Drosophila, canonical dosage compensation is implemented by the male-specific lethal (MSL) complex functioning in all male somatic cells. This complex contains acetyl transferase males absent on the first (MOF), which performs H4K16 hyperacetylation specifically in the male X chromosome, thus facilitating transcription of the X-linked genes. However, accumulating evidence points to an existence of additional, non-canonical dosage compensation mechanisms operating in somatic and germline cells. In this review, we discuss current advances in the understanding of both canonical and non-canonical mechanisms of dosage compensation in Drosophila.Dosage compensation equalizes gene expression in a single male X chromosome with that in the pairs of autosomes and female X chromosomes. In the fruit fly  Drosophila melanogaster with abnormal chromosome ploidy have shown that for several X-linked genes analyzed, amount (per cell) of the protein product in 3X;2A metafemales was almost the same as in 2X;2A  females and in XY;2A  males [Since heterogametic males of mammals and of most insects have an XY karyotype while females have an XX karyotype, there is a problem of imbalance of X-chromosomal gene expression between the two sexes. To eliminate this imbalance, special mechanisms of dosage compensation (DC) have emerged to ensure equal expression of X-linked genes in males and females. Early studies in l) males ,2. Howevl) males ,3,4,5,6.l) males ,8,9,10. l) males . The imbDrosophila, this twofold activation is partially mediated by the male-specific lethal (MSL) complex, which specifically binds to the male X chromosome and performs its H4K16 hyperacetylation, thus promoting transcription of the X-linked genes [Drosophila cells [Drosophila have been published recently [Drosophila, focusing on the hypotheses explaining canonical and non-canonical mechanisms.In different organisms, DC is realized via somewhat similar principles . In mammed genes ,21,22,23ed genes ,25. Moreed genes , but pared genes ,28,29,30ed genes . Finallyed genes ,32. Basela cells ,34,35. Arecently ,37,38, tDrosophila MSL complex were initially identified in the genetic screening for male-specific lethality [roX1 or roX2 long non-coding RNAs [roX RNAs as well as JIL-1, CLAMP, and TopoII are localized very close to each other on the male X chromosome [The components of ethality ,39. Lateing RNAs ,48,49,50ing RNAs ,54,55,56romosome . Thereforomosome ,59.roX1 and roX2 RNAs are functionally nearly redundant since double mutations of roX1 and roX2 genes cause male lethality and loss of coverage by the MSL complex of the male X chromosome, whereas mutations of either roX1 or roX2 genes are phenotypically neutral [roX1 or roX2 RNAs affects expression of different gene sets, thus showing their incomplete interchangeability [roX RNAs into the MSL complex [Brief characteristics of the core MSL complex components are provided below. The  neutral ,48,49,60eability . MSL1 ineability ,63,64. Meability ,66,67. Deability ,69. MLE eability  necessar complex ,71. MOF  complex ,19,21. A complex ,46,72,73 complex .msl3, mle, and mof null and roX1 and roX2 double-null mutant males retain occupancy of these sites by the MSL1-MSL2 subcomplex [The current model explaining the delivery of the MSL complex to virtually all expressed genes on the male X chromosome includes at least two stages . At the bcomplex ,77,82. Ebcomplex ,77, yet bcomplex . This rabcomplex ,73. Recebcomplex . Moreovebcomplex . Therefobcomplex ,86. Howebcomplex ,86. Yet,bcomplex . Hence, bcomplex ,88. Morebcomplex . Therefobcomplex ,88. In abcomplex ,89.roX1 and roX2 gene positions on the X chromosome coincide with two HAS/CES, roX RNAs were supposed to be incorporated in the incomplete MSL complex at the sites of their nascent transcription. Apart from this, roX RNAs may be incorporated in the MSL complex in the nucleoplasm (before it is bound with chromatin), or already on chromatin of other HAS/CES [roX RNAs, making them accessible for interactions with the MSL2 subunit [roX RNAs in the MSL complex triggers its spreading from HAS/CES to the nearby regions [roX transgenes create new HAS/CES at the sites of insertions from which the MSL complex spreads bi-directionally at hundreds of kilobases into autosomal chromatin [roX RNAs were involved in this process. Recent work has shed light on this puzzle. It was found that MSL2 contains intrinsically disordered regions (IDRs) at the C-terminus, which are able to form phase-separated droplets with either roX2 or, less efficiently, with roX1 RNAs [roX RNAs, MSL2, and other MSL subunits within these droplets, which envelope HAS/CES and nearby chromatin, may facilitate binding of the MSL complex to the low-affinity sites and its further spreading to H3K36me3-modified chromatin of active genes. Additionally, the acetyltransferase activity of MOF is necessary for the MSL spreading, since the mof mutation specifically eliminating this activity also blocks the spreading [Since  HAS/CES ,75,90. F subunit ,93,94,95 regions ,71,75. FoX1 RNAs . The incroX RNAs in the initial binding of the MSL1–MSL2 subcomplex to HAS/CES [roX RNA involvement in the MSL targeting HAS/CES is that the replacement of the C-terminal domain of Drosophila MSL2, carrying IDRs, with its counterpart from mouse MSL2, lacking IDRs, results in the loss of binding of the hybrid MSL complex to HAS/CES [Drosophila MSL2 also contained the CLAMP-binding domain, which participates in the cooperative recognition of HAS/CES by MSL2 and CLAMP [roX2 expression level and MSL2 occupancy at HAS/CES [roX2 overexpression partially restored the MSL complex recruiting to the X chromosome upon simultaneous mutations in the CXC and CLAMP-binding domains of MSL2 [roX RNAs may enhance MSL2 binding to HAS/CES has gained support in the studies of MSL targeting the male X chromosome in D. virilis [D. melanogaster, while roX RNAs are more instructive in specifying MSL2-binding sites. The authors suppose that interactions between roX RNA and MSL2 may allosterically modulate MSL2 conformation, thus enhancing MSL2 binding capacity towards HAS/CES.There are somewhat contradictory results from different groups regarding the involvement of  HAS/CES ,96. One nd CLAMP ,88; this of MSL2 . The ide virilis . In thisroX2 transgene insertion by ~2.6 Mb, with no detectable binding within a ~1.7-Mb region in between these sites [Recent works analyzing genome architecture have uncovered additional layers of regulation of the MSL complex recruiting and spreading within the male X chromosome ,101,102.se sites . Furtherse sites . These dA more detailed analysis of genome architecture in sex-sorted embryos revealed some differences in the X chromosome organization in males and females ,101. TheDrosophila males differs from autosomes and from X chromosomes of females by a drastic increase in H4K16 acetylation [mof gene demonstrate the male-specific lethality [msl mutants, thus pointing to the causative role of MOF and H4K16 acetylation in the canonical DC. Finally, upon lack of maternally supplied H4K16ac, caused by MOF depletion in oocytes, DC was compromised in late embryos [How does the MSL complex perform transcriptional activation of the male X-linked genes? Almost 30 years ago, it was found that polytene X chromosome of tylation ,106. Lattylation ,107,108.tylation , and H4Ktylation ,111. Bastylation ,21,112. tylation ,25. Moreethality ,113,114, embryos .Whether the increased H4K16 acetylation facilitates initiation or elongation of transcription, or both, has been a matter of debate, since when using ChIP-seq, the 1.2-fold increase in the Pol II occupancy was revealed at both the promoters and gene bodies of the X-linked genes compared to autosomal genes ,117, wheDrosophila males [If the increased H4K16 acetylation enhances transcription of the X-linked genes, then what is the mechanism that restricts this enhancement to only a 2-fold value? Recent work has shown that nucleoporin Mtor is involved in this process . Mtor wala males . Howeverla males . Importala males . Therefola males , thus shla males .roX RNAs, the formation of phase-separated droplets induced by binding between roX RNAs and MSL2, the creation of “hard-wired” 3D organization of the X chromosome shaped by CLAMP, the spreading of the MSL complex in 3D from HAS/CES to the active X-linked genes due to MSL3 association with their H3K36me3 mark, the stimulation of transcription of the X-linked genes by MOF , and, finally, the restriction of transcriptional activation to a twofold value by Mtor.Altogether, these studies draw the complicated picture of how the MSL complex realizes its mission in the canonical DC . This piDrosophila embryos, the MSL complex is not yet bound with the X chromosome, whereas partial DC is revealed [roX1 and roX2 RNAs) are absent or only barely expressed, and H4K16 acetylation is equally represented on the X chromosome and on autosomes [The canonical DC mechanism which relies on MSL complex activity does not ensure twofold upregulation of the X-linked genes. For example, depletion of MSL complex subunits in the male somatic S2 or SL-2 cells did not lead to complete loss of DC, but only ~1.4-fold downregulated X-linked gene expression ,25. Furtrevealed . Additioutosomes ,29. Neveutosomes ,28,119. utosomes . Collectutosomes ,25,30.What might be the nature of non-canonical mechanisms? It was supposed that non-canonical DC may be mediated by an “inverse dosage” effect  or by “bDrosophila spermatogenesis is a useful model for the analysis of non-canonical DC mechanisms, since male germline cells have the same karyotype as male somatic cells, but the MSL complex does not operate in these cells [se cells . Recent se cells  providesse cells . A similse cells ,100,101.se cells , or by dse cells . What isse cells , may detse cells  and is cse cells , is actise cells . Therefose cells ,101, andse cells  (Figure Another idea is that the enhanced interactions within the active compartment of the male X chromosome may be achieved due to increased plasticity of the single X chromosome stemming from the lack of its paired homologue. Indeed, computer modeling has shown that the absence of a paired homologue makes the X chromosome less rigid and more prone to interactions within the active compartment, which may facilitate transcription . In suppOne more hypothesis is that binding of peripheral chromatin with the nuclear lamina may enhance chromatin interactions between active regions located in the nuclear interior. It was shown that in early germline cells of testes, X chromosome is more strongly bound with the nuclear lamina than autosomes . This efDrosophila cells needs further studies.It should be mentioned that the hypotheses regarding non-canonical mechanisms of DC are not mutually exclusive. However, the question of which of them are actually realized in Drosophila is one of the first organisms where these mechanisms have been studied and where many important results clarifying DC have been obtained. Nevertheless, while the canonical DC has become more clearly interpreted over time, the non-canonical mechanisms are far from being understood. There are many hypotheses, but they were not tested directly. In light of the similarity of DC mechanisms in Drosophila and humans, studies on the Drosophila model become more relevant. The advent of modern technologies opens avenues for future research in this field.Despite several decades of research on DC in various organisms, many questions still remain unresolved.


FOXG1 gene cause a rare neurodevelopmental disorder called “FOXG1-syndrome”. FOXG1 is a key instructor of the developing telencephalon, and patients present with various phenotypes including microcephaly, seizures, and cognitive dysfunctions. We explored the pleiotropy of molecular changes underlying neuronal abnormalities upon loss of FOXG1 and provide the multiomics data set exploring functions of mouse FOXG1 at the chromatin level. We report changes in the epigenetic landscape, impacting the accessibility of chromatin regions and activation of enhancers, upon reduced FOXG1 expression that alter the transcriptome in mouse hippocampal neurons. We identified cooperation of FOXG1 with the proneuronal transcription factor NEUROD1 and HDACs in controlling gene transcription, indicating complex and multimodal FOXG1 functions regarding neuronal maturation and function.Mutations in the  Forkhead box G1 (FOXG1) has important functions in neuronal differentiation and balances excitatory/inhibitory network activity. Thus far, molecular processes underlying FOXG1 function are largely unexplored. Here, we present a multiomics data set exploring how FOXG1 impacts neuronal maturation at the chromatin level in the mouse hippocampus. At a genome-wide level, FOXG1 i) both represses and activates transcription, ii) binds mainly to enhancer regions, iii) reconfigures the epigenetic landscape through bidirectional alteration of H3K27ac, H3K4me3, and chromatin accessibility, and iv) operates synergistically with NEUROD1. Interestingly, we could not detect a clear hierarchy of FOXG1 and NEUROD1, but instead, provide the evidence that they act in a highly cooperative manner to control neuronal maturation. Genes affected by the chromatin alterations impact synaptogenesis and axonogenesis. Inhibition of histone deacetylases partially rescues transcriptional alterations upon FOXG1 reduction. This integrated multiomics view of changes upon FOXG1 reduction reveals an unprecedented multimodality of FOXG1 functions converging on neuronal maturation. It fuels therapeutic options based on epigenetic drugs to alleviate, at least in part, neuronal dysfunction. Forkhead box G1 (FOXG1)-syndrome is a rare, congenital neurodevelopmental disorder. Patients present with a complex phenotypic spectrum including microcephaly, seizures, severe cognitive dysfunction 1, and halIn the mouse, FOXG1 acts as one key determinant in central nervous system (CNS), especially forebrain development . In the 5Foxg1-haploinsufficiency in the mouse hippocampus results in hyperactivity, impaired habituation in open-field tests, reduced performance in contextual fear conditioning  would advance our understanding of FOXG1-mediated molecular alterations. Importantly, delineation of the molecular mechanisms of FOXG1-syndrome was scarce, despite FOXG1 having been recognized as a key transcription factor (TF) of forebrain development for many years. In-depth analyses of chromatin-related and chromatin-independent FOXG1 functions during forebrain development are still limited  to prevent cell death . In the itioning , and autitioning . Altereditioning –18. Thus limited , 19–21.Foxg1 was replaced by the cre recombinase (Foxg1cre/+). Our data show that FOXG1 mainly binds to enhancer regions, reconfigures the epigenetic landscape by altering chromatin accessibility, and alters H3K27ac and H3K4me3 marks at enhancers. Furthermore, FOXG1 cooperates with HDACs and NEUROD1 to regulate expression of genes that affect terminal differentiation/maturation of neurons.Mouse mutants and human features of FOXG1-syndrome indicate that impaired FOXG1 function in the hippocampus might account for some of the phenotypes observed, rationalizing to study mouse hippocampal neurons as model system. To decipher general FOXG1 functions at the chromatin level, we reduced FOXG1 levels through shRNA-mediated KD and used a multiomics approach to unravel FOXG1’s actions at the chromatin level. We exemplarily validated our findings in a mouse model in which one allele of FOXG1-syndrome is associated with impaired hippocampal functions, albeit the underlying molecular mechanism is not well understood. We thus used hippocampal neurons to investigate how FOXG1 controls transcription at the chromatin level.Foxg1 in vivo. As shown in SI Appendix, Fig. S1A and reported by others (Foxg1-heterozygous (Foxg1cre/+) adult hippocampus contained all fields, but was smaller compared with wild type (WT). Foxg1cre/+ reduced Foxg1 transcript levels at E18.5 and in the adult , but only reduced protein levels in the developing hippocampus .The expression of granule cell marker genes in the adult showed neither obvious nor significant quantitative differences between the genotypes , and the trisynaptic neuronal network of the hippocampus was similarly unchanged . Regardless of a compensatory regulation of FOXG1 protein expression in vivo, RNA-seq revealed transcriptomic changes in Foxg1cre/+ adult hippocampus compared with WT, displaying 174 DEGs  within a threshold of log2 fold change (LFC) ±log2(1.5) and an adjusted P-value ≤0.05 . Thus, FOXG1 acts in a cell-type-specific manner in the adult hippocampus, and has presumably different functions/target genes during development.The DG as well as CA field granule neurons expressed y others , 22, 23 ue ≤0.05 A. Comparue ≤0.05  revealedue ≤0.05 , but alscre/+ hippocampal tissue . Notably, the shRNA-mediated KD of FOXG1 in cultivated primary hippocampal neurons in vitro allowed the exploration of general and acute FOXG1 functions in a comparably homogeneous cell context, without confounding developmental effects of Foxg1cre/+. Foxg1cre/+ led to transcriptional changes in E18.5 hippocampus  and in vitro DIV11 primary neurons  .To untangle molecular functions of FOXG1, we favored an earlier, i.e., less complex, developmental time point. Thus, we analyzed the transcriptomes of E18.5 Foxg1l tissue B, cultiv neurons C, and WT neurons D comparecreased) B and C. creased) D. Of theFoxg1, we determined the number of overlapping DEGs between the different transcriptomes , including synapse organization, regulation of membrane potential, and neurotransmitter transport . GO-term analysis of increased and decreased DEGs from the in vivo Foxg1cre/+ (adult) and KD data sets indicated that some functional terms were enriched in the same fractions, while others were enriched in opposite fractions . Synapse organization was enriched in the decrease fraction in both data sets, while extracellular matrix organization was in the increased and decreased fractions in vivo and in vitro, respectively. These differences were also evident when both adult Foxg1cre/+ hippocampus and FOXG1 KD data sets were analyzed separately, to visualize functions that were ranking lower than the top 10 terms in the common analysis .We characterized DEGs in the different model systems determining enriched gene ontology (GO) terms, which are indicative of phenotypic downstream effects of cre/+ in vivo in the adult hippocampus affected similar functions, albeit not always in the same direction, which might be caused by the different FOXG1 protein levels observed. FOXG1 KD in primary hippocampal neurons provided nevertheless a robust and comparable model to further study the molecular functions affected by acutely reduced levels of FOXG1, while circumventing the potential compensatory and developmental effects present in the Foxg1cre/+ adult hippocampus model.Summarizing, FOXG1 KD in DIV11 cultured hippocampal neurons and Foxg1To correlate the transcriptional changes upon FOXG1 KD or genetic reduction in hippocampus tissue or neurons with the presence of FOXG1 at the chromatin level, we performed FOXG1 ChIP-seq in WT samples, in vivo (adult tissue) and in vitro (primary neurons).SI Appendix, Fig. S2 C–F). As we did not concentrate on region-specific FOXG1 functions in further experiments, we merged DG and CA FOXG1 peaks for in vivo ChIP-seq data sets for subsequent analyses.FOXG1 reduction altered transcription in both DG and CA fields, so we used hippocampus tissue subdivided into DG or CA fields for in vivo ChIP-seq. FOXG1 bound to unique loci in both the DG and CA data sets, but also to shared chromatin regions. The pattern of the distribution of FOXG1 peaks within the genome was similar in both DG and CA fields, with a high degree of overlap of functional GO-terms of genes associated with FOXG1 peaks . With the transcription start sites (TSS) as reference points, we analyzed localization of FOXG1 peaks at promoters. GO-term analysis of regions where FOXG1 peaks allocated to specific genes revealed a large overlap between in vivo and in vitro data, with neuronal differentiation and synaptic functions represented within the top enriched terms .For both in vivo and in vitro data, FOXG1 peaks distributed genome-wide mainly to distal intergenic and intronic regions, and a smaller fraction to promoters A. We coned terms B. This waxono- and synaptogenesis both in vivo and in vitro (mitochondrion organization and noncoding RNA biology were represented in clusters 4 (in vivo) and 3 (in vitro). Thus, largely, cluster-wise GO-term analysis retrieved overlapping classifications in vivo and in vitro.Clustering the FOXG1 peaks near the TSS resolved further similarities between binding patterns in vivo and in vitro C, revealin vitro D–F. Genematuration process of neurons. Indeed, we observed developmental dynamics in transcriptional regulation of four genes bound by FOXG1 in the adult hippocampus that affect maturation of the hippocampus . Transcription of Bdnf, Gria1, Gria2, and Syt1 increased in the adult compared with E18.5, and FOXG1 had significantly higher enrichment in Bdnf, Gria1 and Syt1 in the embryonic compared with the adult stage, indicating a repressive FOXG1 function at these loci .The distribution of DEGs upon reduced expression of FOXG1 in the five different clusters of FOXG1 peak locations for in vivo and in vitro data sets revealed DEGs in all 5 clusters, both with increasing and decreasing levels, and statistically significant enrichment of DEGs with either increased or decreased expression in specific clusters E–G. GO-tpocampus . Gain and loss of H3K27ac enriched with genes with increased and decreased expression, respectively, indicating a better correlation between impact of FOXG1 KD on H3K27ac and altered gene expression in these targets , which annotated to axonogenesis and neuron projection extension functions . Specific TF motifs enriched in regions gaining or losing H3K27ac upon FOXG1 reduction . To assess whether genetic reduction in Foxg1 in vivo is also accompanied by altered levels of H3K27ac, we tested two target regions with gain or loss of H3K27ac upon FOXG1 KD, using H3K27ac ChIP-qPCR in Foxg1cre/+ and WT adult hippocampal tissues . The same pattern of gain or loss of H3K27ac was observed in the tissue samples and upon ChIP-seq after FOXG1 KD in vitro. These results confirmed that FOXG1 impacts H3K27ac both upon genetic deletion and after shRNA mediated KD in these two target regions.We also analyzed only the regions that significantly gained or lost H3K27ac upon FOXG1 KD, as differentially enriched regions provide another level of understanding into regulatory mechanisms. Regions with significantly altered H3K27ac levels also localized predominantly to introns and intergenic regions , DEGs with decreased expression were significantly enriched in regions with decreased H3K4me3 , and axon extension enriched as GO-term within the genes that lost H3K4me3 alongside other terms indicative of neuronal differentiation .H3K4me3 associates with enhancers and active/poised promoters. To investigate whether FOXG1 controls transcription also by affecting this epigenetic modification, we generated and analyzed H3K4me3 ChIP-seq data from primary hippocampal neurons. To explore the H3K4me3 enrichment profile at FOXG1-binding sites, we used k-means (k = 3) clustering in FOXG1 KD data set compared with control (shLuciferase), and showed that two clusters displayed mild changes in H3K4me3 levels. Cluster 2 had slightly increased, and cluster 3 had slightly decreased levels of H3K4me3 F. The maclusters H. TF motclusters I. DEGs uclusters J. Focuseaxono- and synaptogenesis . Compared with H3K27ac and H3K4me3, the FOXG1 peaks distributed more sharply around the center of the peak in the regions that changed in accessibility . All clusters contained DEGs  and synaptogenesis enriched as a GO-term within the genes that gained access, while signal transduction was enriched within genes that lost access . bHLH and GATA TF motifs also enriched in regions gaining access upon FOXG1 reduction .The focused analyses of the regions that significantly altered accessibility upon FOXG1 KD affirmed impact at enhancer regions  suggested weak association with the HAT EP300 and HDAC SIRT1 A. Our owWe hypothesized that manipulation of histone deacetylation with epigenetic drugs could be utilized in the development of therapeutic strategies for the human disease. Accordingly, we treated primary hippocampal neurons with the broad HDAC class I/II inhibitor Trichostatin A (TSA) upon FOXG1 KD and assessed transcriptional alterations using RNA-seq. We identified DEGs comparing FOXG1 KD with control (shLuciferase), intersected this DEG set with DEGs from TSA/DMSO upon FOXG1 KD, and excluded DEGs in TSA/DMSO in shLuciferase to eliminate FOXG1-independent effects of HDAC inhibition. This approach retrieved 992 DEGs that changed expression upon TSA treatment in FOXG1 KD D.SI Appendix, Fig. S8A) or recruiting HDACs to the chromatin . The repression model is independent of FOXG1-binding to the chromatin, and predicts that reduced levels of FOXG1 lead to reduced levels of H3K27ac (shFoxg1/Ctrl decreased H3K27ac: 156 loci) and concomitant transcriptional decrease (shFoxg1/Ctrl decreased expression: 1779 DEG). Upon HDAC inhibition with TSA, we expect transcriptional increase of genes (shFoxg1+TSA/shFoxg1 increased expression: 4849 DEG) regulated through the repression model. Intersection of the respective gene fractions showed that 54 genes decreased in expression alongside decreased H3K27ac levels  and concomitantly with increased transcription (shFoxg1/Ctrl increased expression: 729 DEG) at FOXG1-binding regions (FOXG1 peaks: 9066 loci)  I. In thi66 loci) I and J. cre/+ and WT 6-wk-old mice with TSA for 7 d and assessed the transcriptional alterations using qRTPCR : DEGs common in in vivo and in vitro transcriptomes; DEGs with shared/close proximity of FOXG1 and NEUROD1 ChIP peaks in the promoter or gene body (exclusion of intergenic peaks); DEGs linked to reported neuronal function; bHLH and Fkh consensus motifs near or at the ChIP-peak sequence; DEGs linked to neuronal subtype specification; and DEGs with either increased (Ldb2) or decreased  expression upon FOXG1 KD. For our luciferase assay, we used N2A cells that expressed low levels of FOXG1 and NEUROD1, alongside other Fkh or bHLH proteins , and overexpressed either FOXG1  or FOXG1 bereft of its Fkh-domain in the presence of luciferase reporter constructs. FOXG1 activation of Ldb2 transcription was abolished upon deletion of its Fkh-domain, and was dependent on the FOXG1-binding motifs in the reporter construct. Presence of the bHLH/E-box binding sequence seemingly prevented a repressive FOXG1 function. FOXG1 repression of Ncald transcription was also dependent on the presence of its Fkh-domain, and the deletion of the Fkh-domain increased reporter transcription. Deletion of the binding sites for FOXG1 in the regulatory region still resulted in the same response of the reporter transcription as observed for the unmodified construct. The deletion of the bHLH/E-box-binding site silenced the regulatory region, independent of the presence or absence of Fkh motif . Epigenetic alterations were not found to be specifically enriched in any of the clusters, yet FOXG1 unique and shared clusters showed mild changes in accessibility and H3K27ac levels. For example, in the shared cluster, 1,277 regions increased and 1,695 decreased in all epigenetic markers , alongside regions that had only H3K27ac, H3K4me3, or chromatin accessibility alterations, or a combination of these. We filtered and subclustered the FOXG1-NEUROD1 shared cluster that overlapped with dynamic epigenetic landscape clusters from SI Appendix, Fig. S11C).As our data strongly suggested cooperation between FOXG1 and NEUROD1 in transcriptional control, we compared FOXG1 and NEUROD1 ChIP-seq profiles, which returned clustered genes bound by both (shared fraction), and also returned genes that only enriched binding of one of the two TFs  H. Functiogenesis I. For alogenesis J. This fThus, alteration of the epigenetic landscape at regions bound by both TFs was moderate, indicating another mechanism by which these two key instructors affect transcriptional programs important for axono- and synaptogenesis.To analyze the nature of the FOXG1/NEUROD1 crosstalk in more detail, we knocked down either FOXG1 or NEUROD1 in primary hippocampal neurons and assessed the binding profile of the other respective TF using ChIP-seq. We clustered the peak regions into shared fraction and regions that enriched predominantly for either FOXG1 or NEUROD1 A. WithinShared binding regions could reflect binding through one of the respective factors and indirect presence at the site of the other, or usage of adjacent binding sites B. To resSurprisingly, FOXG1 KD decreased NEUROD1 presence at NEUROD1 sites with little enrichment for FOXG1. Similarly, NEUROD1 KD decreased FOXG1 presence at FOXG1 sites with little enrichment for NEUROD1. We concluded that either factor might be also needed before or during recruitment, but not exclusively for stable binding.We next analyzed the changes of NEUROD1 and FOXG1 binding upon the respective KDs, with a focus on regions that showed differential binding of NEUROD1 upon FOXG1 KD. Regions presenting differential binding of NEUROD1 clustered into three main profiles: Cluster 1 (strong enrichment for NEUROD1 and FOXG1), cluster 2 , and cluster 3  D. Upon Fcre/+, we selected four different regions for FOXG1/NEUROD1 ChIP-qPCR. We selected the clustered regions that were differentially bound by NEUROD1 upon FOXG1 KD . Of note, FOXG1 protein levels were globally unchanged upon the genetic reduction in the adult ; thus we first assessed presence of FOXG1 protein at the selected regions in the Foxg1cre/+ hippocampus. For clusters 1 and 2, FOXG1 levels increased at the selected sites, having the opposite appearance compared with the FOXG1 KD model . The cluster 3 region was bound less in the condition of genetic deletion of one Foxg1 allele. Enrichment of NEUROD1, on the other hand, strongly increased at the cluster 1 region. This was opposite to the KD approach, and correlated well with the increased FOXG1 presence at the site, which contained both Fkh and bHLH binding sites. The two cluster 2 regions were bound by increased amounts of NEUROD1 upon FOXG1 KD, and according to increased presence of FOXG1 at the sites in vivo, NEUROD1 levels were decreased in the Foxg1cre/+ hippocampus. Cluster 3 region, which did not contain enrichment of Fkh motifs, had less FOXG1 bound in the Foxg1cre/+ hippocampus, and in accordance with the observation of decreased NEUROD1 presence upon FOXG1 KD, we precipitated less NEUROD1 from the in vivo sample. Taken together, the validation suggests that FOXG1/NEUROD1 cooperation is of functional importance in vitro and in vivo.To validate FOXG1/NEUROD1 cooperation in vivo in Foxg1FOXG1 KD D, shortlIn this study, we reveal diverse and pleiotropic functions of FOXG1 at the chromatin level in maturing neurons. Despite FOXG1 being recognized as a key TF for telencephalic development and neuronal function, insights into the mechanism underlying transcriptional regulation are sparse. Here, we show that i) FOXG1 acts both as repressor and activator, ii) localizes predominantly to enhancer regions, iii) alters the epigenetic landscape, iv) directly affects HDAC functions, and v) acts in concert with NEUROD1 to instruct transcriptional programs necessary for axono- and synaptogenesis.Myc-target regions . On the other hand, in 451 regions that gained NEUROD1, only 1 region had significantly higher H3K27ac levels coinciding with changed gene expression . This observation weakens support for the argument that NEUROD1 acts as a pioneer that alters H3K27ac levels in mature hippocampal neurons upon FOXG1 KD. Because of the reported pioneering potential of NEUROD1, one could hypothesize that NEUROD1 acts upstream of FOXG1, regulating its access to the chromatin. However, our data do not support a clear hierarchy of the two factors in hippocampal neurons; they rather act in concert, and regulate access to each other. Further, FOXG1 interferes with NEUROD1 binding to chromatin, similar to what has been observed for chromatin accessibility of the SMAD/FOXO TF complexes  plasmids, according to the protocol described previously .SI Appendix.RNA-, ChIP-, RELACS-ChIP, and ATAC-seq library preparation and sequencing were done using standard protocols as described in detail in the snakePipes (v. 1.1.1)  . The seq. 1.1.1) . Summaryhttp://hipposeq.janelia.org/ for choosing the dorsal-ventral survey of hippocampal principal cells.Hipposeq  was acceMus musculus.STRING database  was usedNot applicable.Not applicable.Appendix 01 (PDF)Click here for additional data file.


Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 bp resolution and constructed a 3D KSHV genomic structural model with 2 kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by structural maintenance of chromosomes (SMC1) cohesin complex and CCCTC-binding factor (CTCF), and the KSHV transactivator was recruited to those sites during reactivation. Triggering KSHV gene expression decreased prewired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible noncoding RNA regions moved toward the inner space of the structure, resembling the configuration of a “bird cage” during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help facilitate viral gene transcription.IMPORTANCE The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi’s sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 bp resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral life cycle. The virus is strongly associated with Kaposi's sarcoma (KS), an endothelial cell-derived tumor, and two rare lymphoproliferative disorders, multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL). KSHV exhibits a broad host range and can infect a variety of cell types al cells –3. KSHV l cells –, 5. The l cells –, 7. FollA great deal of effort has focused on defining the epigenetic mechanisms that regulate latency, reactivation, and commandeering of the host cell by KSHV –11. Alth–Caenorhabditis elegans (Saccharomyces cerevisiae (Schizosaccharomyces pombe (Caulobacter crescentus (Bacillus subtilis (Advances in chromosome conformation capture (3C)-based studies have uncovered the existence of spatially insulated genomic regions that are now considered the invariant building blocks of chromosomes –17. When– elegans , Saccharrevisiae  and Schies pombe . In addiescentus  and Bacisubtilis , 26 havesubtilis , may be The existence of TAD-like structures within the genomes of large double-stranded DNA viruses such as herpesvirus family has not been reported. For KSHV there are numerous reports concerning the role of the structural maintenance of chromosomes (SMC) cohesin complex and CCCTC-binding factor (CTCF) in control of viral latency and reactivation –36. As b–37–40–in situ and employs a restriction enzyme cocktail coupled with fill-in reactions to incorporate a biotinylated deoxynucleotide to mark the digested ends, which facilitates the enrichment of the DNA ligation products  prior to the KSHV lockdown probe capture step  at which there was an only minimal increase in viral DNA copies, as measured by qPCR  . CaptureNext, insulation scores were calculated to reveal changes in TRDs before and after reactivation. A lower score suggests a higher insulation effect, which is indicative of the position of the boundary. The border strength at the majority of genomic regions was decreased in reactivating samples , suggestWith 3,565,321 (latent) and 5,967,349 (lytic) valid Hi-C di-tags  covering the ~140 kb KSHV genome, we next constructed a theoretical 3D KSHV genomic structural model utilizing TADbit, which was then visualized with the UCSF Chimera program . The modiSLK BAC16-Wt). This overall similarity suggests that the prominent viral genome contacts established during latency appear to be conserved among different KSHV infected cell lines and are stable properties within the infected cell population. Although we note that the overall KSHV genomic similarity is maintained when comparing between BC-1 (KSHV+/EBV+) and BC-3 (KSHV+/EBV-) PEL cell lines  that occupy approximately one sixth of the KSHV genome  was also not considered in our analyses due to difficulties in mapping sequence reads. Further studies are therefore needed to confirm where and how a TR interacts with a unique region, which would have significant impact in KSHV reactivation through recruitment of LANA protein complex . In addi imaging .In summary, this study presented 3D KSHV genomic structure models of episomes during latency and early lytic phase of reactivation and revealed the dynamics of genomic interactions by transcription activation. The 3D genomic structures bestow another element to the effectiveness of herpesvirus gene regulation, a system which may provide an opportunity to further study genomic loop regulation through the intersection of viral and host proteins.in situ CHi-C protocol with kitted reagents from Arima Genomics  based on well-accepted methods described for in situ Hi-C (–KSHV Capture Hi-C (CHi-C) analysis was performed using a robust itu Hi-C , 73, CHIitu Hi-C –76, and tu Hi-C –. BrieflyThe KSHV CHi-C library was then prepared by target enrichment of the libraries for KSHV genomic content by solution hybridization with a custom-designed KSHV genomic capture probe library  as previously described . BrieflyNC_009333.1) reference genomes. Quality reports showed percentage duplicates at less than 15% while >95% di-tags were valid and aligned to restriction fragments . The duplicates were removed and reads mapping only to the KSHV genome were extracted by using Pysam 0.14.1.Sequence alignment and quality check of reads were performed using HiC-Pro 2.11.1 pipeline , each reThe KSHV genome was analyzed and visualized for frequencies of chimeric genomic reads, and finally modeled for 3D Structure using TADbit . The KSHMatrix modeling potential (MMP) score , ranging2. beads/cells complexes, were washed 3 times with digitonin wash buffer , aliquoted, and incubated with specific antibodies in 250 μL volume. The antibodies used in this study were: rabbit polyclonal anti-SMC1 , and rabbit monoclonal anti-CTCF . After incubation, unbound antibody was removed by washing with digitonin wash buffer 3 times. Beads were then incubated with recombinant pAG-MNase, which was purified from E.coli  in 250 μL digitonin wash buffer at 1.0 μg/mL final concentration for 1 h at 4°C with rotation. Unbound pAG-MNase was removed by washing with digitonin wash buffer 3 times. Prechilled 2 mM CaCl2 containing digitonin wash buffer (200 μL) was added to beads and incubated on ice for 30 min. The pAG-MNase digestion was halted by the addition of 200 μL 2× STOP solution . The beads were incubated with shaking at 37°C for 10 min in a tube shaker at 500 rpm to release digested DNA fragments from the insoluble nuclear chromatin. The supernatant was collected after centrifugation  and placed on a magnetic stand. DNA was extracted using the NucleoSpin kit . Sequencing libraries were then prepared from 3 ng of CUT&RUN DNA with the Kapa HyperPrep kit (Roche) according to the manufacturer’s standard protocol. Libraries were multiplex sequenced  on an Illumina HiSeq 4000 sequencing system to yield ~15 million mapped reads per sample. When necessary, E. coli genomic DNAs contaminating purified pAG-MNase were used to normalize sequence reads, as described previously (Cleavage Under Targets and Release Using Nuclease (CUT&RUN)  was perfeviously .NC_009333.1) genome. Valid interaction products called by HiCUP were converted into Juicebox (FASTQ files for the capture Hi-C experiments were processed through the HiCUP (v0.7.4) pipeline  using thJuicebox  input foJuicebox . JuiceboGSE123897), H3K27Ac, H3K4me3, and H3K27me3 (GSE163695). Peaks and read alignments were visualized using the Integrated Genome Browser (IGB)  . Heatmaper (IGB) .iSLK cells that were infected with BAC16 HA-ORF57 WT virus, were maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin-L-glutamine solution, 1 mg/mL hygromycin B, and 250 μg/mL G418. iSLK cells were obtained from Dr. Don Ganem  and were maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin-L-glutamine solution, 400 μg/mL hygromycin, 250 μg/mL G418, and 10 μg/mL puromycin.TREx-F3-H3-K-Rta BCBL-1 cells (TREx-BCBL-1) cells were maintained and reactivated, as previously described . BC-1 anGSE163695.The data sets supporting the conclusions of this study are available in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) repository with the accession code


The current collection of articles in this special issue appreciates the cutting-edge technologies to study chromatin dynamics. A comprehensive article from Anna Chanou and Stephan Hamperl elegantly highlights single-molecule methods to study the positioning of nucleosomes, the basic unit of chromatin organization, DNA replication, transcription, and chromatin modifications. The authors discuss advances in the development of third-generation sequencing, force spectroscopy, imaging, and methylation footprinting (SAMOSA) - current efforts to address the fundamental challenges in studying chromatin dynamics, such as averaging parameters within a population of molecules or cells.Chromatin organization is a function of time, regardless of the biological processes. Development, circadian rhythms, and cellular and environmental stress each influence chromatin architecture. The thriving development of technology to study the temporal 3D chromatin organization with time as its 4th dimension is facilitated by the multiple collaborative efforts in the field generating scientific knowledge hubs dedicated to temporal and spatial chromatin organization within the nucleus. For example, the 4DNucleome consortium , one of the key players in the immune compartment, which regulates T, B, and NK cell development. The combined omics analysis presented in this study revealed a novel role of Zbtb1 in modulating the T lymphoblast chromatin landscape.Combined omics analysis is instrumental for our understanding of chromatin dynamics and functional outcomes during differentiation. The manuscript by Shahrbanou Hossein and co-authors within our topic collection. Their study focuses on the epigenetic regulation of phenotypic sexual plasticity in zebrafish as a versatile biological model organism in which genetically defined sex that can be influenced by environmental factors. A skewed sex ratio might negatively affect population development during rapid environmental changes. This evolutionary process motivated the authors to investigate the regulatory role of epigenetic control in the process. The authors identified increased DNA methylation in male-biased families compared to female-biased families, as reflected by a higher number of methylated positions in testes versus ovaries. Environmental factors such as higher incubation temperatures resulted in higher DNA methylation, providing new insights into sex plasticity in response to the environment.The impact of the ecological macro-environment on epigenetic regulation is a fascinating facet of 4D chromatin organizations. This important topic was tackled in the research article by Tartour and Padmanabhan. The authors discuss how circadian TADs  shift from active euchromatin regions (compartment A in Hi-C experiments) to inactive heterochromatin regions (compartment B) during transitions from active to repressive phases of the circadian rhythm. Current findings in the field indicate a striking “breathing” of chromatin domains on the time scale of hours depending on cellular demands for gene expression. During the “day-night” shift, the intra-TADs also undergo dynamic rearrangements at the level of promoter-enhancer loop organization. Compartment B-targeted circadian TADs are localized at the nuclear periphery, which represents one of the two principal locations for heterochromatin domains  that undergo dynamic alterations during cellular differentiation  on chromatin organization remains reported . The extth dimension of 3D chromatin organization covers aspects of chromatin dynamics, including current methodologies, organismal and tissue development, the impact of circadian rhythms, environmental factors, and inflammatory stresses on chromatin dynamics. These topics represent critical directions that exemplify the multifactorial influence of time on the chromatin landscape. An impressive suite of technologies, such as single-molecule and single-cell omics, allows for investigations of chromatin dynamics with unprecedented detail and addresses the pervasive problem of population averaging. Although current progress is cause for optimism, the integration of the complex omics data sets obtained with different methods across multiple time-dependent biological processes remains a formidable task.In summary, the topic collection we have procured to illustrate the 4


The 2278 bp, 1917 bp, and 1989 bp sequences of zip10, zip13, and zip14 promoters, respectively, were subcloned into pGL3-Basic plasmid for promoter activity analysis. The pcDNA3.1 plasmid coding EGFP tagged pfZip10, pfZip13, and pfZip14 were generated for subsequent confocal microscope analysis. Several potential transcription factors’ binding sites were predicted within the promoters. In vitro promoter analysis in the HEK293T cells showed that high Zn administration significantly reduced the transcriptional activities of the zip10, zip13, and zip14 promoters. The −2017 bp/−2004 bp MRE in the zip10 promoter, the −360 bp/−345 bp MRE in the zip13 promoter, and the −1457 bp/−1442 bp MRE in the zip14 promoter were functional loci that were involved in the regulation of the three zips. The −606 bp/−594 bp KLF4 binding site in the zip13 promoter was a functional locus responsible for zinc-responsive regulation of zip13. The −1383 bp/−1375 bp STAT3 binding site in the zip14 promoter was a functional locus responsible for zinc-responsive regulation of zip14. Moreover, confocal microscope analysis indicated that zinc incubation significantly reduced the fluorescence intensity of pfZip10-EGFP and pfZip14-EGFP but had no significant influence on pfZip13-EGFP fluorescence intensity. Further investigation found that pfZip10 localizes on cell membranes, pfZip14 colocalized with both cell membranes and lysosome, and pfZip13 colocalized with intracellular ER and Golgi. Our research illustrated the transcription regulation of zip10, zip13, and zip14 from P. fulvidraco under zinc administration, which provided a reference value for the mechanisms involved in Zip-family-mediated control of zinc homeostasis in vertebrates.Zip family proteins are involved in the control of zinc (Zn) ion homeostasis. The present study cloned the promoters and investigated the transcription responses and protein subcellular localizations of three LIV-1 subfamily members ( Each of the three Zip proteins usually forms a heterodimer with another protein and thus plays a functional role. For example, Zip10 forms a heterodimer with Zip6 to facilitate zinc flux to trigger mitosis [zip10 mRNA level was not influenced by Zn2+ deficiency, but the protein level of Zip10 were downregulated in response to zinc deficiency in mouse testes. Zheng et al. [zip10 and that zebrafish Zip10 acts as a zinc importer for X. laevis oocytes. Lichten et al. [zip10 mRNA expression level in mouse hepatocytes, the AML12 cell line, and the neuro 2a cell line. Further investigation revealed that the mouse mZip10 localized to the AML12 cell membrane and functioned as a Zn2+ importer, and that Zn deficiency enriched its localization in the cell membrane [zip13 mRNA and protein level in HeLa cells. Xu et al. [zip10, zip13, and zip14 mRNA levels in both the hepatocytes and the intestinal epithelial cells of yellow catfish. Thus, all of these studies indicated that Zip10, Zip13, and Zip14 were regulated by Zn. However, the underlying regulatory mechanisms have received very little attention and remain confusing.Micronutrient zinc (Zn) is an essentially functional and structural element for all living organisms, including fish . Both a vesicles . For exavesicles  and pigmvesicles . In rainvesicles . Zn tran mitosis . Zip13 p mitosis  reported mitosis  reportedn et al.  reportedmembrane . Lee andmembrane  reportedmembrane  reportedmembrane  reportedu et al.  reportedu et al.  reportedu et al.  reportedu et al.  found thtroc1 gene and play repressing roles in troc1 expression [fam13a gene through the binding region in its promoter. MTF-1, which possesses six Cys2-His2 zinc finger structures specializing in the binding of Zn, is a crucial transcription factor for inducing the transcription of some genes that are associated with Zn homeostasis [znt1, znt2, znt6, znt8, zip3, and zip8 transporters of P. fulvidraco [zip10 transporter of zebrafish [znt1, znt2, and the znt5 transporter in mammals [zip4 promoter of mice and, thus, regulated the adaptive expression of zip4. Other studies concerning KLF4 and zip13 led us to speculate that there is an enigmatic connection between KLF4 and Zn homeostasis that is mediated by zip13 [zip14. Thus, it is valuable to investigate whether these transcription factors mediate the transcriptional regulation of zip members zip10, zip13, and zip14.In the eukaryotes, transcription factors initiate and regulate gene expression via their binding locus in the promoter. For example, the TFs of NRF1 and the Smad3 binding site were found in the promoter region of the pression . Liang epression  reportedeostasis . MTF-1 feostasis ,26. MRE lvidraco ,28, the ebrafish , and the mammals ,30,31. K mammals . Liuzzi  mammals  found thby zip13 ,34. The by zip13 . Studiesby zip13  but suppby zip13 . Cousinsby zip13  reportedPelteobagrus fulvidraco, are found broadly across Asian countries and provide an excellent model for nutrition research, due to their delicious flavor, high nutritional value, and economic value [zip10, zip13, and zip14 of yellow catfish [P. fulvidraco. Our study illustrated the zinc-responsive transcription regulation mechanisms of zip10, zip13, and zip14 and provides some views regarding Zip-family-mediated zinc homeostatic control in vertebrates.Compared with terrestrial animals that sequester Zn from their diets, aquatic fish possess two pathways for obtaining Zn from diets or from a water environment . Thus, iic value ,41. Rece catfish . The cur catfish , ER stre catfish , and the catfish  of P. fuzip10, zip13 and zip14 promoters, respectively, were cloned successfully, and several putative TFBSs were predicted, including the binding sites of MRE, PPARα:RXR, FOXO4, FOXO6, SREBP, CREB1, AP1, ZEB1, RREB1, SP1, KLF4, STAT3, STAT5a:STAT5b, STAT6, CEBPα, ATF4, CCAAT-box, and TATA-box. The relative positions of the predicted putative TFBSs are listed in zip10, zip13, and zip14 promoters are presented in The 2278 bp, 1917 bp, and 1989 bp sequences of yellow catfish zip10, zip13, and zip14 promoters using in vitro cultured HEK293T cells and characterized their functional regions  markedly increased the luciferase activity of the zip10 promoter after 100 µM Zn2+ incubation  or the KLF4 binding site (−640 bp/−651 bp) significantly increased the luciferase activity of the zip13 promoter after 100µM Zn2+ incubation  site or the mutation of the STAT3 (1) binding site (−1383 bp/−1375 bp) markedly increased the luciferase activity of the zip14 promoter after 100 µM Zn2+ incubation. However, the mutation of the STAT3 (2) binding site (−1358 bp/−1348 bp) showed no remarkable effects  binding site could not participate in the Zn-responsive zip14 transcription.We carried out site-mutation analysis to determine the functional TFBS in the 3T cells . The seqcubation A, suggescubation B, indica effects C. Surprizips promoters. For the MRE from −2017 bp to −2004 bp on the zip10 promoter, when the MRE sequences were labeled by the biotin as the probe, we found a bright binding band , the lysosome tracker (red), the ER tracker (red), and the Golgi tracker were applied to mark the positions of the cell membrane, the lysosome, the endoplasmic reticulum, and the Golgi apparatus, respectively. The green fluorescence from the pfZip10-EGFP overlapped with the red fluorescence released by the membrane tracker (red), indicating that pfZip10 was localized to the plasma membrane . These TFBSs have been reported in previously published studies as being within the promoter regions of other genes [zip10, zip13, and zip14 promoters.The promoter region is a short 100–1000 bp DNA fragment upstream from the transcription start site . Chen etriptions ,52. In og et al.  reportede et al.  reportedd et al.  reported by NF-Y . Our stulvidraco ,40. In aer genes ,31,57,58mt promoter and was essential for the upregulation of mt expression. Song et al. [znt1, znt2, znt6. and znt8 and upregulate their expression. As in our study, the site mutation analysis implied that the mutation of MRE alleviated the decrease in luciferase activity that was caused by high Zn administration. Here, we found that the −2017 bp/−2004 bp MRE in the zip10 promoter, the −360 bp/−345 bp MRE in the zip13 promoter, and the −1457 bp /−1442 MRE in the zip14 promoter were indispensable for the downregulation of zip10, zip13, and zip14 expression induced by high Zn administration. Similarly, Chen et al. [zip10, zip13, and zip14 promoters in both hepatocytes and intestinal epithelial cells. Interestingly, in the zip10 promoter, the MRE deletion showed different activity with the mutated MRE. Deletion mutation deleted a sequence of approximately 400 bp, while site mutation disrupted only the putative binding sequence. Therefore, we speculated that the rest sequence in the deleted 400 bp~ was functionally antagonistic to the MRE in response to zinc. Furthermore, our EMSA results showed that those MREs were capable of binding nuclear protein. High Zn enhanced the binding effect in the zip10 promoter. However, high-Zn administration showed a relatively low binding affinity in the zip13 and zip14 promoters, and a similar phenomenon was observed in the zip3 and mt promoters [zip10 promoter, implying that MTF-1 was a functional repressor of the zip10 promoter under high-Zn treatment. Similarly, Chen et al. [zip3 and zip8 promoters. Taken together, these results indicated the zinc-responsive regulation roles of these MREs in the transcriptional regulation of the zip10, zip13, and zip14 promoters.MTF-1 is an intracellular Zn sensor that can bind to the MRE of the promoters of its target genes and activate target genes associated with Zn homeostasis . Recentlg et al.  found thn et al.  reportedromoters ,40. Lichn et al.  found a zip13 promoter. We found that the mutation of the KLF4 binding site alleviated the Zn-induced decrease in the activity of the zip13 promoter, suggesting that this KLF4 binding site is necessary for the decrease in zip13 expression activity in a high-Zn environment. Our EMSA showed that high Zn significantly enhanced the binding effect between nuclear proteins and the KLF4 binding sites. Thus, we confirmed that the KLF4 binding sites could regulate the zip13 expression in response to a high zinc environment, implying a crucial mechanism whereby KLF4 maintains Zn homeostasis. It was also reported that the KLF4 binding site is responsible for the upregulation of mtf-1 expression [zip13 promoter. Liuzzi et al. [klf4 restrained the upregulation of the zip4 promoter in a zinc-depleted environment. Similarly, our results indicated that KLF4 play crucial roles in maintaining intracellular Zn homeostasis in a high-Zn environment through regulating zip13 expression.In our study, we predicted −606 bp/−594 bp KLF4 binding sites within the pression . It seemi et al.  reportedzip14 promoter. The site mutation analysis indicated that STAT3 (1), not STAT3 (2), was responsible for the downregulation of the activity of the zip14 promoter, and our EMSA confirmed that the effect of the −1383 bp /−1375 bp STAT3 (1) binding site on the zip14 promoter was the functional one. Thus, our results indicated that STAT3 mediated the maintenance of Zn homeostasis by regulating the zip14 expression via the −1373/−1383 bp STAT3 (1) binding site on its promoter. Interestingly, Du et al. [zip2 expression. Kitabayashi et al. [zip14 promoter. In addition, other studies suggested that the zip6 promoter served as the STAT3 target [zip14 promoter Thus, these results, together with ours, suggest that STAT3 plays a crucial role in maintaining Zn homeostasis and that the STAT3 binding site participated in the control of the zip14 promoter of P. fulvidraco.We also predicated the −1383 bp /−1375 bp STAT3 (1) and the −1358 bp /−1348 bp STAT3 (2) binding sites within the u et al.  discover3 target ,61. CousP. fulvidraco.Within the body, there could be enhancer (or silencer) DNA fragments far away from the transcription start site, and the formation of a chromosome loop makes it possible for the enhancer (or silencer) to participate in enhancing (or repressing) transcription. In our study, we confirmed that the predicted zinc-related TF binding sites were functional and involved in zinc-responsive regulation. Most TF binding sites are a 4 bp- to 30-bp wide integral DNA sequence . FurtherThe SLC39A Zn transporters carry Zn into cytoplasm from intracellular organelles or extracellular space, and their specific subcellular localization determines the specific function of the Zn transport. For example, Kong et al.  found thzip10, zip13, and zip14 promoters [Several studies have established that high Zn reduced the mRNA and protein expression of the romoters ,18,21. Sromoters  reportedromoters  reportedP. fulvidraco, used for genomic DNA extraction, were purchased from a local fishery farm . The HEK293T cell lines were from the Cell Resource Center in the Fishery College of Huazhong Agricultural University . The Dulbecco’s modified eagles medium (DMEM), the 0.25% trypsin-EDTA, and the fetal bovine serum (FBS) were from Gibco . Lipo293 for transfecting plasmid into HEK293T were from Beyotime Biotechnology, Wuhan, China. Passive lysis buffer and the dual luciferase activity assay reagents were from Promega, Madison, WI, USA. Nuclear protein extraction kits were from Thermo Fisher Scientific, Waltham, MA, USA). We made sure that the protocols for animal experiments and the use of cell lines followed the institutional ethical guidelines of HZAU for the care and use of laboratory animals, and that they were approved by the university’s ethics committee (I.D. code: Fish-2020-09-24).The tail fins of zip10, zip13, and zip14 promoters obtained in our laboratory [zip10, zip13 and zip14 promoters were subcloned into the pGL3-Basic between the Sac Ⅰ and Hind Ⅲ restriction cloning sites with Clone press II One Step Cloning Kit , according to the distance from the TSS. Those full-length promoters (wild type) were pGL3-2182/+96 (zip10), pGL3-1853/+64 (zip13), and pGL3-1854/+135 (zip14). The unidirectional deletion mutation products of corresponding promoters were randomly generated using the full-length promoters as templates. The primer sequences are listed in zip10 promoter; pGL3-1279/+64, pGL3-861/+64, pGL3-489/+64, and pGL3-295/+64 for zip13 promoter; and pGL3-1409/+135, pGL3-1153/+135, pGL3-654/+135, and pGL3-266/+135 for zip14 promoter. For constructs coding EGFP-tagged pfZips , the complete cDNA sequences of the zip10, zip13 and zip14 promoters were generated by the same nested PCR principle. The primers are listed in Based on the full-length complementary DNA (cDNA) of the boratory , we idenboratory . For conhttp://www.genomatix.de/, accessed on 31 August 2021), with a minimum threshold of 0.85, and Jaspar , with minimum threshold of 9. The MegAlign Pro Sequence Alignment Software was used to evaluate whether the sequences were successfully cloned, according to NCBI. .For the function characterization of these promoters, we predicted the putative transcription factor bind sites (TFBSs) with the online database MatInspector  or DMEM (10% FBS) + 100 μM Zn2+. The Zn concentration was selected according to our recent study [The transfection of the constructed promoter plasmids into the HEK293T cells and the assay of the dual luciferase activities were carried out according to the methods of Xu et al. . Brieflynt study . Four huzip10, zip13 and zip14 promoters, we used the QuickChange II Site-Directed Mutagenesis Kit  to direct the site mutation. The corresponding full-length promoters were used as templates. The specific primers for the site mutation are listed in zip10, Mut-MTF-1-zip13, Mut-KLF4-pGL3-zip14, Mut-MTF-1-pGL3-zip13, and Mut-STAT3-pGL3-zip14. Then, the plasmids were transfected and the cells were harvested for dual luciferase activity analysis.To identify the corresponding binding sites on the zip10, zip13, and zip14 promoters. Briefly, the HEK293T cells were seeded into 6-cm dishes and maintained in the DMEM with or without 100 μM Zn2+. The incubation lasted for 24 h. Then, the cells were lysed for the extraction of nucleoprotein (NP) and the protein concentration was determined with a bicinchoninic acid assay (BCA) method. Ten μg of nucleoprotein was added and mixed with each oligonucleotide duplex of the binding sequence of the MTF-1, KLF4, and STAT3, using the EMSA kit according to manufacturer’s protocols . The oligonucleotide sequences of the EMSA are presented in An EMSA was conducted to verify the MTF-1, KLF4, and STAT3 binding on the The transfection protocols of the constructed pcDNA3.1-pfZip10-EGFP, pcDNA3.1-pfZip13-EGFP, and pcDNA3.1-pfZip14-EGFP were the same as that of the promoter plasmids indicated in the We further investigated the pfZips’ subcellular localization. The HEK293T cells were seeded on coverslips, and the transfection of the constructed pcDNA3.1-pfZip10-EGFP, pcDNA3.1-pfZip13-EGFP, and pcDNA3.1-pfZip14-EGFP and the cell-washing were as indicated in the t-test between two treatments; p < 0.05 was considered to be statistically significant.We carried out the statistical analysis with SPSS 22.0 software . All of the data were presented as means ± standard error of mean (SEM). First, we analyzed the data for normality via the Kolmogorov–Smirnov test, and for the homogeneity of the variances among the treatments via Bartlett’s test. Then, we used one-factor ANOVA and Duncan’s multiple range test to analyze the significance of the differences among ≥3 treatments, and Student’s zip10, zip13, and zip14 promoters from P. fulvidraco in the heterologous expression host HEK293T. We discovered that a high concentration of Zn decreased the activities of the three zip promoters. The MRE in the zip10 promoter, the MRE and KLF4 binding sites in the zip13 promoter, and the MRE and STAT3 binding sites in the zip14 promoter mediated the zinc responsive transcription regulation of their activities. We found that the pfZip10 located on the cell membrane, the pfZip13 colocalized with ER and Golgi, and the pfZip14 colocalized with lysosome. A high Zn administration reduced the protein level of pfZip10 and pfZip14. We illustrated the transcription regulation of zip10, zip13, and zip14 promoters under zinc administration and provided a novel mechanism for Zip-family-mediated zinc homeostatic control in vertebrates.In summary, for the first time, we characterized the structure and function of the