV5 but not in the negative control . In the search for BRD-8V5’s interactors, no statistical tests were taken, because we worried that this would rule out certain real targets due to their potential digestion or/and coverage issues in mass spectrometry analysis. Instead, we validated the identified interactors individually by immunoprecipitation assays with epitope-tagged proteins  or FLAG M2 resin  as indicated in the figures, rotating at 4 °C for 2 hrs. The agarose beads were then washed twice with the protein extraction buffer  and eluted with 50 µL of 5 × SDS sample buffer at 99 °C for 5 min.IP was performed as previously described62. Primer sets against C-box and pLRE were the same as in a previous publication8. Primer sets against the frq locus used in Fig. 94. “A” in the first “ATG” of the full-length frq coding region is designated as “+1” and the upstream nucleotides are labeled “-“ accordingly (without “0”). frq C-box is from −2,878 to −2,624 nt, the amplicon with the frq1 primer set is from −2,754 to −2,644 nt, while that by the C-box primer set is from −2,790 to −2,678 nt. ChIP using Neurospora tissue: Neurospora tissue was cross-linked with 3% formaldehyde for 15 min and quenched with 0.25 M glycine for 5 min. Tissue was then washed with PBS three times and vacuum-dried; 0.4 g was weighed, cut into 9 pieces, and each piece soaked in 0.5 ml SDS lysis buffer  containing Roche protease inhibitors . The soaked tissue was first sonicated for 8 sec for three times at 30% amplitude with a Bronson sonicator equipped with a microtip, and then further sonicated in a water-bath sonicator 5 times for 5 min each with an interval of 30 sec on and 30 sec off. The sonicated cell lysate was cleared of cellular debris by an 8,000 rpm centrifugation twice for 5 min each. 200 μl supernatant was saved and added to 1.8 ml RIPA buffer; 15 μL protein A magnetic beads and 2 μL V5 antibody  were added to the mixture followed by rotation at 4 °C overnight. The following morning, immunoprecipitated DNA was washed with buffers A-D  once each, eluted with Elution buffer , reverse crosslinked with NaCl at the final concentration of 0.2 M, and purified with Qiagen PCR purification kit.ChIP experiments were done as previously described using fresh tissuesThe purified ChIP DNA was end-repaired and ligated to Ion-compatible barcode adapters using Ion XpressTM Plus Fragment Kit  and Ion XpressTM Barcode Adapters . The final libraries were purified with two rounds of AMPure XP Bead capture to size select fragments between 160 to 340 bps in length. The emulsion clonal bead amplification to generate bead templates for the Ion Torrent platform was performed on the Ion Chef System (Thermo Fisher Scientific) with the Ion PI™ Hi-Q™ Chef Kit  and Ion PI™ Chip Kit v3 . Sequencing was done using the Ion PI™ Hi-Q™ Sequencing 200 Kit  on Ion Proton sequencer with sequencing data processing using the Torrent Suite TM Software (Ver. 4.0.2) on the Torrent server.Neurospora crassa genome (from GenBank) using TMAP . Mapping efficiency and read quality were visualized using htseq-qa (http://htseq.readthedocs.io/en/release_0.9.1/). Peaks were called using MACS2 (https://github.com/taoliu/MACS), and peaks were annotated with ChIPseeker . In addition to MACS2, the SICER software (https://home.gwu.edu/~wpeng/Software.htm) was used to identify peaks and identify differentially abundant peaks between WT and mutant strains. For comparison of WT vs. control in histone and acetylated histone, three runs apiece were performed with window sizes of 200, 400 and 600, and gap sizes of 600, 1,200 and 1,800, respectively. Redundant peaks between the three runs  were eliminated with a simple R script. Another script was used to identify genes within 1,000 bp from either border of the peaks.Samples were mapped to the 95. Briefly, ESA-1 was immunoprecipitated from the esa-1V5 strain as described in the section of “IP” with V5 antibody-conjugated resins. Immunoprecipitated- ESA-1V5-bound resins were washed twice with phosphate buffered saline containing 0.1% Tween 20 and once with the acetyl-transferase assay buffer . 50 μL of the acetyl-transferase assay buffer containing 10 μM acetyl CoA (Sigma-Aldrich) was added to the washed resins. Recombinant human histone H2A  or H4  respectively was added to the mixture at a final concentration of 0.1 mg/mL. Acetylation reactions were incubated at 30 °C for 1 hr, followed by adding 50 μL of 5 x SDS-PAGE sample buffer, heated at 99 °C for 5 min, electrophoresed in a 4-12% bis-tris SDS-PAGE gel, and transferred to a PVDF membrane. Acetylated histones H4 and H2A were detected by immunoblotting with rabbit anti-acH4  and anti-acH2A K9 , respectively.The histone acetylation assay was performed as previously described with modificationsNeurospora mRNA was isolated with TRIzol reagent (Life Technologies) and cDNA synthesis was done using SuperScript III first strand synthesis kit (Life Technologies) with 3 µg of purified RNA47. Real-time PCR was performed with QuantiTect SYBR green RT-PCR kit (Qiagen) in an ABI 7500 Fast system. Primers against frq and wc-1 genes were from previous publications96. Normalization for RT-qPCR was to NCU08964, a gene identified previously as among the most constantly expressed under different conditions and times or day, and least responsive to changes in media40.62. Luciferase assays were performed as previously described81. If QA was used in the luciferase assay, it was indicated in the figures. Nuclear preparation was performed as reported92.Mass spectrometry was performed as previously describedFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary