Fusarium is a species-rich group of mycotoxigenic plant pathogens that ranks as one of the most economically important fungal genera in the world. During growth and infection, they are able to produce a vast spectrum of low-molecular-weight compounds, so-called secondary metabolites (SMs). SMs often comprise toxic compounds  that contaminate precious food and feed sources and cause adverse health effects in humans and livestock. In this context, understanding the regulation of their biosynthesis is crucial for the development of cropping strategies that aim at minimizing mycotoxin contamination in the field. Nevertheless, currently, only a fraction of SMs have been identified, and even fewer are considered for regular monitoring by regulatory authorities. Limitations to exploit their full chemical potential arise from the fact that the genes involved in their biosynthesis are often silent under standard laboratory conditions and only induced upon specific stimuli mimicking natural conditions in which biosynthesis of the respective SM becomes advantageous for the producer. This implies a complex regulatory network. Several components of these gene networks have been studied in the past, thereby greatly advancing the understanding of SM gene regulation and mycotoxin biosynthesis in general. This review aims at summarizing the latest advances in SM research in these notorious plant pathogens with a focus on chromatin structure. Fusarium belongs to a diverse group of phytopathogenic fungi that collectively can infect virtually all agricultural important crop plants worldwide [F. graminearum and F. oxysporum, are even ranked amongst the most detrimental plant pathogens worldwide [F. oxysporum and F. solani [Fusarium comprises about 300 species found amongst 23 different Fusarium species complexes [F. fujikuroi species complex (FFSC), F. graminearum species complex (FGSC), F. solani species (FSSC) complex and the F. oxysporum species complex (FOSC) [F. verticillioides, F. proliferatum as well as by some F. fujikuroi isolates [F. graminearum and F. culmorum [Fusarium species [The genus orldwide . Two proorldwide . Some ge. solani . In geneomplexes ,5. The ex (FOSC) ,7. Next x (FOSC) . Promineisolates ,10,11,12culmorum , or enni species ,15. This species ,17. In lFusarium species via whole-genome sequencing [In the last decades, vast efforts have been made to obtain more insight into the genetic capacity of various quencing . This haquencing . In the quencing . This ilquencing ,19, and TRI5, which encodes the deoxynivalenol (DON) biosynthetic key enzyme. This deletion led to attenuated disease symptoms in wheat due to F. graminearum [The biosynthesis of individual SMs is often accompanied by an additional set of enzymes. These are comprised of either tailoring enzymes, which are able to post-translationally modify nascent products such as glycosyltransferases, P450 monooxygenases or oxidoreductases or else transcription factors and/or multidrug transporters involved in regulation and transport, respectively. These genes are often clustered together in the fungal genome, forming SM biosynthetic gene clusters (SMBGCs) and thereby facilitating co-expression under the respective SM-inducing conditions. SMs are mostly dispensable for the survival of the producer but may provide a selective advantage and, as such, may very well become essential for fungal adaption to certain environmental niches ,21. Thisminearum ,23. Propminearum .Fusarium spp. are, for example, the components of the VELVET complex and the associated putative methyltransferase Lae1, which coordinates light, fungal development and secondary metabolism [Fusarium.The timely expression upon reception of inducing stimuli implies a tight regulatory network. Well-known regulators affecting the biosynthesis of many SMBGCs in tabolism ,33,34,35tabolism ,37,38,39DNA is present in a highly condensed state in the eukaryotic cell nucleus, mainly to physically package the whole genome, to protect it from damage as well as to control DNA accessibility in a highly regulated manner. The compacted form of DNA is called chromatin, and it consists of about 150 bp of DNA which is tightly wrapped around an octamer of the core histone proteins H2A, H2B, H3, and H4. Chromatin structure differs in their degree of compaction, being either loosely packed and open for transcription (euchromatin) or densely packed and transcriptionally inert (heterochromatin). Heterochromatin can be further distinguished as constitutive heterochromatin (static) and dynamic facultative heterochromatin, which is prone to remodeling processes . As suchF. graminearum and F. fujikuroi. As described for several other organisms, H2A.Z is essential in both fusaria. Intriguingly, lethality is rescued in F. graminearum by compensatory mutations in other regulatory subunits of complexes associated with changes in the chromatin landscape, e.g., SWR1, which is important for the dynamic exchange of H2A-H2B dimers against H2A.Z-H2B dimers or the catalytic subunit of Rpd3 complex essential for histone deacetylation [The chromatin landscape is subject to a diverse set of modification mechanisms, which results in the alteration of its structure. These regulatory circuits control interactions with the underlying DNA, ensuring the correct biological readout. Already known mechanisms comprise the direct exchange of histones against non-canonical core histones or the removal or addition of whole nucleosome units by energy-dependent chromatin remodeling enzyme complexes . While ftylation ,43, thertylation ,45.The first histone mark was already identified by Allfrey and colleagues in 1964 , and witFusarium, and will thus be the focus of this review. Whereas histone acetylation is tightly associated with active gene transcription, histone methylation has ambivalent functions. Depending on the exact methylation site and methylation degree, it can either repress or stimulate gene expression [Fusarium species, the number of predicted histone methyltransferases/demethylases and histone acetyltransferases/deacetylases varies as depicted in However, so far, only the methylation and acetylation of histone N-termini has been studied in greater detail in filamentous fungi, including pression . NotablyAspergillus nidulans. Here, the deletion of the histone acetyltransferase-encoding gene hdaA led to the upregulation of two SM-related genes [Fusarium. Deletion of the putative H3K9me3 reader protein-encoding gene FgHEP1 in F. graminearum resulted in an altered chemical profile [Efforts to alter the chromatin landscape by the manipulation of histone marks to trigger an expressional change of the underlying genes have been manifold across the fungal kingdom. First experiments performed as early as over a decade ago were described for ed genes . Only a  profile . Since tFusarium spp. These include, for example, the perithecial pigments (fusarubins) [F. fujikuroi but more seldomly by some F. proliferatum or F. oxysporum isolates [F. graminearum clade and some closely related species [Fusarium metabolites is ever-growing, we are still only at the beginning of understanding the prerequisites of their expression. Fusarium SMs for which biosynthesis and/or gene expression responds to the loss and/or overrepresentation of enzymes involved in altering the chromatin structure in chosen fusaria.Fusaria are able to produce numerous SMs, and a great effort has been made regarding the identification of novel compounds as well as the linkage of already known SMs to their respective SMBGC. Notably, only a few SMBGCs are conserved among the different arubins) , the cycarubins) , the sidarubins) , fusarinarubins)  and gibearubins)  which arisolates ,57,58. T species . Interes species  is only  species . While tHistone marks are roughly categorized into groups according to their associated function, i.e., induction of euchromatin formation accompanied by gene expression or gene silencing through compaction and the formation of facultative or constitutive heterochromatin. Thus, some marks are considered heterochromatic, e.g., H3K9me3 or H3K27me3, while others are prone to be associated with euchromatin such as H3K4me3 and histone acetylation on H3/H4. As such, it is noteworthy to mention that, despite the rigid “classical” categorization of histone marks, some own ambivalent characteristics .Fusarium.The next paragraphs summarize the current knowledge on chromatin regulatory mechanisms, i.e., histone marks, provide an overview on their mechanisms and shed light on the dynamic interplay between so-called global regulators and histone marks with a focus on SM gene regulation in the genus Su(var)3–9, enhancer of zeste and Tri-thorax) domain [Fusarium is still enigmatic, some proteins have been studied in greater detail in filamentous fungi, including fusaria. Those are Kmt1 (H3K9me3), Kmt2 (H3K4me3), Kmt3 and Ash1 (H3K36me3), as well as Kmt6 (H3K27me3)  domain . DOT1 st) domain . In cont) domain . Dependi) domain . Fusaria3K27me3) .Schizosaccharomyces pombe. SpLsd1 function is limited by the removal of the mono- and dimethylation (me1/me2) states of H3K4/H3K9 residues via its amine oxidase domain, at least in S. pombe [Fusarium: Kdm4 (H3K36me) [To safeguard balanced methylation states, methyltransferase’s function can be antagonized by the targeted demethylation of histone lysine residues by another class of enzymes: histone lysine demethylases . The firS. pombe . DespiteS. pombe ,101. ThuH3K36me)  and Kdm5H3K36me) ,104  complex [S. cerevisiae, i.e., Set1, Bre2, Swd1, Spp1, Swd2, Swd3, Sdc1 and Shg1, with ScSet1 , which is established by components of the so-called COMPASS ( complex . The COMely Kmt2 ) as the ely Kmt2 . H3K4 meely Kmt2 ,109. HomF. fujikuroi and the cereal pathogen F. graminearum, the Set1 orthologs FfSet1 and FgSet1, respectively, are detrimental for H3K4 di- and trimethylation, while remaining levels of H3K4 monomethylation (H3K4me1) were observed for both species [FfSET1, which revealed an increase in the biosynthesis of the two red pigments bikaverin (BIK) and fusarubins (FSR), as well as fusarin C (FUS) and, to some extent, also fusaric acid (FUB), while production of the plant hormone and virulence factor GA was completely abolished in axenic culture [ffset1 revealed reduced H3K4me2 and me3 levels at the GA cluster genes, the only SMBGC in F. fujikuroi shown to harbor H3K4me marks under inducing conditions thus far [ffccl1 strains that also resulted in reduced GA production and increased BIK and FUS biosynthesis, while FUB biosynthesis remained unaffected [GA but also at the BIK genes, levels remained unaffected at the FUS SMBGC [In the rice pathogen  species ,110. SM  culture . In linethus far . This phaffected . NotablyUS SMBGC , suggestUS SMBGC ,111.F. graminearum, the lack of FgSet1 and associated COMPASS components, i.e., FgBre2 or FgSdc1, led to an impaired DON biosynthesis. Again, ChIP-qPCR revealed drastically reduced levels of H3K4me2 and decreased levels of me1- and me3 at the TRI cluster genes in ∆fgset1 [AUR cluster genes [FgBRE2, here designated FgCCL1 [F. fujikuroi, H3K4me2/me3 levels are not strictly correlative with the chemical profile. In contrast, global H3K4me2 distribution studies determined by ChIP-seq in F. graminearum performed earlier showed that the FUS genes are overall devoid of H3K4me2 in inducing and non-inducing conditions [ffccl1  and ∆fgccl1, respectively, suggesting that plant signals can still deduce full pathogenicity [F. fujikuroi and F. graminearum, deletion of FvSET1 also altered the chemical profile in F. verticillioides. Here, biosynthesis of the potent mycotoxin fumonisin B1 (FB1), the only SM investigated in this study, was significantly impaired upon loss of FvSet1 [Similarly, in  ∆fgset1 . Next toer genes . These fd FgCCL1 . Next tod FgCCL1 . Similarnditions . These rgenicity . Next tof FvSet1 . To sum f FvSet1 ,112, theF. fujikuroi and F. graminearum [FfKDM5 showed a decrease of the overall H3K4me3 levels and an increase in global H3K4me2, while deletion of FfKDM5 led to the opposite [F. graminearum: H3K4me3 was increased, while H3K4me2 was decreased in ∆fgkdm5. The amount of the global H3K4me1 was influenced neither by the overexpression nor the deletion of FfKDM5. As shown for other organisms, it is most likely that FfKdm1 is involved in the reversal of the me1. This, however, awaits further proof. Next to this, SM analysis supports the antagonizing effects of FfKdm5. Here, deletion of FfSET1, as well as constitutive overexpression of FfKMD5, showed increased pigment (FSR/BIK) and mycotoxin (FUS) biosynthesis, while ∆ffkdm5 behaved contrarily.To ensure transcriptional balance, H3K4 methylation can be antagonized by Kdm5 , a JmjC minearum ,104. ConFusarium species, deletion of KDM5 accompanied by a global increase in H3K4me3 led to the downregulation of most of the SMBGCs investigated here. Though this is in contrast with the proposed activating gene function of H3K4me3, it may very well reflect the fact that the analyzed Fusarium SMBGCs are not enriched for H3K4me2/me3 and thus not regulated by H3K4 methylation directly. Another possibility is that Kdm5 has an additional role in regulating SM gene expression apart from its histone demethylase activity as proposed for A. nidulans [It is noteworthy that, for both nidulans ,114.S. pombe [Neurospora crassa [N. crassa [DIM-5/-7/-9/CUL4/DDB1 complex) complex, which guides DIM-5 to the methylation site and facilitates H3K9me3 [N. crassa, HP1 acts as a scaffold for the recruitment of the DNA methyltransferase DIM-2. Here, HP1 can directly interact with DIM-2 through its chromoshadow domain, guiding the methyltransferase to remaining cytosines, which can then be methylated [Fusarium.A prominent mark important for the formation of constitutive heterochromatin—the transcriptionally inert form of chromatin—is histone H3 lysine 9 trimethylation (H3K9me3). H3K9me3 is established by the H3K9-methyltransferase Kmt1 , also knS. pombe  or NcDima crassa . In gene. crassa . Briefly H3K9me3 . Here, H H3K9me3 , which i H3K9me3 . Next tothylated ,121. DNAF. mangiferae and F. verticillioides. For the latter, deletion of the histone methyltransferase-encoding gene FvDIM5 showed a strong upregulation of two melanin biosynthesis-related genes on the transcript level and led to an elevated biosynthesis of the mycotoxin FB1 [F. mangiferae, FmKmt1 represses beauvericin (BEA) biosynthesis in nitrogen-limited conditions in basal culture milieu only, while BEA biosynthesis remained unaffected in nitrogen-sufficient and nitrogen-limiting conditions in an acidic milieu. Next to this, a functional FmKmt1 appears essential for wild type-like expression of some PKS-related genes in this fungus. Deletion of FmKMT1 resulted in downregulation of FmPKS8 and FmPKS40, two (at that time) cryptic PKS genes in F. mangiferae. The latter was recently identified as being involved in fusapyrone (FPY) biosynthesis [F. fujikuroi thus far [Fusarium.The function of the DIM-5 homolog Kmt1 has so far only been studied in two fusaria, oxin FB1 . For theynthesis . Notablythus far . Here, Hthus far . Thus, iF. graminearum. Here, loss of the HP1 ortholog FgHep1 had no impact on global H3K9me3 levels, but SM analysis showed decreased DON biosynthesis, while AUR biosynthesis was significantly increased in axenic cultures [FgDIM-2 and FgRID, were identified and characterized in F. graminearum [fgdim-2 and ∆fgRID, revealed elevated levels of 15-acetylated DON (15-ADON) for both, and a strain deleted for both, ∆∆fgdim-2/fgRID, led to an even more pronounced biosynthesis of this mycotoxin. Next to this, transcriptomic analysis of the double deletion strains suggests that additional SM-associated genes are also affected by the loss of FgDim-2 and FgRID. Concomitantly, SM measurement showed a high correlation between metabolic and transcriptomic data. In detail, the ∆∆fgdim-2/fgRID double deletion mutant was enriched in DON, fusaoctaxin and gramilin biosynthesis, while a decrease in AUR, FUS and fusaristatin production was observed [fgdim-2/fgRID) did not completely abolish DNA methylation in this fungus, suggesting the presence of more DNA methyltransferases in F. graminearum. Other components involved in constitutive heterochromatin formation have been characterized in cultures . Recentlminearum . Chemicaobserved . NotablyFusarium in some way. However, future research on the different components involved in the formation of constitutive heterochromatin in one organism combined with genome-wide H3K9me3 distribution analysis is required to shed some light on this issue [To sum up, H3K9me3 is involved in SM gene regulation in the genus is issue .Enhancer of zeste (E(Z)) in Drosophila melanogaster [N. crassa [polycomb repressive complex-2 (PRC2), which in D. melanogaster consists of four core proteins: E(Z), suppressor of zeste 12 (SU(Z)12), extra sex combs (ESC) and Nurf55. All components of the PRC2 complex are present in the genus Fusarium; however, the Nurf55 ortholog most likely acts outside the PRC2 complex, at least in N. crassa and F. graminearum [F. graminearum [D. melanogaster [N. crassa [The tri-methylation of histone H3 lysine 27 (H3K27me3) is a mark of repressive chromatin domains and is typically associated with chromosomal regions subject to a dynamic regulation, also called facultative heterochromatin . This manogaster  and Set7. crassa . Kmt6 isminearum ,126. Nexminearum , which anogaster  or N. crN. crassa [F. fujikuroi and F. graminearum [FgKMT6 as well as the knock-down of FfKMT6 in F. graminearum and F. fujikuroi, respectively, resulted in the de-repression of 15–30% of all genes, including many formerly silent and cryptic SMBGCs in both species [F. graminearum, 35 out of 45 SMBGCs are enriched in H3K27me3 in axenic culture. Lack of FgKmt6 led to the de-repression of 32 of these 35 SMBGCs. Similarly, for F. fujikuroi, 20 out of 48 putative SMBGCs were affected by the partial relief of H3K27me3 [FfKMT6 knock-down strains [F. graminearum, a mass spectrometry (MS)-based metabolomics approach identified overall 22 SMs in the fgkmt6 deletion mutant that were absent from cultures of the corresponding wild-type strain [Fusarium.In contrast to . crassa , large gminearum ,128. Int species ,128. In  strains ,128. Sime strain . To sum D. melanogaster [via its PHD domain, thereby facilitating distinct changes to the underlying chromatin landscape. RNA-sequencing (RNA-seq) analysis determined that loss of FgBp1 leads to the de-repression of a gene set, which is about 94% similar to the FgKMT6 deletion mutant. Comparable to ∆fgkmt6, about 74% of all genes associated with fungal secondary metabolism were strongly up-regulated, which goes in line with FgBP1 being involved in reading H3K27me3. Unexpectedly, only 44% of the genes bound by FgBP1 were detected in regions enriched for H3K27me3, entailing putative secondary functions of this protein [PRC1 components are described to “read” H3K27me3 deposited by PRC2 and, by this mechanism, promote chromatin compaction and maintenance of heterochromatic structures, as shown for nogaster . Howevernogaster . Only re protein . This, aSaccharomyces cerevisiae. Here, it is shown that the hyperphosphorylated form of RNA polymerase II (RNA PolII) travels along with ScSet2 and acts as a potent activator but also repressor of gene transcription [As some histone marks can strictly be associated with euchromatic or heterochromatic regions, it is more difficult for histone H3 lysine 36 trimethylation (H3K36me3). H3K36me3 is known to be located in both transcriptional regions in filamentous fungi . H3K36 mcription ,134.Fusarium, the first studies were performed in the maize pathogen F. verticillioides. Here, the absence of FvSet2 led to reduced amounts of the mycotoxin FB1 and the red pigment BIK in axenic culture, accompanied by reduced levels of H3K36me3 at FUM and BIK cluster genes as determined by ChIP-qPCR [FvSET2 in F. verticillioides led to a drastic reduction but not the complete abolishment of H3K36me3 levels in Western blot analysis [F. fujikuroi, where a second SET-domain containing protein, FfAsh1, was identified to be also involved in methylating H3K36 next to FfSet2 [ffset2 or ∆ffash1, H3K36me3 was completely abolished in ∆∆ffset2/ffash1 in this species. While H3K36me3 is found along complete chromosome arms [F. fujikuroi compared to F. verticillioides: while in the latter, FvSet2 is involved in BIK biosynthesis, deletion of FfASH1 but not FfSET2 affects BIK production [FfASH1 deletion mutant. Intriguingly, both clusters are located at subtelomeric regions, generally targeted by FfKmt6 and FfAsh1, but Set2 appears to mediate H3K36me3 at the BIK cluster [F. fujikuroi [For the genus hIP-qPCR . Unexpecanalysis . These ro FfSet2 . While Home arms ,112, botome arms ,112. Micoduction . Chemica cluster . Howeverujikuroi .S. cerevisiae [FfKDM4 in F. fujikuroi did not reveal an overall increase in H3K36 methylation and did not affect the biosynthesis of known SMs, whereas the constitutive overexpression of KDM4 showed reduced genome-wide H3K36me3 as determined by Western blot, thereby confirming FfKdm4 as a H3K36-specific demethylase. Of note, FfKdm4 overexpression is unstable, thereby prohibiting further phenotypic characterizations [F. fujikuroi [H3K36me3 is removed by ScRph1 or Kdm4 ,136, a mrevisiae . Deletioizations . Whetherujikuroi .S. pombe implicated that H4K20me is more restricted to coordinating the DNA damage response than heterochromatin formation [S. pombe [Histone H4 lysine 20 trimethylation (H4K20me3) is a repressive histone mark and, as such, is associated with heterochromatin formation and herein gene silencing in higher eukaryotes . Other iormation ,140. Allormation  in S. poF. graminearum and F. fujikuroi [S. pombe, in fusaria, Kmt5 is solely responsible for the me1, me2 and me3 of H4K20. Deletion of FgKMT5 and FfKMT5 has distinct effects on fungal secondary metabolism, depending on the species. Deletion of FfKMT5 positively affects FUS biosynthesis in F. fujikuroi, while the same pathway was negatively affected by ∆fgkmt5 in F. graminearum. While the loss of FgKmt5 led to strongly reduced amounts of ZEA, constitutive overexpression of FgKMT5 led to a slight but significant upregulation of DON biosynthesis. In F. fujikuroi, FfKMT5 overexpression positively influenced GA and BIK biosynthesis [Fusarium, and hence their role in regulating fungal SMBGCs remains elusive at the moment.Thus far, H4K20 methylation is not well understood in filamentous fungi, and only recently was the role of Kmt5 in secondary metabolism explored in the two plant pathogens ujikuroi . As obseynthesis . NotablyF. fujikuroi, H3K9ac is highly abundant at transcribed genes, including active SMBGCs, while this histone mark is largely absent from non-expressed genes [general control non-de repressible 5 (GCN5)-related acetyltransferase), MYST  and Rtt109 (regulator of Ty1 transposition gene product 109) [Fusarium [Fusarium but included in the following paragraphs, emphasizing their importance regarding SM gene regulation.Histone methylation generally is recognized by reader proteins, determining the putative readout of the newly established mark, i.e., formation of condensed or open chromatin, while histone acetylation itself changes the chromatin formation through neutralization of the positively charged lysine residues, resulting in the overall relaxation of the chromatin structure . The aceed genes . HATs aruct 109) , with thFusarium ,147,148,2+-dependent HDACs as well as Class III NAD+-dependent silent information regulators 2 (Sir2) (“sirtuins”) [2+-dependent HDACs have been characterized in more detail in fusaria  . For the fusaria . So far, fusaria , which iSpt-Ada-Gcn5-acetyltransferase) complex is one of the most studied chromatin-modifying complexes in filamentous fungi, including fusaria. In S. cerevisiae, the SAGA complex consists of 18 highly conserved subunits, including a structural core, the catalytic acetyltransferase Gcn5 (KAT2) [The SAGA (5 (KAT2) , a histo5 (KAT2) ,152.F. fujikuroi, co-immunoprecipitation (co-IP) determined the presence of all structural elements known from yeast, except the DUB submodule Chd1 as well as the physical interaction of FfGcn5 and the transcriptional adaptors FfAda2 and FfAda3. Demonstrating the manifold substrate recognition, deletion of FfGCN5, FfADA2, and FfADA3 revealed that they are detrimental at least for H3K4, H3K9, H3K18, H3K27 and H3K36 acetylation [F. graminearum a similar but still distinct Gcn5-dependent acetylation pattern was observed. Here, loss of FgGcn5 resulted in the dramatic reduction of H3K9, H3K14, H3K18 and H3K27 acetylation [GCN5 led to the de-regulation of a large set of genes, including primary and secondary metabolism in F. fujikuroi, F. graminearum and F. oxysporum [F. graminearum, 202 genes belonging to 62 SMBGCs were affected in the ∆fggcn5 mutant, including TRI gene expression. Concomitantly, DON biosynthesis was almost abolished upon deletion of FgGCN5 [Fusarium-related disease on crop plants is tempting, but since the SAGA complex is conserved from yeast to humans, the effect of PCN on non-target organisms must be evaluated in detail. For F. fujikuroi, FfGCN5 deletion resulted in the deregulation of 61% of all SM key enzyme-encoding genes. Here, the majority of these genes were down-regulated in at least one condition, going in line with the activating role of histone acetylation [ffgcn5 mutant. As expected, H3K9, H3K14, and H3K27 acetylation levels were barely detectable at the 5′-end of those SMBGCs. Interestingly, transcriptomic data suggests decreased GA and BIK biosynthesis under these normally repressing conditions, while deletion of FfGCN5 had no impact on FUB biosynthesis. For GA and BIK, it is doubtful that under non-inducing conditions, histone acetylation is present anyways, while for FUB biosynthesis, loss of these acetylation marks had no impact on biosynthesis, suggesting the involvement of another regulator. Genome-wide ChIP analyses of each acetylation mark under inducing/non-inducing conditions would be more conclusive at this point [Fusarium, and additional target sites are likely. Not surprisingly, SM gene regulation is affected by the loss of Gcn5. However, further studies are required to dissect the impact of single acetylation marks on SM gene expression and thus unravel how Gcn5 and the SAGA complex mediate SM gene expression in this fungus.For tylation . In F. gtylation . Overallxysporum . In detaf FgGCN5 . Four cof FgGCN5 ,153. In f FgGCN5 . Notablytylation . To studis point . To sum S. cerevisiae [A. nidulans or F. graminearum [S. cerevisiae, ScEsa1 is the catalytic subunit of the NuA4  complex, important for global acetylation levels on H4 [The NuA4 complex consists of 13 unique subunits important for nucleosome acetylation, transcription and recruitment of other factors . Esa1 berevisiae . This isminearum ,158. In ls on H4 . Thus faF. graminearum [FgFNG1 revealed that it is integral for the function and stability of the NuA4 complex, and as such, FgFng1 is detrimental for the acetylation of H4K5, H4K8 and H4K12 and to a lesser extent also for H4K16. Localization studies showed that FgFng1 is enriched in euchromatic regions co-localizing with H3K4me2. Next to this, transcriptomic data of a ∆fgfng1 deletion strain showed a strong upregulation of FgKmt2 (FgSet1) and FgKmt6. However, no direct correlation between H4 acetylation and H3K4- or H3K27 methylation has been confirmed so far. Notably, the FgFNG1 deletion strain is defective in DON biosynthesis [One conserved component of the NuA4 complex is ScYng2, which is responsible for transcriptional activation in yeast. Contrary to FgEsa1, the ScYng2 ortholog, Fng1, is dispensable in minearum . Deletioynthesis .something about silencing protein), also known as KAT6 [nucleosome acetyltransferase of histone H3). It is important for gene transcription, HAT activity and integrity of the complex in S. cerevisiae. [F. graminearum, FgSas3 is important for the acetylation on H3K4 and H3K14, but the loss of FgSas3 implicated no change on global H3 acetylation levels. Deletion of FgSAS3 revealed the deregulation of important SM genes involved in AUR, ZEA and DON biosynthesis [Sas3 , or KAT8, [S. cerevisiae [F. graminearum, and its deletion also had no significant impact on the biosynthesis of other known SMs [Sas2  is the minearum . It is tujikuroi ,148, sugregulator of Ty1 transposition 109)/KAT12 [Aspergillus flavus and Beauveria bassiana imply acetylation activity at H3K9 and/or H3K56 residues, respectively [F. graminearum [Fusarium spp.Differently to Sas2 and Elp3, Rtt109 (9)/KAT12  is a fun9)/KAT12 . Recent ectively ,170. Losminearum . It remaThe yeast ortholog ScHat1 belongs to the class B of histone acetyltransferases and is shown to solely post-translationally modify lysine 5 and 12 of the free histone H4 ,172. NexFusarium species, the Hat1 (KAT1) [F. fujikuroi. Here, localization studies revealed, that FfHat1 is mainly localized to the nucleus, unlike what has been proposed for class B HATs. This, however, is in agreement with reports from S. cerevisiae [F. fujikuroi, as targeted deletion and constitutive overexpression of FfHAT1 showed reduced and elevated GA levels, respectively. In contrast, FUB biosynthesis appears to be negatively affected by FfHat1, as FUB levels were elevated in the FfHAT1 deletion strain, while the opposite was observed in the overexpression strain [Fusarium.For 1 (KAT1)  functionrevisiae ,171. FfHn strain . It rema2+-dependent HDACs of Class I and II are well characterized in yeast and humans and sport many important functions regarding gene expression [F. fujikuroi and F. graminearum and, to some extent, also in F. asiaticum. Hda1, Hda2 and Hda4 are dispensable, while the homolog of Rpd3, Hda3, appears essential in both species [F. fujikuroi, respectively. Microarray analysis for ffhda1 deletion mutants showed a strong down-regulation of all investigated genes, including 10 out of 48 SMBGCs. Subsequent SM analyses confirmed microarray analysis for selected SMs. Similar results were observed for the ∆ffhda2 mutant, while deletion of FfHDA4 did not reveal changes in its SM profile. Interestingly, constitutive overexpression of FfHDA1 showed a similar chemical profile as the respective deletion strain, suggesting that balanced acetylation levels are crucial for wild type-like secondary metabolism. Notably, in F. graminearum, deletion of FgHDF1 (Hda2 ortholog) led to an overall reduction of 60% of HDAC activity, possibly implying different roles for the HDACs in both species [F. graminearum, lack of FgHdf1 positively influenced AUR biosynthesis, while slightly decreased DON levels were detected [F. asiaticum resulted in significantly increased DON production, while no impact on DON production was observed for the FgHDF1 deletion strain [Histone deacetylases counteract histone acetyltransferase function by the dynamic removal of acetyl residues, not only altering chromatin structure but leading to the addition or exclusion of alternative histone marks. Znpression . This cl species ,175. A H species ,175. In detected . Interesn strain ,176.FfHDA1 induced expression of the otherwise silent NRPS22 gene cluster, which was later identified to be responsible for the biosynthesis of the toxic depsipeptide BEA [BEA gene cluster revealed that loss of FfHda1 was accompanied by increased H3K27 acetylation at BEA cluster genes [BEA gene expression under standard laboratory conditions [F. fujikuroi, H3K9ac, present under inducing conditions in the wild type, was found to be reduced at the GA gene cluster in ∆ffhda1, in line with a decreased biosynthesis under inducing conditions. Similarly, H3K9ac was enriched at BIK genes under non-favorable conditions accompanied by BIK production upon loss of FfHda1. On the contrary, decreased BIK production was observed under favorable conditions, suggesting that additional factors are involved and necessary for the correct biological read-out. HAT complexes are known to recruit global transcription factors (TFs); the disturbance of the acetylation level could partly overrule this phenomenon [Next to already known compounds, deletion of tide BEA ,69. Detanditions ,175. Whi2+-dependent HDACs are largely enigmatic, and their overall targets are unknown. In the case of sirtuin-domain HDACs, no information is available at all in the genus Fusarium, giving the incentive to analyze both classes of histone-modifying enzymes and their interplay with other histone marks in the future to shed light on their mode-of-action in vivo.Thus far, the functions of ZnChanges in the chromatin structure are mediated through altering the composition of the histone marks, thereby giving way to other factors to act on the underlying DNA, such as narrow- or broad-domain TFs. This illustrates a complex gene network involving the establishment and removal of histone marks, the recruitment of TFs and, finally, accessibility for PolII, thereby ensuring the appropriate changes in the chromatin structure and the correct biological readout of the underlying DNA. Few examples exist that shed light on this complex topic.F. fujikuroi [GA or BIK [FfAREA and FfAREB led to abolished GA biosynthesis and suggested synergistical activation [GA gene cluster, which was accompanied by drastically reduced levels of H3K9ac at this cluster [FfAREA and FfAREB and HATs/HDACs [F. fujikuroi is regulated by an intrinsic cluster-specific TF (FUM21) and depends on the presence of both FfAreA and FfAreB. Here, constitutive overexpression of FUM21 in the ∆∆ffareA/ffareB double mutant showed partial restoration of FUM biosynthesis, indicating positive regulation by nitrogen availability [In general, filamentous fungi can utilize a diverse set of nitrogen sources but use energetically favored substrates, e.g., ammonium or glutamine, before scavenging alternative substances . Two GATujikuroi . Next toA or BIK ,179. GA A or BIK ,38. Heretivation . Next, d cluster . This suTs/HDACs . Next tolability .Fusarium species. Furthermore, interaction between VeA/Vel1 and the putative methyltransferase Lae1 were determined [F. oxysporum, the combinatorial roles of FoAreA and velvet complex proteins was investigated. Down-regulation for BEA biosynthesis was observed in FoAREA, FoVEA and FoLAE1 deletion strains; however, interaction studies could not determine physical interactions between FoAreA and velvet complex components [F. oxysporum, modification of chromatin accessibility in different SMBGCs was tested. Here, for triacetylfusarinine C and ferricrocin chromatin, accessibility in the ∆fovelA and ∆fovelB was completely abolished, while for ∆folae1, a strong reduction in accessibility was observed, similarly to what was seen for BIK biosynthesis [FvLAE1 led to a dramatic change in global gene expression, where about 55% of all genes were de-regulated; most were classified as SM-regulatory [F. fujikuroi transcriptomic data suggested different SM-regulation mechanisms in the ∆ffvel1 deletion strain [FUM, FUS, FUB, GPY and GA genes and upregulation of BIK biosynthetic genes was observed [FfVEL2 caused reduced GA, GPY and increased BIK gene expression [The velvet protein family is a fungal-specific conserved complex important for the regulation of secondary metabolism in filamentous fungi . The comtermined ,181,182.mponents . For F. ynthesis . Deletiogulatory . Notablyn strain ,55,73. Dobserved ,55,73 Sipression ,55.F. fujikuroi, BIK biosynthesis depends on the presence of the global regulators FfPacC and FfAreA. PacC is a Cys2His2-zinc finger transcription factor involved in the pH-dependent expression of SM genes [BIK promoters [F. fujikuroi, FfVEL1 deletion overruled FfPacC/FfAreA dependency, since BIK is accumulated under unfavorable pH conditions and sufficient nitrogen conditions, despite unchanged transcript levels of FfPacC and FfAreA/FfAreB in the deletion strains [FfHDA1 deletion mutant, while the impact of the nitrogen-responsive TF FfAreA was abolished for inducing conditions [BIK gene cluster, demonstrate the versatile regulatory network underlying SM gene expression. As Vel1/Lae1 was shown to be involved in the modulation of chromatin accessibility in F. oxysporum, it is evident that similar mechanisms take place in F. fujikuroi [For SM genes . PacC isSM genes ,183. FfPromoters . Interes strains . Howevernditions . Studiesujikuroi . In the F. fujikuroi, showed that 22 out of 48 SMBGCs were affected by transcriptomic analysis. Intriguingly, overexpression led to the activation of clusters of thus far unknown products, i.e., STC7 and PKS9 [BEA gene cluster [FfLAE1 led to the circumvention of other regulatory circuits, e.g., nitrogen or pH repression, which was shown before for BIK biosynthesis in the ∆ffvel1 strain [FfHAT1 was able to partially restore the chemical profile of ∆fflae1 [An extensive characterization of FfLae1, in and PKS9 ,55,73, a cluster ,70. For 1 strain ,34. HoweF. fujikuroi ∆ffsge1 showed the de-regulation of 12 histone-modifying enzymes in nitrogen-sufficient conditions, which are preferred for FfSGE1 expression [ffvel1 and ∆ffsge1 led to increased and decreased BIK production, respectively. However, constitutive overexpression of FfSGE1 in ∆ffvel1 could not rescue BIK biosynthesis. Next, a ffareA-deficient strain showed loss of GA and FUM biosynthesis; however, overexpression of FgSGE1 in this background could partially restore GA biosynthesis, while this was not the case for FUM. Similarly, overexpression of FfSGE1 in the ∆ffapf2 background (apicidin F (APF) cluster-specific TF), which abolishes APF biosynthesis, was able to partially restore APF biosynthesis [Sge1 is considered a transcriptional regulator in higher eukaryotes, revealing important functions as a morphological switch between saprophytic and pathogenic lifestyle as well as for the regulation of fungal secondary metabolism ,185. TraFusarium.To sum up, the dynamic interaction between regulators, TFs and/or chromatin-modifying enzymes appears manifold. Currently, the complex hierarchy of SM gene expression is still opaque, giving rise to a platform for future work in order to further unravel the exact mode of action of the different regulatory components and how they are interconnected to ensure the timely and reversible expression of SM genes in the genus Fusarium. Furthermore, the versatile interplay in changes of the chromatin structure and the recruitment of transcription factors, or “regulators”, will help to deepen our understanding of the complex regulatory networks behind SM gene expression. Some histone marks seem to be more important than others for fungal secondary metabolism, e.g., Kmt6 (H3K27me3) or GCN5 , both considered important mediators for the expression of secondary metabolism-associated genes. However, even with histone methylation and acetylation, which appear to be already well-characterized in the genus Fusarium, their molecular function is not well-understood. Genome-wide distribution studies, combined with detailed molecular and chemical characterizations, are due to shed further light on their role and their functional network in detail. Additionally, there is still a series of predicted proteins with unknown function, as well as other possible histone marks such as phosphorylation, ubiquitinoylation or crotonylation, which are so far not examined at all. Furthermore, global regulators such as Lae1 and Sge1 were shown to either modify DNA accessibility directly or by interacting with other histone-modifying enzymes. However, little is known so far about the crosstalk between histone marks and “regulators”, providing an incentive for future research. Knowledge about the molecular aspects that govern SM gene regulation thus constitutes a powerful tool to gain broader insights into the deduction of novel compounds and to provide tools for novel cropping strategies.This review summarizes the latest advances in understanding chromatin regulatory mechanisms and underlines the importance of the chromatin structure on SM biosynthesis in the genus


Long non-coding RNAs (lncRNAs) play a vital role in a variety of biological functions in plant growth and development. In this study, we provided an overview of the molecular mechanisms of lncRNAs in interacting with other biomolecules with an emphasis on those lncRNAs validated only by low-throughput experiments. LncRNAs function through playing multiple roles, including sponger for sequestering RNA or DNA, guider or decoy for recruiting or hijacking transcription factors or peptides, and scaffold for binding with chromatin modification complexes, as well as precursor of microRNAs or small interfering RNAs. These regulatory roles have been validated in several plant species with a comprehensive list of 73 lncRNA–molecule interaction pairs in 16 plant species found so far, suggesting their commonality in the plant kingdom. Such initial findings of a small number of functional plant lncRNAs represent the beginning of what is to come as lncRNAs with unknown functions were found in orders of magnitude more than proteins. Non-coding RNAs (ncRNAs), transcribed in over 90% of eukaryotic genomes of fungi, plants, and animals  refer to those ncRNAs longer than 200 nucleotides (nt) in length , Northern blot, real-time quantitative polymerase chain reaction (RT-qPCR), overexpression, RNA interference (RNAi), clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9), RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP)  is complementary to miR399, but it contains a mismatched loop to interrupt the pairing at the miRNA cleavage site , which subsequently reduces the Pi content of the shoot  that are small ncRNAs (21–23 nt in length) play vital roles in diverse biological processes such as root development  locus in rice, is cleaved by miR2118 to generate 21-nt phasiRNAs. The abundance of phasiRNAs is associated with the photoperiod-sensitive male sterile  and MSPPL2, are also cleaved by miR2118 to yield phasiRNAs that play a role in the posttranscriptional regulation during meiosis in rice . In eukaryote, lncRNAs or other RNAs are complemented and cleaved by miRNA to generate single-stranded RNAs first, and then to form double-stranded RNAs from the lysed RNA  Komiya, . As showMIR genes mainly located in the intergenic regions are first transcribed into primary miRNAs, then modified into the precursor miRNAs, forming miRNA duplexes, and finally forming mature miRNA to guide the posttranscriptional gene silencing  in rice is an antisense transcript of R2R3-MYB transcription factor gene, namely, MYB60 locus. The RNA interference and the overexpression experiments indicate that TL cis-suppresses the MYB60 expression in regulating leaf blade flattening in rice by altering the chromatin structure  was shown essential for the lateral root development by regulating the transcription level of auxin-responsive genes. APOLO controls these auxin-responsive genes in cis or trans by modulating chromatin loops  after treating with auxin and cis-activates the neighboring gene PID through formatting a chromatin loop with the promoter of PID by reducing H3K27me3 in the loop  to deposit the DNA methylation on the genomic region of APOLO-PID   to repress its transcription in trans  and CBF1. RNA Pol II read-through transcription of SVALKA leads to the expression of antisense CBF1 (asCBF1), and the cascade of SVALKA-asCBF1 represses CBF1 transcribing through RNA Pol II collision stemming, leading to maximize the low-temperature tolerance of plants with minimal adaptation changes  to deposit the repressive histone marks  at the target chromatin and to repress the auxin-responsive gene transcription   , respectively  to enrich H3K27me3 at FLC chromatin  gene discovered in Arabidopsis, and it activates MAF4 transcription involved in H3K4me3 deposition. Further assays showed that MAS binds to the locus of MAF4 directly and recruits the COMPASS-like complexes  to enhance H3K4me3. The cold-induced formation of the MAS–WAR5a complex suppresses the premature flowering in vernalization  lncRNAs, which is sensitive to osmotic stress in Populus simonii, through binding with MYBs transcription factors to interrupt them from interacting with DNA and lead to an increase in the expression of target genes in cis and trans  and is important in root nodule organogenesis . When MtENOD40 expressed in root tissues, the ENOD40-MtRBP1 complex would relocate from the nucleus to the cytoplasm , when co-expressed with MtENOD40, also lead to the relocation of AtNSRs from the nucleus to the cytoplasm, indicating the conserved relocation activity of MtENOD40 , is induced by auxin. This lncRNA would competitively bind AtNSRs with AS targets in vitro and in vivo, regulating transcriptome diversity in the formation of lateral root development  is derived from the antisense strand of neighboring gene LRK (leucine-rich repeat receptor kinase) cluster. Its overexpression leads to the upregulated expression of LRK genes and increased rice grain yields  and OsWDR5 in vivo, which in turn gives a rise in the enrichment of H3K4me3 and H4K16 acetylation (H4K16ac) at chromatin to active LRK1  activates PATHOGENESIS-RELATED GENE1 (PR1) by directly binding with Mediator subunit 19a (MED19a) and negative transcription regulator FIBRILLARIN2 (FIB2) to enrich MED19a on PR1 promoter  and MtSNARP2  (Zhou et al., The finding of plant lncRNAs and their functional mechanisms is still in its early phase, and all the experimentally validated interaction pairs in plants are shown in What are the patterns in the secondary structure of lncRNAs in recognizing interacting molecules?What are the key sequence or structural factors that affect lncRNA functions?What is the degradation mechanism of lncRNAs in organisms?These questions highlight the importance of RNA structures in providing functional clues. Recent advances in deep learning of protein structure prediction (Senior et al., As illustrated in Currently, plant lncRNAs were shown to function through the formation of eTM with miRNA to regulate the expression of miRNA target genes indirectly , acting A. thaliana and O. sativa are the most studied plants (Taken together, understanding the functions of plant lncRNAs is just at the beginning stage. Model plants such as d plants . Signifid plants . It willQC conceived the topic of the article. QC, KL, RY, PH, and ZC wrote the manuscript. BZ, YZ, and JW reviewed and revised the manuscript. All authors have directly contributed to the work and also read and approved the final published version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The centromere is a specialized chromosomal structure essential for chromosome segregation. Centromere dysfunction leads to chromosome segregation errors and genome instability. In most eukaryotes, centromere identity is specified epigenetically by CENP-A, a centromere-specific histone H3 variant. CENP-A replaces histone H3 in centromeres, and nucleates the assembly of the kinetochore complex. Mislocalization of CENP-A to non-centromeric regions causes ectopic assembly of CENP-A chromatin, which has a devastating impact on chromosome segregation and has been linked to a variety of human cancers. How non-centromeric regions are protected from CENP-A misincorporation in normal cells is largely unexplored. Here, we review the most recent advances on the mechanisms underlying the prevention of ectopic centromere formation, and discuss the implications in human disease. To achieve this, the spindle, a micro-machine composed of microtubules, is formed when a cell starts to divide. Spindle microtubules attach to chromosomes via the kinetochore, a large protein complex consisting of over 100 proteins, and physically pull the two chromatids of the same chromosome towards opposite poles . The ce. 2Schizosaccharomyces pombe, Candida albicans, Drosophila melanogaster, and chicken and human cells [The most common types of centromere are the large and complex regional centromeres. Regional centromeres contain multiple microtubule attachment sites per centromere and can span a few kilobases to several megabases of DNA. DNA sequences underlying regional centromeres vary from species to species ,11. Neocan cells –24. Thesan cells –27.Box 1. Glossary.Acentric:lacking a centromereAneuploidy:abnormal number of chromosomesCentromere:a specialized chromatin region where the kinetochore is assembledEpigenetic:relating to heritable phenotypic changes that do not involve alteration of the DNA sequenceHeterochromatin:tightly condensed chromatin region with relatively low gene transcriptionHistone variant:non-canonical variant of a histoneHolocentric species:species with holocentric chromosomes, of which the entire length functions as the centromereKinetochore:a large multi-protein complex that interacts with microtubules to facilitate chromosome segregationNeocentromeres:new centromeres that form at non-canonical centromeric regionsPoint centromere:the centromere in budding yeast that is genetically defined by a 125 bp DNA sequenceProteolysis:enzymatic breakdown of proteins or peptides into amino acidsRegional centromeres:large and complex centromeres that form multiple microtubule attachment sites per chromosomeCID in Drosophila results in mislocalization of CENP-A in non-centromeric regions, which promotes ectopic kinetochore assembly [CID using lacO/LacI system to chromosome arms in Drosophila cells leads to the formation of functional centromeres. The ectopic CENP-ACID chromatin can self-propagate even without lacO/LacI system [The conserved centromere-specific histone H3 variant, CENP-A, plays an essential role in centromere identity by acting as a key epigenetic mark for centromeres. CENP-A was first identified as a human autoantigen in human scleroderma patients, and was subsequently found to copurify with histones and nucleosomes –30. The assembly ,42. ArtiI system . MislocaI system ,44–46. NDrosophila that guides the deposition of CENP-A [Drosophila somatic tissues [CENP-A proteins across species share a conserved C-terminal histone fold domain (HFD), which carries the CENP-A targeting domain (CATD). CATD containing the first loop (L1) and the second α-helix (α2) is necessary and sufficient for centromeric localization of CENP-A in vertebrates ,48. On tf CENP-A ,63. Intef CENP-A . Severalf CENP-A ). The ti tissues –68. Howe tissues –71. The  tissues –78.per se appears antithetical to CENP-A chromatin [S. pombe and Drosophila [Another notable feature of regional centromeres is that they are often embedded in epigenetically distinct heterochromatin. The gene-poor chromatin domain is transcriptionally silenced, enriched with the histone H3K9 methylation. Although heterochromatin hromatin ,80, the hromatin ,42,81–85osophila ,41,42,84osophila ,87. In cosophila . Interesosophila . Removinosophila . The exaSaccharomyces cerevisiae, contain genetically defined ‘point centromeres'. The point centromere in budding yeast is 125 bp long, forming a single microtubule attachment site. The DNA sequence is necessary and sufficient for centromere formation [Caenorhabditis elegans demonstrated that CENP-A is incorporated at low density in non-repeated regions across approximately half of the genome [In contrast with regional centromeres, some budding yeasts, such as ormation ,90,91. Uormation –94. Anotormation . A genome genome . Neverthe genome .. 3CID in Drosophila induces ectopic assembly of CENP-ACID chromatin at non-centromeric regions. Mislocalized CENP-ACID chromatin can promote the formation of functional kinetochores. As a result, cells exhibit mitotic delays, anaphase bridges, chromosome fragmentation and spindle disorganization, which lead to cell and organism lethality [Cnp1 in fission yeast also results in the assembly of CENP-ACnp1 chromatin at non-centromeric regions. The mispositioned CENP-ACnp1 is able to recruit kinetochore proteins. Consequently, fission yeast cells overexpressing CENP-ACnp1 also display chromosome missegregation during mitosis and meiosis [Overexpressed CENP-Aethality . Similar meiosis ,98. Over meiosis ,45,46. I meiosis . Overexp meiosis –101. IntIt has been shown that in a variety of organisms, excess CENP-A preferentially targets the region near heterochromatin ,98,102. . 4Upregulation of CENP-A expression has been found in more than 20 different human tumours. CENP-A overexpression has thus been recommended for use as a biomarker of poor patient prognosis and as a predictive biomarker for chemotherapy ,106–112.MYC [In cancer cells with elevated CENP-A level, CENP-A-containing nucleosomes are assembled at non-centromeric regions. Similar to yeasts and flies, overexpressed CENP-A in human cancer cells can result in mislocalization to both highly accessible open chromatin regions and heterochromatic loci, such as subtelomeres . EctopicMYC . How CEN. 5Cse4 degradation by targeting its CATD domain [Cse4 proteolysis [Cse4 to prevent its mistargeting. A ubiquitin protease, Ubp8, is able to deubiquitylate CENP-ACse4, and functions in concert with Psh1 to mediate the CENP-ACse4 proteolysis [Cse4 and Psh1 requires a proline isomerase, Fpr3. It is proposed that the structural change between the cis and trans form of CENP-ACse4 regulated by Fpr3 may be important for the CENP-ACse4 degradation by Psh1 [Cse4 proteolysis. The study further showed that the role of DDK in CENP-ACse4 proteolysis is independent of its role in DNA replication [One of the best-studied mechanisms involved in the prevention of CENP-A mispositioning is ubiquitin-dependent proteolysis of CENP-A . The rolD domain ,119. Itsteolysis . A numbeteolysis , Doa1  in CENP-ACse4. However, Slx5-mediated CENP-ACse4 proteolysis acts in a manner that is independent of Psh1 [Cse4 in budding yeast [Small ubiquitin-like modifier (or SUMO) proteins are a family of small proteins similar to ubiquitin that is covalently attached to other proteins. Sumoylation is involved in many cellular processes, including ubiquitination. The SUMO-targeted ubiquitin ligase (STUbL) links SUMO and ubiquitin modification pathways to facilitate proteolysis of cellular substrates . Sumoyla of Psh1 ,129. Intng yeast ,131.Drosophila to prevent the formation of ectopic CENP-A chromatin [Drosophila, the F-box protein Ppa, the ubiquitin E3 ligase APC/C and the E3 ligase CUL3/RDX have been found to regulate the proteolysis of the CENP-ACID [CID is phosphorylated by casein kinase II. The phosphorylation regulates the stability of prenucleosomal CENP-ACID via the Ppa-proteasome pathway and promotes the removal of CENP-ACID from ectopic sites [Ubiquitin-mediated CENP-A degradation has also been shown in fission yeast and hromatin ,132,133.ENP-ACID –136. A ric sites .Ubiquitin-dependent proteolysis of CENP-A has also been used in vertebrates to regulate centromere functions. In human cells, the herpes simplex virus type 1 protein ICP0 has been shown to be able to induce the proteasome-dependent degradation of CENP-A . UbiquitCse4 is lethal in budding yeast cells lacking Psh1 [Drosophila, causes severe chromosome missegregation and growth defects [Cse4 compared to other species which contain large regional centromeres; CENP-ACse4 may thus be subject to more efficient ubiquitination, leading to its rapid degradation [Cse4 chromatin at ectopic regions in budding yeast may be less stable, which can be quickly removed during the cell cycle.Overexpression of CENP-Aing Psh1 ,119, buting Psh1 ,144. By  defects ,44,46. Oradation . Anotherradation ,43,44. T. 5.1S. cerevisiae has been suggested to associate with CENP-A to inhibit its degradation [Drosophila, was also implicated in protecting CENP-ACID from degradation [Cnp1 from ubiquitin-mediated degradation [Cnp1 in fission yeast, indicating that additional pathways also participate in the process [These exciting discoveries also raise another important question: how is CENP-A at centromeres protected from ubiquitin-mediated degradation? HJURP/Scm3, the CENP-A chaperone, in human cells and radation ,118. CALradation ,135. Addradation ,145. A rradation . The higradation . Defectsradation –149, and process .. 6Proper centromere assembly depends on the tight regulation of the CENP-A level. CENP-A level is subject to not only post-translational regulation, but also transcriptional control. Reduction of CENP-A RNAs causes lagging chromosomes ,150. A ret al. [Cnp1 transcription in fission yeast. CENP-ACnp1 transcription in fission yeast occurs at G1, leading to an almost twofold increase in CENP-ACnp1 during the S-phase [et al. demonstrated that the periodic transcription of CENP-ACnp1 is lost in MBF mutants, resulting in the increased level of CENP-ACnp1 and consequently CENP-A mislocalization to non-centromeric regions and mitotic defects  complex is a conserved chromatin remodelling complex, composed of two subunits, Spt16 and SSRP1 (Pob3 in yeast). The complex is responsible for recycling dissociated histones during transcription elongation when nucleosomes are transiently disassembled in front of RNA polymerase II. Dysfunctional FACT results in the loss of histone H3 nucleosome across the genome in revisiae ,171. In promised . Howeverradation . Interestromeres –175.. 8.1.2Cse4 at euchromatin corresponding to the sites of rapidly exchanging nucleosomes in wild-type cells. It was thus proposed that CENP-ACse4 mislocalization primarily results from decreased eviction of CENP-ACse4 at euchromatic regions mediated by both HIR and CAF-1 [Cse4 assembly is drastically diminished in the absence of CAF-1 in budding yeast. This opposite effect led them to propose that CAF-1 interacts with CENP-ACse4 to facilitate the deposition of CENP-ACse4 to non-centromeric regions [Cse4 is mispositioned in euchromatin in the mutant of HIR2, another subunit of the HIR complex, when CENP-ACse4 is overexpressed. The study further showed that HIR2 mediates CENP-ACse4 proteolysis by facilitating the interaction of CENP-ACse4 with Psh1 [Cse4 mislocalization through regulation of ubiquitin-mediated CENP-ACse4 degradation.The HIR/HIRA complex is a conserved histone chaperone that facilitates the deposition of the histone variant H3.3 into chromatin in a replication-independent manner . CAF-1, nd CAF-1 . Howevernd CAF-1  has foun regions . Further regions  showed tith Psh1 . Thus, t. 8.1.3Cse4 has been found to associate with non-centromeric regions in a mutant of Snf2, the catalytic subunit of SWI/SNF. But the overall level of CENP-ACse4 does not change in the mutant, suggesting that the abnormal distribution of CENP-ACse4 in the mutant is not due to defects in the regulation of CENP-ACse4 protein level [Cse4, but not H3 nucleosome in vitro [Cse4 to centromeres by removing it from ectopic sites. It is unclear why SWI/SNF prefers to destabilize CENP-ACse4 nucleosomes. It is speculated that the CENP-ACse4 nucleosomes may be inherently less stable than H3 nucleosomes [The SWI/SNF complex is a large, multi-subunit chromatin remodelling complex that plays important roles in the regulation of transcription, the cell cycle, and DNA repair and replication. The conserved complex uses the energy of ATP hydrolysis to mobilize and restructure nucleosomes . In buddin level . It has in vitro . This stleosomes .. 8.1.4Drosophila and human cells. The HFD-containing protein, CHRAC14, is a subunit of the CHRAC complex. CHRAC14 is important for DNA damage response in Drosophila. In the absence of CHRAC14, DNA damage is unable to be efficiently repaired [CID is mislocalized to damage DNA sites in the Chrac14 mutant, resulting in the formation of ectopic kinetochore and genome instability. CHRAC14 also interacts with CENP-ACID [CID observed in the Chrac14 mutant results from DNA repair defects in the mutant, or whether CHRAC14 plays a direct role in preventing the assembly of ectopic centromeres.The chromatin accessibility complex (CHRAC) is an ISWI-containing chromatin remodelling complex found in repaired . NotablyENP-ACID . It has ENP-ACID . It is s. 8.2et al. [Cnp1 in fission yeast. CENP-ACnp1-GFP forms ectopic foci in the ccp1Δ mutants, whereas overexpression of Ccp1 results in removal of mistargeted CENP-ACnp1-GFP. Ccp1 preferentially associates with CENP-ACnp1 both in vitro and in vivo. Crystal structural analyses indicated that, like other NAP family proteins, Ccp1 forms a homodimeric ‘headphone’ architecture with a pronounced cleft at the centre of the dimer. The dimerization of Ccp1 is required for its anti-CENP-A loading activity [Cnp1 from the chromatin is not clear.The Nucleosome Assembly Protein (NAP) family proteins are found in many organisms, and often act as histone chaperones, serving a crucial role in the assembly and disassembly of nucleosomes . Using aet al.  identifiactivity . But howet al. [Cnp1 loading within centromeres. Thus, both CENP-A loading and anti-loading factors are recruited to centromeres, which may explain how CENP-A and histone H3 levels are properly maintained in centromeres. Consistent with this idea, both Ccp1 and the CENP-A chaperone Scm3 are recruited to centromeres at the end of mitosis, and dissociated from centromeres in metaphase [Interestingly, Ccp1 also plays an important role in modulating epigenetic stability in centromeres ,187. CENet al.  demonstretaphase . Why Ccp. 8.3Cnp1-GFP in the pht1 deletion mutant is mislocalized to non-centromeric regions [Cnp1 chromatin, with the chromosome arms [pht1 ccp1 mutant shows synthetic defects in CENP-ACnp1 distribution, suggesting that H2A.Z corroborates with Ccp1 to mediate the prevention of CENP-A mislocalization [Cse4 chromatin [H2A.Z is a conserved variant of the core histone H2A, which shares about 60% sequence similarity. H2A.Z nucleosome occupancy has been implicated in both transcription activation and repression . H2A.Z i regions . It appeome arms . H2A.Z alization . A recenhromatin .. 8.4et al. [cnp3-1) of a gene encoding the homologue of the mammalian CENP-C in fission yeast. The mutant became temperature-sensitive when CENP-ACnp1 was overexpressed. The authors further showed that the cnp3-1 mutant was prone to promiscuous accumulation of CENP-ACnp1 at non-centromeric regions at the restrictive temperature when overexpressing CENP-ACnp1 [Cnp1 at centromeres by preventing mislocalization of CENP-ACnp1 to ectopic sites. But how cnp3-1 causes misincorporation of CENP-ACnp1 across the genome remains unclear (CENP-C is a key subunit of the inner kinetochore complex, CCAN. CENP-C associates with CENP-A and contributes to the stability of CENP-A nucleosomes ,191. A ret al.  identifiNP-ACnp1 . This wo unclear .Table 1. 9Recent exciting findings have provided substantial insights into mechanisms inhibiting the formation of ectopic centromeres. Many important questions remain unanswered. One of the key questions is how factors involved in prevention of CENP-A mislocalization specifically recognize misincorporated CENP-A? It has been proposed that CENP-A-containing chromatin may be ‘licensed’ by additional modifications that may provide specificity for kinetochore assembly . Further


NAALADL2, one of the most frequently altered fragile sites in the cancer genome. The copy-number variations (CNVs) which occur at fragile sites, such as NAALADL2, may span many genes without typical DNA repair defects and could have a large impact on cell signalling.Recent developments in sequencing the cancer genome have provided the first in-depth mapping of structural variants (SV) across 38 tumour types. Sixteen signatures of structural variants have been proposed which broadly characterise the variation seen across cancer types. One signature shows increased duplications and deletions at fragile sites, with little association with the typical DNA repair defects. We discuss how, for many of these fragile sites, the clinical impacts are yet to be explored. One example is  In this Perspective, Simpson, Pye, and Whitaker discuss recent research identifying structural genomic variants in human cancers with a particular focus on deletions and duplications at genomic fragile sites. They argue that tumours with predominantly fragile site structural variants represent a distinct mutational signature that warrants further research. Recently, Li et al. provided the most detailed account of structural variants (SV) in the cancer genome to date where researchers derived 16 distinct signatures of structural rearrangement. The signatures were characterised by an over-representation of a particular SV class, size, replication timing and genomic location. They also compared the co-occurrence of these signatures with known pathogenic mutations in key DNA repair genes . The fragile sites signature showed only moderate co-occurrence with alterations in DNA repair genes, instead being characterised by deletions and tandem duplications at chromosomal fragile sites2. The genes in closest proximity to the most commonly affected fragile sites highlighted by Li et al. are shown in Table 12. While the mechanism underpinning these sites is not fully understood, there are a number of proposed mechanisms for why these fragile sites are so vulnerable to breaks.Fragile sites are specific loci that are vulnerable to breaks and constrictions when chromosomes are exposed to replication stress, acting as genomic ‘fault lines’3, with the most common fragile sites (CFS) corresponding to the largest actively transcribed genes or transcription units (TU) in both human and mouse cells4. Active TUs >1 Mb are known to be reliable predictors of chemically induced CNV hotspots4. In the most affected fragile sites described by Li et al., all genes were ≥0.89 Mb with an average length of 1.54 Mb (Table 2. The transcription-dependent double-fork failure (TrDoFF) model proposes that genomic instability may arise from cellular stress induced by transcription during replication7. Curiously, the increased transcription in large TUs does not necessarily increase the instability and may even increase the stability at these sites8. The TrDoFF model suggests that large TUs could promote simultaneous failure of two converging replication forks through the formation of RNA:DNA hybrid structures known as R loops5. Alternatively, large TUs may create late-replicating domains, which prolong transcription into the S-phase and disrupt the initiation of DNA replication at origins (origin firing)5. As large TUs fail to replicate the DNA in the S-phase, these regions have also been shown to uniquely exhibit mitotic DNA synthesis (MiDAS)10. Sites of MiDAS may be defined through a method known as MIDAS-seq and are evident as well-defined twin peaks that merge into a single peak as the M-phase progresses11. These peaks are conserved between cell lines and encompass all known CFSs as well as regions resembling CFSs11. Consequently, the presence of MiDAS is an indicator that cells are experiencing DNA replication stress9. Within these unreplicated regions, fragile site breaks occur, creating a deletion CNV in the DNA that spans the TU5. This is supported by experimental evidence in primary cells that shows clusters of double-stranded gene breaks and translocations that localise to the gene bodies of longer genes12. The alternate possibility is the formation of a copy-number gain.Large genes are considered more likely to harbour fragile sites13. This is known as the fork stalling and template switching (FoSTeS) model14. In more contemporary work, the FoSTeS model is superseded by the microhomology-mediated break-induced replication model, which proposes that a single double-strand end results from replication fork collapse in a cell under stress and as part of the stress response, repair molecules RecA/Rad51 become downregulate preventing double-stranded repair. As a consequence, the 3′ end from the collapsed fork anneals to any single-stranded template with sufficient microhomology. This annealing typically occurs in front of, or behind the position of the fork collapse, leading to gene deletion or duplication, respectively14.Okazaki fragments are short sequences of DNA nucleotides (150–200 base pairs long in eukaryotes) that are synthesised discontinuously on the lagging strand. At fragile sites, duplications (CNV gains) may also occur, theoretically following fork-stalling, when the 3′ end of a nascent Okazaki fragment disengages and anneals with the lagging strand template of a nearby replication fork undergoing replication13. However, the exact mechanism behind these gene duplications remains unclear and a number of other plausible models exist13.The alternative breakage–fusion–bridge cycle model proposes that double-strand breaks between the DNA are bridged, joining the Watson and Crick strands, and that, over progressive cycles of breakage and fusion, create a series of tandem inverted gene duplications14. In cell models, genome instability occurs in cells treated with DNA replication-stress-inducing agents, eventually resulting in CNVs in the genome15. Mapping of the resulting CNVs follows these genomic fault lines and large genes, including those identified in the Li et al. study15. While deletions at these loci are more common with chemically induced replication stress, gains have also been observed in cells15. If these alterations provide a fitness advantage, then it seems feasible that the frequency of alterations may increase through clonal selection.Irrespective of the mechanism, experimental evidence shows a clear correlation between fragile sites and copy-number changesFHIT and WWOX16. However, some sites are poorly understood, such as the site at the N-acetylated alpha-linked acidic dipeptidase like-2 (NAALADL2) gene.Many of the genes that harbour these fragile sites and CNVs have already been implicated in oncogenesis and have well-established roles in cancer development and/or progression, e.g. the tumour suppressors NAALADL2 was identified as the fifth most altered site in a pan-cancer analysis by Li et al. It is a giant gene spanning 1.37 Mb, approximately 45 times larger than the average gene, which is usually between 10–15 kbp18. The biological role of NAALADL2 and its relevance in oncogenesis are relatively understudied. However, data exist implicating NAALADL2 in tumour development and progression23.NAALADL2 to risk in breast and lung cancers and several studies have identified SNPs within the NAALADL2 locus that are associated with prostate cancer risk or aggression25. A GWAS of 12,518 prostate cancer cases identified rs78943174 within the NAALADL2 locus as one of two loci associated with a high Gleason sum score,22 leading to suggestions that NAALADL2 could be a potentially valuable therapeutic target21. Other SNPs in NAALADL2 have been found in TP53 and GATA2 binding sites and associated with reduced time to biochemical recurrence in patients undergoing radical prostatectomy25. SNPs within the NAALADL2 locus have been shown to be in linkage disequilibrium (LD) with SNPs associated with an increased risk of PCa, suggesting possible synergy or, alternatively, that one of these genes represents a false-positive association26.Genome-wide association studies (GWAS) have linked single-nucleotide polymorphisms (SNPs) in 27. Its overexpression in prostate cancer cell lines can lead to altered extracellular matrix binding, increased growth and invasive capabilities. Cell lines overexpressing NAALADL2 had altered transcription of genes in pathways involving the cell cycle, cell adhesion, epithelial to mesenchymal transition and cytoskeletal remodelling, suggesting a potential functional role in tumour progression; however, the specific nature of its mechanism remains elusive27.NAALADL2 protein expression has previously been shown to be increased in higher-stage and grade cancersNAALADL2 locus with an aggressive prostate cancer phenotype28. Copy-number gains in NAALADL2 were found to occur in 15.99% (95% CI:13.02-18.95) of primary prostate cancers with increasing frequency in metastatic, castrate-resistant and neuroendocrine disease. This contrasts the pattern of NAALADL2 CNVs across all tumour types, where the loss occurred more frequently than gains2. Gains in NAALADL2 were associated with clinical hallmarks of aggressive prostate cancer, including tumour stage, Gleason grade, reduced time to disease recurrence following radical prostatectomy, increased likelihood of a multi-focal tumour, positive surgical margins and lymph node metastasis28. Importantly, of the 465 genes that were frequently co-amplified with this locus, 47.5% of the genes displayed a significant increase at a transcriptional level compared to just 2.36% that were downregulated28. This suggests that a gain or loss may have a predictable effect on transcription and therefore the function of any affected gene is important. Copy-number gains in the locus co-occurred with 67 nearby oncogenes, including BCL6, ATR, TERC and PI3K family members, and are associated with the altered transcription of 473 oncogenes, activating pro-proliferative transcription processes28. Therefore, the consequences of potential breakage at these fragile sites can be highly significant.We recently published a report on the association of somatic copy-number gains at the 29. We observed significant  increases in the corresponding mRNA transcripts of the genes encoding these proteins: MKI67 , CCNE1 , POLD3  and FANCD2  in patients with NAALADL2 gain compared to diploid carriers (no changes in H2AFX expression)28. This supports the hypothesis that those patients with gains in this region have increased replication stress. In the case of NAALADL2, this correlates with a CNV in a potentially clinically significant fragile site as summarised in Fig. NAALADL2 gene, replication stress at this site may increase the chance of breakage and the formation of an SV. Alternatively, it may be that once a duplication event has occurred, transcription of such large transcripts could be responsible for increasing the replication stress.Ren et al. proposed a small signature of five proteins  associated with DNA replication stress across several tumour typesFHIT and WWOX, it currently remains unclear whether the associations between the NAALADL2 fragile site and this gene signature are related to the protein function of NAALADL2. This seems plausible given that as the locus surrounding the NAALADL2 fragile site is rich in oncogenes, upon breakage, gains frequently co-occur, leading to concurrent changes in expression in pro-proliferative genes that could drive clonal expansion30. This raises the possibility that the location of a fragile site and the proximity of any oncogenes may be used to predict its significance in disease. The majority of research into the NAALADL2 fragile site has been in prostate cancer. Furthermore, just as fragile site–CNA interaction is often cell-type specific, it is likely that fragile site SV signatures are specific to certain tumour types, and this could prove to be a worthwhile area of research. This is supported by the findings of Li et al., who noted that tumours of the gastrointestinal tract such as colorectal and oesophageal adenocarcinomas showed higher rates of the fragile site signature and overall prostate cancer showed little enrichment for the fragile site signature. It may prove useful to further assess these sites in theses specific tumour types2.Importantly, unlike NAALADL2 that has only recently attracted scientific interest. Research into this gene has highlighted the possibility that the function of the encoded protein may not be the only factor influencing the impact of structural variants. Given the broader effects and scale of the CNVs that may occur along these fault lines in the absence of significant DNA repair defects, fragile sites are likely to represent important sites in the cancer genome that have so far been largely overlooked.Tumours with copy-number changes that occur predominantly at fragile sites represent a distinct class of structural variation in the cancer genome. The clinical significance of many of these sites remains unexplored, as evidenced by the frequently altered fragile site within Peer Review File


Neural stem cells (NSCs) in the adult and aged brain are largely quiescent, and require transcriptional reprogramming to re‐enter the cell cycle. However, the mechanisms underlying these changes and how they are altered with age remain undefined. Here, we identify the chromatin accessibility differences between primary neural stem/progenitor cells in quiescent and activated states. These distinct cellular states exhibit shared and unique chromatin profiles, both associated with gene regulation. Accessible chromatin states specific to activation or quiescence are active enhancers bound by key pro‐neurogenic and quiescence factors. In contrast, shared sites are enriched for core promoter elements associated with translation and metabolism. Unexpectedly, through integrated analysis, we find that many sites that become accessible during NSC activation are linked to gene repression and associated with pro‐quiescence factors, revealing a novel mechanism that may preserve quiescence re‐entry. Furthermore, we report that in aged NSCs, chromatin regions associated with metabolic and transcriptional functions bound by key pro‐quiescence transcription factors lose accessibility, suggesting a novel mechanism of age‐associated NSC dysfunction. Together, our findings reveal how accessible chromatin states regulate the transcriptional switch between NSC quiescence and activation, and how this switch is affected with age. Quiescent neural stem cells in the adult mammalian brain overhaul their transcriptome as they activate for proliferation and neurogenesis. We report that distinct modes of chromatin dynamics at promoters and enhancers support stem cell activation, and that key metabolic genes become less accessible with age. Studies in rodents show that adult‐born neurons contribute to learning and memory, sensory functions, and mood regulation  to map genomic chromatin states in quiescent and activated NSCs with age. We observe a strong association between chromatin accessibility, gene expression, and binding of key transcriptional regulators. Interestingly, we observe that in quiescent, but not activated NSPCs, accessibility is reduced with age at genes supporting proliferation and metabolic functions, suggesting a chromatin‐level mechanism for decreased neurogenesis in the aged brain.22.1p < 0.001, Student's t‐test)  Figure A,B. Cons) Figure  we found) Figure A. Lastly) Figure .Using this quiescence model system, we generated ATAC‐seq libraries in quiescent and activated NSPCs to map the genomic changes in chromatin accessibility in the two cellular states Figure . InitialOlig2 and Fgf1 , and another site that was more “Accessible in Quiescent,” or “AQ” (Peak 2)  Figure , bottom.s Figure  in two is Figure  and 2) cs Figure . Furthers Figure D. Globals Figure . Togethe2.2in vivo 20.4% and in vitro 20.0%)  Figure . Overall) Figure  and S2A.) Figure . These f) Figure . Notably) Figure , suggest) Figure   Figure . Interesin vivo NSCs and in vitro NS5 cells , although comparison of genes downregulated in aNSCs versus qNSCs in vivo and in our primary culture did not reach statistical significance. This may be due to additional quiescence‐promoting signals in the SVZ niche that are absent in the in vitro culture system, as the overlap between downregulated genes in BMP4‐treated NS5 cells and NSPCs was significant . Our finding that both dynamic and stable chromatin states could be linked to transcriptional activation suggests that transcriptional changes in NSC activation are supported by at least two distinct mechanisms. Consistent with this possibility, we observed that dynamic and stable chromatin sites had different distributions relative to transcription start sites (TSSs)   Figure . Most st) Figure . Moreove) Figure , indicat) Figure B.−7) revealed that they were enriched with the TATA box or human TCT elements   Table . Intrigus) Table C,D (Parrs) Table . Thus, s2.4in vivo gene expression, although it did not reach statistical significance  reside in distal regions Figure , suggeste Figure B,C. Thise Figure . In distAqp4, which is expressed in quiescent NSCs and astrocytes and involved in NSC proliferation, migration, and differentiation   near s Figure E. We fou) Figure . These rin silico motif analyses to identify enriched consensus sequences  and ASCL1 (basic helix‐loop‐helix) in AA sites (p = 1 × 10−372) , and we confirmed CTCF binding with an available dataset  Figure . Interes3in vitro quiescence system utilized in this study has been shown to be a reliable tool to study the transcriptional profiles of NSCs  and aged (20–24 month) mouse NSPCs were isolated as previously described . After washing five times with PBS/0.05% Tween‐20, coverslips were incubated with the appropriate secondary antibody for 1 h at room temperature . Cells were imaged with a Zeiss Axiovert 200 M Fluorescence microscope.NSPCs were plated at 5 × 104.3ATAC‐seq libraries were generated from early passage (2–4) NSPCs cultured in activated or quiescent conditions, with two biologically independent replicates per condition. Library preparations and quality analyses were performed as described  using Bowtie2 (Version 2.2.5) , and aligned to the most recent 4.5The DiffBind package in R was used (Version 3.3.1)  in R was used  for 15 min on ice, and harvested by scraping. Nuclei were pelleted and resuspended in RIPA buffer , and chromatin was sheared with a Covaris S220 Focused‐ultrasonicator, at peak power 140 W, duty factor 10% and 200 Cycles/burst at 4°C. 5 μg H3K4me3 antibody (Abcam ab8580) coupled to anti‐Rabbit Dyna beads IgG (Invitrogen) was used per ChIP. Beads were pelleted and washed 1X with low salt, 2X with high salt, 3X with LiCl wash buffers (Millipore Sigma), and 2X with TE buffer . DNA was eluted using elution buffer  at 65°C for 2 h. Beads were removed, and crosslinks reversed overnight at 65°C. DNA isolation was performed using 25:24:1 phenol:chloroform:isoamyl alcohol, ethanol precipitation of DNA for 1 h at −20°C, and resuspension in 100 μl water. Libraries were generated by Genewiz .H3K4me3 ChIP‐seq libraries were generated from 1 × 104.8GSE48336 (ASCL1), GSE29184 (H3K4me3 in adult and embryonic mouse tissues), GSE85185 .Raw sequencing reads (fastq files) were processed as previously described  . Samples were collected in triplicate for each age group and condition. PolyA libraries were generated using the NEB Next Ultra II library kit and sequenced by Genewiz.Total RNA from quiescent and activated NSPCs was isolated from 2 × 104.11Transcripts were processed as previously described in the “new Tuxedo protocol”   were generated using GT‐Scan  for 6 days. dCas9‐KRAB‐expressing NSPCs were induced into quiescence as described above, and simultaneously infected for 24 h with 1:1 ratio of growth media to gRNA lentiviral supernatant. RNA was collected 7 days after infection.gRNAs targeting a putative enhancer region upstream of 4.15Total RNA was isolated using the QIAGEN RNeasy kit with on column DNase digestion (QIAGEN). 250–500 ng RNA was used for reverse transcription, which was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCRs were performed using Powerup SybrGreen Master Mix (Invitrogen) and run on an Applied Biosystems ViiA 7 Real‐Time PCR System. See Supplemental Table 4.16P‐values from the Homer Known Motif Enrichment Results are reported. In cases where a motif appeared multiple times, only the top  P‐value is reported.Motif analysis was performed using the Homer findGenomeMotifs.pl tool . To detect element motifs, −50/+50 bp window around stably open TSS was analyzed. For each element, the positions from the input start position and corresponding PWM scores were reported. For enrichment analysis, −50/+50 bp window around TSS for all constitutively accessible gene promoters was used as background. Enrichment significance was calculated using the hypergeometric distribution and adjusted by Bonferroni correction. Only genes with element motifs in biologically relevant positions  were highlighted.Core promoter analysis was performed using ElemeNT command line version 13  (Qin et al., Unique motifs in genes upregulated and downregulated with A.E.W. is a scientific co‐founder of Bolden Therapeutics, a company founded to develop treatments to replenish neurons in the adult brain.S.M‐L. and A.E.W. conceived and designed the study. S.M‐L. and S.D. harvested primary mouse NSPCs for experiments. S.M‐L. performed ATAC‐seq and ChIP‐seq with the supervision of A.E.W. S.M‐L. performed processing and analysis of all datasets. A.K.B. performed immunocytochemistry experiments. M.Y. set up the dCas9‐KRAB system. All remaining experiments were done by S.M‐L. A.S. and T.J‐G provided guidance on core promoter analysis, as well as helpful comments on the manuscript. S.M‐L and A.E.W. prepared the manuscript.Fig S1‐8Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.Table S8Click here for additional data file.Table S9Click here for additional data file.Table S10Click here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Auricin is produced in a narrow interval of the growth phase after entering the stationary phase, after which it is degraded due to its instability at the high pH values reached after the production phase. The complex regulation of auricin BGC is responsible for this specific production by several regulators, including the key activator Aur1P, which belongs to the family of atypical response regulators. The aur1P gene forms an operon with the downstream aur1O gene, which encodes an unknown protein without any conserved domain. Homologous aur1O genes have been found in several BGCs, which are mainly responsible for the production of angucycline antibiotics. Deletion of the aur1O gene led to a dramatic reduction in auricin production. Transcription from the previously characterized Aur1P-dependent biosynthetic aur1Ap promoter was similarly reduced in the S. lavendulaeaur1O mutant strain. The aur1O-specific coactivation of the aur1Ap promoter was demonstrated in a heterologous system using a luciferase reporter gene. In addition, the interaction between Aur1O and Aur1P has been demonstrated by a bacterial two-hybrid system. These results suggest that Aur1O is a specific coactivator of this key auricin-specific positive regulator Aur1P. Bioinformatics analysis of Aur1O and its homologues in other BGCs revealed that they represent a new family of transcriptional coactivators involved in the regulation of secondary metabolite biosynthesis. However, they are divided into two distinct sequence-specific subclasses, each of which is likely to interact with a different family of positive regulators.We previously identified the  Streptomyces  are characterized by their ability to produce a wide range of bioactive secondary metabolites, including many known antibiotics. They undergo an exceptional process of morphological differentiation, initiated by germination of spores to form a network of branched multinucleoid hyphae . In response to various signals, it differentiates into white air-grown hyphae , which eventually undergo septation into unigenomic spore chains. Secondary metabolite production is coordinated with morphological differentiation and often occurs at the time of aerial mycelium formation or at the end of the exponential growth phase during growth in a liquid medium  ATP (producing approximately 106 dpm/pmol of probe) and treated with 120 U of S1-nuclease . The DNA probe for the aur1Ap promoter (274 bp DNA fragment) was prepared by PCR amplification using chromosomal DNA from S. lavendulae subsp. lavendulae CCM 3239 as a template and the 5′ end-labelled primer aur1AS1Rev from the aur1A coding region and the unlabeled primer aur1AS1Dir from the aur1A upstream region. The control hrdBp2 promoter DNA probe was described in [32P] ATP  and T4 polynucleotide kinase  as described in  of m/z = 542.2 with positive mode detection [Spores (4 × 10eviously . Brieflyent agar  containietection . The ESI