The oocyte faces a particular challenge in terms of gene regulation. When oocytes resume meiosis at the end of the growth phase and prior to ovulation, the condensed chromatin state prevents the transcription of genes as they are required. Transcription is effectively silenced from the late germinal vesicle (GV) stage until embryonic genome activation (EGA) following fertilisation. Therefore, during its growth, the oocyte must produce the mRNA transcripts needed to fulfil its protein requirements during the active period of meiotic completion, fertilisation, and the maternal-to zygote-transition (MZT). After meiotic resumption, gene expression control can be said to be transferred from the nucleus to the cytoplasm, from transcriptional regulation to translational regulation. Maternal RNA-binding proteins (RBPs) are the mediators of translational regulation and their role in oocyte maturation and early embryo development is vital. Understanding these mechanisms will provide invaluable insight into the oocyte’s requirements for developmental competence, with important implications for the diagnosis and treatment of certain types of infertility. Here, we give an overview of post-transcriptional regulation in the oocyte, emphasising the current knowledge of mammalian RBP mechanisms, and develop the roles of these mechanisms in the timely activation and elimination of maternal transcripts. For correct and timely function, each cell regulates the expression of its genes to suit its specific protein requirements at any given moment. A cell’s requirements differ according to cell type and depend on an interaction of extracellular and intrinsic cues. A cell can execute gene regulation at a number of stages along the protein production pathway: (1) At the transcription level, via transcription factors and chromatin accessibility, (2) at the mRNA level by controlling the processing, translation, and degradation of transcripts, and (3) at the protein level, where protein activity can be modulated via post-translational modifications and degradation.The most prominent form of regulation for the majority of genes is executed at the level of transcriptional control . The bincis-regulatory elements typically within the 3′UTR of the mRNA sequence. Such sequences are recognised by trans-acting RNA-binding proteins (RBPs), which are capable of recruiting or preventing recruitment of key actors of the synthesis machineries.Post-transcriptional regulation is an essential component of the cell’s ability to regulate protein expression as it represents a secondary layer of control that is independent from the transcription machinery. This type of regulation allows for rapid response to external stimuli  and becoIn the oocyte, a long growth phase during which chromatin is in an ‘open’ conformation and transcriptional activity is high is succeeded by a phase of resumed meiotic activity and transcriptional silencing. Meiotic resumption is triggered in fully grown GV oocytes in response to the LH surge, entailing chromatin condensation, completion of meiosis I (with extrusion of the first polar body), and progression through to metaphase of meiosis II before ovulation. At fertilisation, the oocyte completes the final stages of meiosis II (extruding the second polar body) whilst undergoing nuclear reprogramming and fusion of the male and female pronuclei as part of the maternal-to-zygote transition (MZT), followed by the first embryonic divisions and activation of the embryonic genome (EGA) . These nTo meet these requirements, a number of specialised RBPs exist, making up a specialised and multi-layered mRNA processing system. The oocyte environment is a particularly stable one, with a very low intrinsic decapping rate. Rather than hours, maternal oocyte mRNAs have an average half-life of around two weeks in growing mouse oocytes ,6. The pThroughout an mRNA’s lifetime, its movements and fate are governed by the proteins and other molecules that bind to it. An mRNA molecule is constantly bound by a multitude of RNA-binding proteins (RBPs) that are necessary for facilitating intracellular transport, entry to and exit from translational activity, and recruitment of or protection from the cell’s degradation machinery. For this reason, it is better to refer to the synthesis and movement of messenger ribonucleoprotein (RNP) particles. RBPs typically bind to elements within the mRNA sequence, often located in the 3′ UTR, through RNA-binding domains. Several such domains have been characterised, including zinc-finger (Znf), RNA recognition motif (RRM), and K homology (KH) domains. The affinity of these binding interactions is usually relatively low and the recognised sequence variable, leading to a range of possible affinities and interactions .In eukaryotes, protein-coding genes with accessible promoter regions are transcribed by RNA polymerase II, which is recruited and bound to the promoter through transcription factors . The polOnce in the cytoplasm, the nuclear CBC is replaced by the trimeric cytoplasmic cap-binding complex, eIF4F, comprising eukaryotic translation initiation factors eIF4E (cap-binding), eIF4G, and eIF4A. eIF4F binds the 40S ribosomal subunit via eIF3. eIF4G associates with PABP, creating a pseudo-circular structure that favours efficient translation and protects the mRNA from degradation . AnotherA long poly(A) tail is correlated with translational efficiency. Deadenylation slows translation and, in most cell types, initiates mRNA degradation by disrupting the translation initiation complex and exposing the 5′ end to decapping enzymes. Deadenylation and exoribonuclease degradation is the pathway by which most mRNAs undergo decay. It is thought that first the PAN2/3 (poly(A)-nuclease) complex shortens the poly(A) tail to around 80 residues, after which the CCR4-NOT complex or PARN (Poly(A)-specific ribonuclease) takes over and removes most or all of the remaining tail . DeadenyThe above-described mRNA decay and interference machinery as well as translationally inactive mRNAs tend to concentrate in discrete cytoplasmic foci known as P-bodies (processing bodies). P-bodies are membrane-less organelles that are sites of translational repression, mRNA degradation, and also mRNA storage, since mRNAs can subsequently return to translation . They arIt is estimated that 30-45% of the mRNAs transcribed during oocyte growth in vertebrates are translationally repressed until meiotic maturation or fertilisation . These sXenopus, due to the quantity of oocyte material that can easily be obtained in comparison with mammalian models. This model has allowed the characterisation of many RNPs involved in translational control. RBPs whose mammalian orthologues have been shown to have important roles through mouse KO studies as well as any association to human reproductive disease are listed in The most widely studied model organism for oocyte transcript dynamics is Xenopus, Drosophila, C. elegans, and zebrafish), germ granules are present in oocytes of all stages. In mammals, however, germ granules have been described to exist only in primordial follicle oocytes of mice under two weeks of age . T. T55]. TXenopus acts through the prevention of the interaction between cap-binding eIF4E and eIF4G via an intermediary protein, thus preventing formation of the eIF4F complex . In Xenopus oocytes, 4E-T was shown to interact with eIF4E1b: An ovary-specific close homologue of eIF4E (70% identity) that weakly binds the 5′ cap ..Xenopus Another member of the CPEB family, CPEB4, has also been found to play an important role in oocyte meiotic progression. CPEB1 controls the translation of CPEB4, activating it at a later stage of meiosis. CPEB4 likely replaces CPEB1, driving the transition of metaphase I to metaphase II in a positive translational loop .Xenopus oocytes. They bind to the 3′ PUM-binding element (PBE: UGUAX2-4UA) as well as the Nanos response element (NRE: UGUA) [Xenopus oocytes [The Pumilio proteins (PUM1/2), members of the PUF protein family, are involved in translational regulation in E: UGUA) . Pum1/2 E: UGUA) . Pum2 fo oocytes ,63. PUM1 oocytes . There a oocytes .DAZL and EPABP can be considered co-repressors of translation when they are associated with PUM2. However, DAZL itself is as an activator of translation thought to be able to interact with a large number of oocyte RNAs through uracil-rich 3′ UTR sequences. In mice, DAZL is expressed until the zygote stage, plays an essential role in primordial germ cell differentiation, and is thought to regulate mRNAs important for spindle assembly, meiosis, and maternal mRNA degradation . The proXenopus, mouse, and human [Xenopus oocytes. In Xenopus, Epabp is both part of the Cpeb1-CPSF and Dazl-Pum2 complexes. EPABP is required for oocyte maturation and also for oocyte-granulosa cell communication in mice [While PABP acts as a global translational regulator, EPABP is specific to and is the predominant poly(A)-binding protein in oocytes and early embryos in nd human . Epabp p in mice ,65.Pat1 (protein associated with topoisomerase II) proteins, conserved across eukaryotes, are RNA-binding proteins that have been shown to be involved in numerous aspects of mRNA metabolism. They participate in large regulatory RNP complexes and localise to P-bodies in somatic cells. The current hypothesis is that Pat1 proteins act as scaffold or platform proteins, interacting with both mRNA and other proteins of various functions. Pat1 proteins have been implicated in mRNA decay, translational repression, and, more recently, in pre-mRNA processing. A single Pat1 protein (Pat1p) exists in yeast and invertebrates, whereas in vertebrates, two orthologous proteins have evolved (PatL1/Pat1b and PATL2/Pat1a) with distinct regulatory functions in the soma and germline. Somatic PATL1 appears to be primarily involved in mRNA decay and PATL2, specific to oocytes, in translational repression.Xenopus PATL2 (xPat1a) was identified as a cytoplasmic RNP in growing oocytes. It was found to be an abundant binding partner of Cpeb1, able to selectively bind RNA and to repress the translation of specific mRNAs. xPat1a is rapidly degraded at meiotic resumption and ectopic expression of xPat1a significantly impaired the ability of oocytes to mature upon progesterone stimulation [Patl2 invalidation in mice. We showed that Patl2 invalidation leads to subfertility in females with oocytes showing dysregulation of a number of key transcripts expressed at various stages of oocyte maturation [Patl2 KO females mature to the MII stage and the rate of progression through the early embryo stages is significantly impacted. PATL2 therefore appears to be a crucial factor for oocyte maturation, although its precise mode of action and molecular interactions remain to be elucidated in mammals.mulation ,66,67. Ituration . Fewer oXenopus Frgy2) is highly abundant in mouse oocytes until fertilisation, comprising around 2% of total oocyte protein in mice [Xenopus orthologue Frgy2 is a partner of Cpeb1 and a principal component of maternal RNPs [Msy2-/- compared to WT oocytes, and there was a global decrease of 25% in mRNA quantity  [Germ-cell specific RNA-binding protein MSY2  . Since nXenopus oocytes, its orthologue Xp54 was shown to mask maternal mRNAs as a binding partner of Cpeb1, associating with mRNAs encoding proteins necessary for meiotic progression [DEAD-Box Helicase 6 is an RNA helicase with a diverse range of functions. It is both a decapping activator and translational repressor and is a component of all known types of RNP granules: P-bodies, stress granules, and germ cell granules . In Xenogression ,71. In tgression .Drosophila, Musashi has been well-characterised in Xenopus oocytes where it was found to be part of the Pumilio and Cpeb1 pathway. RINGO/Spy-dependent Cdk1 activity phosphorylates Musashi, activating translation of its target mRNAs including Mos [Musashi RBPs have a double role as translational repressors and activators. They contain RNA recognition motifs in their N-terminals and bind to a Musashi binding element (MBE) in the mRNA 3′ UTR. Originally described in ding Mos . This acding Mos . MusashiXenopus oocytes and interact directly with Xp54 [Xenopus oocytes of all stages as well as in embryos, but was shown to be phosphorylated in matured oocytes, indicating a potential mode of action. In mouse oocytes, it has been identified in Balbiani bodies and found to colocalise with DCP1a and DDX6 [The RAP55  family consists of two paralogues in vertebrates: RAP55A and RAP55B. Conserved in eukaryotes, they contain several domains (including a LSm domain: A domain found in many RBPs conferring a specific 3D structure) allowing for translational repression, mRNP inclusion, and localisation to P-bodies and stress granules. Originally discovered as an RNA-binding in newt oocytes, xRAP55 has been found to repress translation of maternal mRNAs in ith Xp54 . It is eand DDX6 ,75. Studand DDX6 . It was and DDX6 .Xenopus kinesin xklp2) whose translation is controlled in a spatial as well as temporal manner [As is well established, the crosstalk between the oocyte and its surrounding cumulus cells is vital for the oocyte developmental competence. It is possible that this signaling affects translational regulation in oocytes. The cumulus-cell-dependent PI3K/PTEN/mTOR signaling pathway influences the accumulation of certain oocyte proteins. During GVBD, mTOR kinase (TORC1) downstream of cumulus cell growth factor/Kit Ligand stimulation phosphorylates 4E-BP: A cap-dependent translational repressor expressed in mammalian oocytes. This leads to 4E-BP’s detachment from eIF4E allowing for translational activation. Transcripts that are controlled via this pathway include those required for spindle assembly, chromosome alignment, and segregation, such as TPX2  and endogenous small interfering RNAs (endo-siRNAs) are important actors of post-transcriptional control and are expressed in mammalian oocytes. These small RNAs degrade or repress the expression of target mRNAs through full or partial sequence complementarity. miRNAs are transcribed by RNA polymerase III in a precursor form and processed by the Drosha/DGCR8 complex before being exported to the cytoplasm and cleaved by DICER to around 21 nucleotides. They then combine with Argonaute (AGO) proteins to form an RNA-induced silencing complex (RISC) that recognises the 3′ UTR of targets. siRNAs differ to miRNAs in that they bypass Drosha/DGCR8 processing, require full sequence complementarity, and associate with AGO2, which is unique in having endonucleolytic activity.Dicer or Ago leads to disrupted meiotic maturation and spindle formation defects [Dgcr8 invalidation (interfering with miRNA but not siRNA levels) did not affect oocyte or embryo development [miRNAs have been found to play a critical role in deadenylation and clearance of maternal mRNAs in zebrafish zygotes . In mice defects , Dgcr8 ielopment . This inelopment . Based oXenopus oocytes, in which meiotic progression can be induced through treatment with progesterone. The chain of events leading to translational activation is not as well characterised in mammals as in amphibians due to difficulty in obtaining sufficient material. Although largely similar, certain differences, especially in the timing of events, have been reported between the amphibian and mammalian mechanisms, including later phosphorylation of CPEB1 in mammals.Oocyte maturation is governed by waves of translation leading to the cascade of molecular events that are associated with meiotic resumption. CPEB1-mediated activation and polyadenylation of dormant mRNAs is thought to play a primary role. Many elements of this cascade have been elucidated in Xenopus, this mechanism is as follows: upon progesterone stimulation, Pum2 (Pumilio) dissociates from the Pumilio Binding Element present in the 3′ UTR of RINGO/Spy mRNA as well as from Dazl and Epabp. The Dazl–Epabp complex then activates RINGO/Spy translation and RINGO/Spy recruits Cdk1, initiating the phosphorylation of Aurora A as well as Musashi. Aurora A kinase phosphorylates Cpeb1, and Musashi induces early activation of Mos and cyclin B5 [Meiotic progression requires activation of kinases MAPK (mitogen-activated protein kinase) and CDK1 (cyclin-dependent kinase 1). The MAPK cascade is activated by Mos, which also combines with CDK1 to form the Maturation Promoting factor. Translation of both Mos and CDK1 is activated by CPEB1 . CPEB1 ayclin B5 ,73,83. POne mouse oocyte study analysed polysome-bound mRNA and revealed a switch in the translation program of mouse oocytes before and after GVBD (in MII versus GV oocytes) . One-thicis-elements and their trans-binding proteins whereby the state and timing of translational repression or activation is determined by a play-off between repressive and activating factors. Piqué et al. [Differences have been found in the timing of polyadenylation of CPE-containing mRNAs during maturation such as for Mos and Cyclin B1. Such differences could be explained by a combinatorial code of 3′UTR é et al.  describeé et al. . Differeé et al. . In thisThe degradation of maternal mRNAs begins at meiotic resumption. In mouse oocytes, meiotic resumption is accompanied by a decrease in total poly(A) RNA quantity from 80 pg to around 50 pg per oocyte. This degradation continues in the zygote and two-cell embryo, where it drops to 10 pg . As showXenopus, the maternal component of the decay machinery mainly involves RNA-binding proteins that recognise 3′ UTR sequences such as AREs (AU-rich element) and EDENs (embryonic deadenylation element). The EDEN binding protein (EDEN-BP) is activated and induces deadenylation upon fertilisation. Degradation of targeted maternal mRNAs then depends on the zygotic component that largely relies on small RNAs [Drosophila is the maternal RBP Smaug, which deadenylates maternal mRNAs through recruitment of the CCR4-NOT complex [Degradation of maternal transcripts is highly regulated both temporally and spatially according to a species-specific program . Generalall RNAs . A well- complex .In mice, there is a strong wave of transcript elimination that occurs shortly after fertilisation triggered by maternal factors, and a second round that coincides with the major EGA mediated by zygotic factors . The proMaternal proteins are also targeted for degradation during the maternal-to-zygote transition (MZT) by the ubiquitin–proteasome pathway . The remAs shown in CPEB1 gene, indicating CPEB-haploinsufficiency as a rare cause of POI [DAZL in cohorts of women with POI and men with azoospermia (few or no sperm in the ejaculate). PUM2 SNPs have been associated with POI in a genome-wide association study (GWAS) [EIF2B genes, part of the 43S translation preinitiation complex  . Variant complex  , a type of primary infertility characterised by the repetitive production of immature oocytes that do not mature in vitro. Invalidation of ntinents ,42,43,44FMRP and LIN28, whose dysregulation have been associated with POI and germ cell tumours respectively [Several other oocyte RBPs with important roles in human oogenesis exist, for example ectively . These hIt is clear that RBPs play an indispensable role in oocyte maturation and developmental competence across species. Given their importance, these complex regulatory networks merit further clarification, particularly in mammalian systems for which knowledge is often lacking. There are many aspects of oocyte translational regulation that require further elaboration, for instance, the spatial aspect of mRNA regulation, the exact role of small non-coding RNAs and the mammalian networks orthologous to those described in amphibians or invertebrates. The species differences that have been found between orthologous mRNA regulatory systems show that, if we want to improve our knowledge of human oogenesis, it is important to identify models whose mechanisms most closely resemble the human system despite the challenges involved.The existence of oocyte-specific RBPs and their importance for oocyte maturation would make them good candidates for association with female infertility in cases of gene invalidation or with oocyte ageing. Infertility is a global public health issue affecting over 48.5 million couples worldwide. The elucidation of these RNA regulatory networks generates knowledge that could therefore be used to improve our diagnostic capabilities for human infertility and may aid in the development of new treatment strategies. Beyond this, the knowledge generated can be used to inform the improvement of existing assisted reproductive techniques such as in vitro oocyte maturation (IVM).IVM is a technique currently applied to immature retrieved oocytes for IVF (in vitro fertilisation) or ICSI (intracytoplasmic sperm injection) in which oocytes are cultured to induce oocyte maturation to make fertilisation possible. The success of this technique is intimately dependent on culture conditions, and its effectiveness is currently limited to oocytes derived from larger follicles. A better understanding of the processes that occur during oocyte growth and maturation could inform the optimisation of culture conditions, improving success rates and potentially paving the way for expansion of this technique to the maturation of younger, more immature oocytes. This would have far-reaching applications, including creating the possibility for fertility preservation for young cancer patients whose oocytes may be cryopreserved ahead of reprotoxic therapy.In this review, we have highlighted the importance of oocyte RNA-binding proteins in post-transcriptional regulation during and beyond oocyte maturation. The correct accumulation of maternal RNAs and RBPs during the growth phase and the finely balanced control that recruits RNAs to the translation apparatus or initiates their degradation in a timely fashion are processes that are central to the production of developmentally competent oocytes. We must strive to understand the oocyte’s complex regulatory mechanisms if we are to understand the pathologies linked to their disruption, and to recreate conditions favorable to correct oocyte development in reproductive therapy.


Primary effusion lymphoma (PEL) has a very poor prognosis. To evaluate the contributions of enhancers/promoters interactions to PEL cell growth and survival, here we produce H3K27ac HiChIP datasets in PEL cells. This allows us to generate the PEL enhancer connectome, which links enhancers and promoters in PEL genome-wide. We identify more than 8000 genomic interactions in each PEL cell line. By incorporating HiChIP data with H3K27ac ChIP-seq data, we identify interactions between enhancers/enhancers, enhancers/promoters, and promoters/promoters. HiChIP further links PEL super-enhancers to PEL dependency factors MYC, IRF4, MCL1, CCND2, MDM2, and CFLAR. CRISPR knock out of MEF2C and IRF4 significantly reduces MYC and IRF4 super-enhancer H3K27ac signal. Knock out also reduces MYC and IRF4 expression. CRISPRi perturbation of these super-enhancers by tethering transcription repressors to enhancers significantly reduces target gene expression and reduces PEL cell growth. These data provide insights into PEL molecular pathogenesis. Primary effusion lymphoma (PEL) has a very poor prognosis. Here, the authors perform H3K27ac HiChIP in PEL cells and generate the PEL enhancer connectome, linking enhancers and promoters in PEL, as well as super-enhancers to dependency factors. Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8) and Epstein-Barr virus (EBV) are tightly linked to this malignancy. A small subset of KSHV- or EBV-encoded proteins as well as non-coding RNAs and miRNAs play essential roles in PEL pathogenesis. The expression of KSHV genes is important for PEL growth and survival5. vFLIP (KSHV) and LMP1 (EBV), each activate NF-kB7. EBV superinfection of KSHV only in PELs increases tumorgenicity in vivo8. EBV and KSHV co-infection causes tumor formation in a humanized mouse model, and results in greatly reduced mice survival9. EBV can enhance the KSHV genome maintenance in PELs10. Analysis of PEL cell genomes revealed that coinfection with EBV is associated with fewer host genomic alterations compared to PELs that are only KSHV infected11. PELs that are coinfected with KSHV and EBV differ in gene expression profiles compared with KSHV positive and EBV negative PELs12.Primary effusion lymphoma (PEL) is a rare non-Hodgkin’s lymphoma with very poor prognosis that often occurs in HIV infected people. Even in the highly active antiretroviral therapy (HAART) era, a PEL patient’s median survival is ~6 months, as there is no specific therapy13. These genes include MYC, IRF4, CCND2, MCL1, and CFLAR, which are important for transcription, cell cycle progression, and survival13. The expression of these oncogenes is controlled by enhancers and promoters. Enhancers are marked by unique histone modifications including H3K27ac and H3K4me1, and can be readily identified by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). However, it is difficult to assign these enhancers to their direct target genes as they can be anywhere in the genome, relative to their direct targets. In EBV transformed lymphoblastoid cell lines (LCLs), chromatin interaction analysis followed by deep paired-end sequencing (ChIA-PET) links enhancer–enhancer, enhancer–promoter, and promoter–promoter interactions genome wide14. LCL enhancers hundreds kb upstream loop to the MYC promoter to activate MYC expression. Enhancers upstream of IRF4 skip the nearest gene DUSP22 and interact with IRF4 that is further away from the enhancer. Some enhancers even interact with genes on other chromosomes15. Frequently, important oncogenes are linked to multiple enhancers14. However, ChIA-PET is technically challenging as hundreds of millions of cells are needed. The recently developed HiCHIP method can achieve the same goal with much higher efficiency and much less input DNA16.PEL cells depend on a group of genes for their continuous proliferation, identified by genome-wide CRISPR screens17. Viral proteins can form new enhancers by directly binding to enhancer DNA or induce cell TF DNA binding (such as NF-κB) to control key oncogene expression20, including that of EBV and KSHV proteins21. Human papilloma virus integration can function as a super-enhancer (SE) to drive viral oncogene expression22. Oncogenic TFs such as NOTCH can assemble SEs to control oncogene expression23. SEs can also be acquired by genome amplification17. EP300, a HAT, is essential for PEL growth and survival13, probably through enhancer activation.Transcription factors (TFs) and co-factors bind to enhancer DNA to assemble active enhancers. These TFs also recruit histone acetyl transferases (HATs) and histone methyl transferases (HMTs) to modify the histone tails at these enhancers. In different cancers, through altering enhancer DNA sequence, oncogenes can acquire new enhancers through de novo enhancer formation24. Alterations in histone modifications are often associated with oncogenesis25. ChIP-seq and ChIP-on-ChIP analysis of PEL cells identified active and repressed regions in the KSHV genome29. H3K4me3 and H3K27me3 ChIP-seq analysis of PEL cells identified active promoters and repressed regions genome wide31.Combinations of different histone modifications mark different genomic regulatory elements32. SEs play critical roles in development and oncogenesis. SEs are co-occupied by many TFs and co-factors, and proposed to form phase-separated nuclear subdomains and are more sensitive to perturbations. SEs frequently target key oncogenes such as MYC14. Importantly, SEs are more sensitive to perturbations than typical enhancers24. Characterization of the SE components may lead to interventions.SEs are enhancers with extraordinarily high and broad H3K27ac ChIP-seq peaks compared to average enhancersHere we generate the PEL enhancer connectomes using H3K27ac HiChIP, linking all PEL enhancers to their direct target genes in both EBV positive and negative PEL cell lines. We then integrate these data with the H3K27ac ChIP-seq data. We find that PEL dependency factors are linked to PEL SEs. CRISPRi validate the functional significance of these PEL SEs in gene expression and cell growth.16. PEL cell lines BC1, JSC, BC3, and BCBL1 were chosen for the analyses. BC1 and JSC are EBV positive while BC3 and BCBL1 are EBV negative. PEL cells were first cross-linked with formaldehyde. The DNA was cut with Mbo I. The DNA ends were filled with biotinylated dATP and other unlabeled nucleotides and ligated in situ. H3K27ac ChIP was used to enrich the DNA interactions mediated by H3K27ac. Ligated DNA was captured by avidin beads and paired-end deep sequenced. The sequencing reads were then mapped to the human genome using HiC-Pro33. Significant genomic interactions were called using hichipper, normalized by total valid paired interaction reads34. H3K27ac HiChIP identified 12,511, 13,821, 8184, and 12,129 significant interactions between H3K27ac peaks in BCBL1, BC3, BC1, and JSC cells.In order to fit the 2 m long genomic DNA into the tiny nucleus, genomic DNA is packaged in a very complexed yet ordered way. Transcription regulatory elements such as enhancers and silencers are positioned in close proximity to their direct target genes by looping out intervening sequences, even though they can be hundreds of kb away from each other. Next-generation sequencing-based assays now can efficiently identify long-range chromatin interactions to generate connectomes. To link PEL enhancers to their direct target genes, H3K27ac HiChIP was usedPRDM1(BLIMP1), IRF2, MYB, and MIR21 miRNA 45. Here, 21,166, 26,197, 25,546, and 39,610 significant H3K27ac peaks were identified in BC1, JSC, BC3, and BCBL1 cells, respectively; 56.2%, 50.4%, 49.8%, and 38.3% of the H3K27ac peaks were at promoters (within ± 2 kb of the transcription start site (TSS)) and 43.8%, 49.6%, 50.2%, and 61.7% were in intergenic regions or within gene bodies. Out of 49010 total peaks, 11506 were common for all four cell lines; 208 peaks were common in BC1 and JSC cells but not significant in BC3 and BCBL1 cells; 2247 peaks were common in BC3 and BCBL1 cells but not significant in BC1 and JSC cells  enhancer–enhancer interactions, ~678–2563 (~35–42%) enhancer–promoter interactions, and ~326–1776 (~18–27%) promoter–promoter interactions . These data suggested that CTCF and cohesins may mediate the PEL enhancer looping.Looping factor CTCF is a sequence-specific DNA binding protein. When CTCF molecules form a homo-dimer, the distant DNA elements are brought to close proximity and loop out the DNA between two CTCF sites. This allows enhancers to contact other enhancers or promoters57. In LCLs, EBV TFs form SEs ~525 and 428 kb upstream of the MYC promoter and activate MYC expression by looping to the MYC promoter14. In centroblast B cells and high-grade B cell lymphomas, more H3K27ac signals are at the MYC 3′ downstream region58. In peripheral blood B cells, mantle cell lymphomas, and small lymphocytic leukemia cells, the H3K27ac peaks are predominantly at the 5′ enhancer regions58. In PEL cells, multiple SEs 400–600 kb downstream of MYC promoter were found in most PEL lines evaluated  which drives EBNA1 expression, and microRNA , 100 unit/mL streptomycin, and 100 mg/mL penicillin (Gibco). HEK293T cells purchased from ATCC were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (Gibco), 100 unit/mL streptomycin, and 100 mg/mL penicillin. All the cells were maintained at 37 °C in a 5% COAntibodies used in this study are: H3K27Ac , GAPDH , MYC , MEF2C , IRF4 , and CAS9 .19. PEL cell H3K27Ac HiChIP libraries were prepared following16. In brief, 15 million cells were cross-linked with 1% formaldehyde and lysed with Hi-C lysis buffer . Cross-linked DNA was digested by 375 U Mbo I at 37 °C for 2 h. DNA ends were filled in with biotin-dATP (Thermo Fisher 19524016), dCTP, dGTP, and dTTP mix, and DNA Polymerase I, Klenow fragment  with shaking at 37 °C for 1 h. T4 DNA Ligase was used for proximity DNA ligation at 4 °C overnight. After ligation, DNA was sonicated using a Covaris M220. Fragmented DNA were then diluted 10 times with ChIP dilution buffer  and samples precleared with Protein A Dynabeads at 4 °C for 1 h. DNA–protein complexes were captured with 8 μL H3K27Ac antibody at 4 °C overnight. DNA–protein complexes were captured with Protein A beads with rotation at 4 °C for 2 h. Beads were washed and DNA were eluted twice with 150 μL of freshly prepared DNA Elution Buffer . The eluted DNA was reverse cross-linked. DNA was purified with a PCR purification Kit (Qiagen). Biotin dATP labeled DNA was captured with 5 μL Streptavidin C-1 beads  and DNA was then transposed with 2.5 μL of Tn5. Beads were then washed. After the final wash, beads were re-suspended with 23 μL ddH2O, 25 μL 2X Phusion HF (New England Biosciences), and 1 μL of each of Nextera forward primer (Ad1_noMX) and Nextera reverse primer (Ad2.X) at 12.5 μM. PCR was run at: (1) 72 °C for 5 min, (2) 98 °C for 1 min, (3) 98 °C for 15 s, (4) 63 °C for 30 s, and (5) repeat steps 1–4 for a total of 8 cycles, and final extension at 72 °C for 1 min. After PCR amplification, a two-sided size selection with Ampure XP beads was performed for DNA size selection and purification. Samples were then sequenced on the Illumina Nextseq platform (2 × 75 bp).ChIP-seq was done followingBCBL1 was transduced with CAS9 expressing lentivirus followed by Blasticidin selection. sgRNAs from the Brunello library were used to knock out gene of interest. sgRNAs were packaged into lentiviruses. Lentiviruses were prepared by transfecting HEK293T cells with pCMV-VSVG , psPAX2 , and pLenti-Guide-puro plasmid expressing gRNA with TransIT-LT1 transfection reagent (Mirus) according to the manufacturer’s instructions. Viruses were used to transduce BCBL1 cells; 48 h after infection, cells were selected by 3 μg/mL Puromycin. Cells were then allowed to grow for another 3 days.BCBL1 and JSC stably expressing dCAS9-KRAB-MeCP2 fusion proteins were selected by blasticidin. Cells were transduced by lentiviruses expressing sgRNA. Cells were selected with puromycin and then allowed to grow for 3 days.® Luminescent Cell Viability Assay (Promega) was used to determine the relative number of viable cells in each condition.CellTiter-GloTotal mRNAs were extracted using PureLink RNA mini kit (Life Technologies). Two hundred nanogram of mRNA was used as template for reverse transcription with iScript™ Reverse Transcription Supermix (Bio-rad). cDNAs were then amplified on an CFX96 Touch real-time PCR detection system (Bio-Rad), and SYBG Green (Thermo Fisher) was used to detect cDNA amplification. GAPDH was used to normalize gene expression. RNA relative expression was calculated using the 2 ΔΔCT method. The value for the cells transduced with non-targeting sgRNA was set to 1.Ten million cells were fixed with 1% formaldehyde. The cells were then lysed and lysates were sonicated with bioruptor (Diagenode) with 30 s on, 30 s off for 45 cycles. Sonicated chromatin was diluted with ChIP dilution buffer and incubated with H3K27ac or control antibodies. Protein–DNA complexes were precipitated with protein A beads. After precipitation beads were washed extensively and eluted protein–DNA complexes were reverse cross-linked. DNA was purified by using QIAquick Spin columns (Qiagen). qPCR was used to quantify the DNA from ChIP assay and normalize it to the percent of input DNA. Primers used in this study are provided in Supplementary Data 72. Normalized signals of probes targeting the same genes were averaged for each gene. Genes were further categorized into two groups for expression comparison: H3K27Ac peaks within 20 kb upstream of genes’ transcription start site or no peaks otherwise.Gene expression microarray data were download from GEO (GSE1880) for BC1, BC3, and BCBL1 cell lines, followed by normalization using RMAhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc) was performed on both ChIP-seq and HiChIP sequencing reads to ensure sequencing experiments have no considerable flaws such as heavy GC bias and PCR artifacts. ChIP-seq reads were aligned to human (hg19) and EBV (Akata) genomes using Bowtie2 v2.2.373 under default settings except parameter −k was set to 1. Read mappability rate ranges from 94 to 98% across ChIP-seq samples. ChIP-seq peaks were called using MACS v2.1.074 (default settings except reporting criteria was set as FDR < = 0.99) on each replicated sample followed by IDR analysis (v2.0.3), default settings except reporting criteria was set as IDR < = 0.02, which is suggested by ENCODE consortium76 to generate reproducible peaks between replicates. Peaks located in blacklist regions77 were excluded in analysis. The HOMER program46 was then used to detected motifs in ChIP-seq peaks. Genome-wide ChIP-seq coverage were normalized with size factors which were determined using DiffBind78 combined with DESeq279. SEs were called with ROSE v1.0.0 under default settings32 using IDR-reported H3K27ac peaks. HiChIP paired-end reads (17–27 million reads for each sample) were mapped using HiC-Pro v2.11.133 (default settings with LIGATION_SITE set as GATCGATC for Mbo I) and significant loops identified with hichipper v0.7.534 (default settings except parameter—skip-diffloop was set). In detail, HiChIP experiments are performed with Mbo I, which can introduce a biasness to the fragment sizes due to non-uniform distribution of cut sites. hichipper performs a background correction based on the non-uniform distribution of restriction fragments, to better infer anchors and loops. Quality control was performed by ensuring a high percentage of reads were mapped (>90%), within anchors (>65%) and supporting valid interactions (25–35%). Mapped reads were also visually inspected on the WashU genome browser80 to ensure significant enrichment when compared to H3K27ac ChIP-seq reads, general uniformity between replicates, and good signal-to-noise ratio, which are indicative of a successful ChIP experiment. Replicates samples were then merged and loops were identified by (1) merging anchors within 1.5 kb of each other, (2) removing loops that are <5 kb in length. Loops scores were further normalized by total number of valid interaction read pairs in each cell line. A minimum of three normalized read pairs were used to filter strong long-range interactions. Long-range interactions were then annotated using diffloop v1.10.081 (default parameters with enhancer and promoter regions defined as below) to decide enhancer–enhancer loops, enhancer–promoter loops, and promoter–promoter loops. Here, promoters were defined as ± 2 kb regions surrounding gene transcription start sites. Enhancers were defined as identified H3K27Ac binding regions except those located at promoter regions.FastQC (82. Selected KEGG pathways that have P-value less than 0.05 were reportedPathway enrichment analysis was performed with gene names using DAVID version v6.8Statistics were done as stated in the figure legends. No statistical method was used to predetermine sample size. Sample sizes were chosen based on similar studies in the relevant literature. Source data are provided as a Source Data file.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting summary


Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.Transcriptional regulation of the  HO gene. Ash1 is a sequence-specific DNA-binding protein, and we show that it recruits the Tup1 global repressive factor to the HO promoter. Using a method to determine where Ash1 and Tup1 are bound to DNA throughout the genome, we discovered that Tup1 is also present at most places where Ash1 binds. The majority of these sites are in “Nucleosome Depleted Regions,” or NDRs, where the absence of chromatin makes factor binding easier. We discovered that the HO promoter is an exception, in that the two places where Ash1 binds overlap nucleosomes. Activation of the HO promoter is a complex, multi-step process, and we demonstrated that chromatin factors transiently evict these nucleosomes from the HO promoter during the cell cycle, allowing Ash1 to bind and recruit Tup1. Thus, activators must evict nucleosomes from the promoter to allow the repressive machinery to bind.Nucleosomes inhibit both gene expression and DNA-binding by regulatory factors. Here we examine the role of nucleosomes in regulating the binding of repressive transcription factors to the complex promoter for the yeast  Chromatin is generally repressive to transcription, limiting access of regulatory factors and RNA polymerase to the DNA . HoweverSaccharomyces cerevisiae HO gene is an important model for examining the interplay between transcription factors and chromatin. The HO promoter is highly regulated, with a complexity more similar to higher eukaryotic promoters than typical yeast promoters, but with an ease of genetic manipulation Please let us know if you have any questions while making these revisions.Yours sincerely,Geraldine ButlerAssociate EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In this manuscript, Parnell et al. continue the analysis of HO gene regulation in yeast that has been a long-term project of the Stillman lab. The current work focuses on the association of the Ash1 repressor, which is required for repression of HO in daughter cells following cell division, with the HO promoter. Using standard ChIP and ChIP-seq, they identify two regions in the HO promoter with which Ash1 associates, as well as >200 genomic sites. Ash1 co-localizes with the co-repressors Tup1 and Rpd3 at HO and the majority of its other genomic sites, and contributes to their binding in most cases, though the mechanism of binding of Ash1 remains unclear. At HO, Ash1 binds to sites that are nucleosomal through most of the cell cycle, excepting for the window following Swi5 binding, and Swi5 is required for Ash1 binding. In addition, depleting the nucleosomes at which Ash1 binds by inserting Reb1 sites allows Swi5-independent Ash1 association.Technically the work is solid, and the authors are clear in explaining their use of replicates and when they are not needed . The results have novel aspects and provide new insight into mechanisms of cell cycle regulation and repression via Ash1, as well as identifying numerous targets of this repressor about which little is presently known. As to broader significance, based on trends in citations, publications, and grant awards , I think the issues addressed in this work do not hold as much general interest as they once did. Nonetheless I would favor publication, as the work adds significantly to the impressive knowledge of the complex regulation of this paradigmatic promoter and should be of interest to those studying mechanisms by which chromatin regulates transcription.Some minor points:1) Results for an untagged control are presented for standard ChIP but apparently this control was not performed for ChIP-seq experiments. As I imagine the ChIP-seq experiments were performed in Dr. Bai’s lab, I wonder if she has results from any untagged control using the anti-V5 (or other) antibodies that could be shown together with the heat maps in Figure 6? I think it is not likely the results will change but it would be a nice control if available.2) State in Figure 1 legend that replicates are shown in Fig S1.3) Table S5: It would be helpful to provide p-values for enrichment for the various categories shown. What is the source of information for stress responsive genes and for the other categories? I assume they correspond to standard Gene Ontology categories?4) Line 598, “switch” should be “switches”5) Ref 62 is incomplete—in press?6) Methods—presumably perfetto delitto was used for nucleosome replacements? This (or whatever method was used) should be stated.Reviewer #2: In this study, the authors investigate the role of the Ash1 transcription factor in regulation of the budding yeast HO promoter. The HO gene is an important model gene because of its very long promoter (~2.5 kb), which is much more typical of the genes of higher eukaryotes than of other yeast genes. The Stillman Lab has studied the cell cycle dependence of HO regulation in great depth; here they provide additional insight into its control. HO is expressed only in mother cells. Ash1 is a negative transcription factor that accumulates in daughter cells and is primarily responsible for mother cell-specific HO expression. The authors show using ChIP that Ash1 binds to two sites in the HO promoter that appear to be located inside nucleosomes; these sites are atypical in that most Ash1 binding sites are located in nucleosome-depleted regions (NDRs). They go on to show that Ash1 recruits both the Tup1 co-repressor and the Rpd3 histone deacetylase complex to the HO promoter. Finally, Ash1 binding at HO is shown to be dependent on the Swi5 transcription activator. Interestingly, if the nucleosomal sequence where Ash1 binds is mutated to include a pair of Reb1 binding sites, creating an NDR, Ash1 binding becomes Swi5-independent, suggesting that this nucleosome is a direct regulator of Ash1 binding. The authors propose that nucleosomes block Ash1 binding at HO and must be removed in a Swi5-dependent process before Ash1 can bind, thus ensuring appropriate timing of Ash1 binding during the cell cycle.The manuscript is interesting, represents an important advance in our understanding of HO regulation and is clearly written. I have the following comments:1. The nucleosome positions on the HO promoter are derived from MNase-seq data from another study, presented in Fig. 1A. They are critical for the interpretation of the data, but the nucleosome positions were determined using log-phase cells and are therefore averaged over the cell cycle. Since Ash1 binding is cell cycle-dependent, it is possible that the nucleosome positions on the HO promoter change during the cell cycle and that their average positions are misleading. In addition, there is the complication that Ash1 may bind only in daughter cells and that the chromatin structures of mother and daughter cells also differ. The question that arises is whether the Ash1 binding site is always in a nucleosome, except when activated, as proposed? If Ash1 binds at HO for only ~10 minutes of the cell cycle and only in daughter cells, then a transient NDR where Ash1 binds might be expected in only perhaps 5% of cells in a log-phase population, which would be very difficult to detect with any certainty in MNase-seq data.2. An associated point is that the authors represent the nucleosomes as if they cover only 76 bp. This is often done to make the nucleosome map easier to interpret. However, it is a mis-representation of the data since the nucleosome covers 147 bp.3. Page 9: "An rpd3 null single mutant did not change expression of HO in the bulk population, but single-cell analysis demonstrated that HO was expressed in approximately 50% of the daughter cells." Could this be explained if the cells are producing the same amount of HO mRNA but, in rpd3-null cells, it is not being correctly partitioned between mother and daughter?4. The ChIP-seq data for Ash1, Tup1 and Rpd3 appear to be of excellent quality . The authors should show the ChIP-seq data for the HO gene.5. The ChIP data as a function of the cell cycle show that the Swi5 activator binds first, then the Ash1 repressor (recruiting Tup1) and then HO transcription begins . Presumably, Ash1 is only binding and repressing in the daughter cells, while mother cells continue with activation. I think this could be stated more clearly in the Discussion.Minor points:6. The ChIP-seq data have been submitted to GEO but can't be accessed by the reviewers. I think that appropriate biological replicate experiments have been done, but I can't confirm that this is the case.7. Figure 6: It is unclear what is plotted in the heat maps (define nucleosome density).**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the No: Data for qPCR analysis of standard ChIP experiments is missingReviewer #1: Reviewer #2: Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  23 Dec 2020PGENETICS-D-20-01462R1  HO  promoter Requires Activator-Dependent Nucleosome Eviction Ash1 and Tup1 Dependent Repression of the Saccharomyces cerevisiae Dear Dr Stillman,  HO  promoter Requires Activator-Dependent Nucleosome Eviction" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.We are pleased to inform you that your manuscript entitled "Ash1 and Tup1 Dependent Repression of the Saccharomyces cerevisiae The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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Satellite DNAs are tandemly repeated sequences preferentially assembled into large arrays within constitutive heterochromatin and their transcription is often activated by stress conditions, particularly by heat stress. Bioinformatic analyses of sequenced genomes however reveal single repeats or short arrays of satellite DNAs dispersed in the vicinity of genes within euchromatin. Here, we analyze transcription of a major human alpha satellite DNA upon heat stress and follow the dynamics of “silent” H3K9me3 and “active” H3K4me2/3 histone marks at dispersed euchromatic and tandemly arranged heterochromatic alpha repeats. The results show H3K9me3 enrichment at alpha repeats upon heat stress, which correlates with the dynamics of alpha satellite DNA transcription activation, while no change in H3K4me2/3 level is detected. Spreading of H3K9me3 up to 1–2 kb from the insertion sites of the euchromatic alpha repeats is detected, revealing the alpha repeats as modulators of local chromatin structure. In addition, expression of genes containing alpha repeats within introns as well as of genes closest to the intergenic alpha repeats is downregulated upon heat stress. Further studies are necessary to reveal the possible contribution of H3K9me3 enriched alpha repeats, in particular those located within introns, to the silencing of their associated genes.  Drosophila, under standard conditions, transcription factor dATF-2, which regulates expression of stress response genes, recruits heterochromatin protein 1 (HP1) to pericentromeric heterochromatin, while under heat stress, activated MAP kinases such as p38 phosphorylate dATF-2 which is released from heterochromatin, leading to abolishment of HP1 and disruption of heterochromatin [Tribolium castaneum revealed heat-stress induced destabilization of heterochromatin coupled with overexpression of a major heterochromatic satellite DNA TCAST1, followed by processing of satellite transcripts into siRNAs [T. castaneum, heat-stress induced transcription of TCAST1 satellite DNA correlates with an increased level of repressive heterochromatin marks H3K9me2/3 not only on satellite repeats in constitutive heterochromatin but also on dispersed TCAST1 satellite elements and their proximal regions within euchromatin, affecting expression of nearby genes [Satellite DNAs are tandemly repeated sequences assembled within constitutive heterochromatin at the (peri)centromeric and subtelomeric regions . Howeverhromatin . In vivoo siRNAs . Satellio siRNAs ,16,17. Iby genes . Alpha satellite DNA comprises up to 10% of the human genome and is located in the centromeric and pericentromeric regions of all chromosomes . The funIn this paper, using different human cell lines as a model system, we analyze transcription of alpha satellite DNA upon heat stress. In addition, we followed epigenetic changes of histones, in particular the “silent” H3K9me3 and “active” H3K4me2/3 marks at alpha repeats dispersed within euchromatin, in introns and at intergenic regions as well as tandemly arranged alpha satellite which are characteristic for heterochromatin. The results reveal an increase of H3K9me3 level at alpha repeats and spreading of H3K9me3 to the flanking regions up to 1-2 kb from the insertion sites of the euchromatic repeats. The dynamics of H3K9me3 enrichment at alpha repeats correlates with the dynamics of alpha satellite DNA transcription activation upon heat stress, being the most prominent immediately after heat stress. No change in the level of H3K4me2/3 is detected either at heterochromatic or euchromatic alpha satellite repeats. We also followed the dynamics of expression of genes containing alpha repeats within introns as well as of the genes closest to the intergenic alpha repeats, upon heat stress. Expression of the genes is downregulated, being negatively correlated with H3K9me3 levels at the associated alpha repeats. Further studies are necessary in order to reveal the possible contributions of H3K9me3 enriched alpha repeats, in particular those located within introns, to the silencing of their associated genes.Alpha satellite repeats annotated in the human genome assembly GRCh17.hg19 were extracted from the rmsk table in the UCSC Table Browser. Organization of repetitive sequences identified by RepeatMasker at regions near dispersed alpha repeats was analyzed by UCSC Genome Browser.2 at 37 °C. For heat shock experiments cells were incubated for 1 or 2 h at 42 °C in complete medium, followed by recovery at 37 °C. Chaetocin from Abcam was used as a specific inhibitor of the human histone methyltransferase Suv39H1.The following human cell lines were used in the experiments: MJ90 hTERT , 697 (human Pre-B leukemia), MCF-7 (human breast adenocarcinoma), HT29 (colon cancer adenocarcinoma), Cal 27 , HeLa (human cervix carcinoma), Hep2 (human cervix carcinoma HeLa derivative), SW620 (colon adenocarcinoma), SW48 , DLD1 , HCT116 (colon carcinoma), ZR75 (breast carcinoma), HepG2 (liver hepatocellular carcinoma), A431 (squamous carcinoma), 293T (human embryonic kidney), and OV-90 (ovarian adenocarcinoma). Cells were cultured in appropriate medium (DMEM or RPMI1640) supplemented with 10% FBS and 5% COGenomic DNA was prepared using the “GenElute Mammalian Genomic DNA Miniprep Kit” Sigma-Life Science. PCR reactions were performed using 2× DreamTaq Green PCR Master Mix (Fermentas) in a final reaction volume of 20 μL containing 0.2 μM of each primer and 50 ng of genomic DNA. Primers used in the analysis of insertion polymorphism of dispersed alpha satellite repeats were designed in a unique sequence around the insert using Primer3Plus software. Primers are listed in For RNA isolation from cell cultures lysis buffer was added directly after the PBS washing step avoiding trypsin treatment. RNA was additionally digested with Turbo DNase (Ambion) and quantified with the Quant-IT RNA assay kit using a Qubit fluorometer (Invitrogen). Integrity of RNA was checked by gel electrophoresis and the presence of DNA contamination was checked by PCR. Approximately 1 μg of RNA was reverse transcribed using the PrimeScript RT reagent Kit with gDNA Eraser  in 10 μL reaction using random and oligo dT primers mix. For all samples negative controls without reverse transcriptase were used. Glucuronidase beta (GUSB) [GUSB gene (Gene ID: 2990) was stably expressed without any variation among samples after heat stress. The following thermal cycling conditions were used: 50 °C 2 min, 95 °C 7 min, 95 °C 15 s, 60 °C 1 min for 40 cycles followed by dissociation stage: 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s. Amplification specificity was confirmed by dissociation curve analysis and specificity of amplified products was tested on agarose gel. Control without template (NTC) was included in each run. Post-run data were analyzed using LinRegPCR software v.11.1. [qPCR analysis was performed according to the previously published protocol . Primersa (GUSB)  was used v.11.1. ,25 whichMJ90 hTERT, Cal 27, HepG2, and OV-90 cells were grown to subconfluence, washed in PBS, scraped in nuclear isolation buffer  with protease inhibitor cocktail CompleteMini (Roche) and chromatin immunoprecipitation was performed according to the published protocol . The antAlpha satellite repeats (ARs) annotated in the human genome were downloaded using the UCSC table browser. The human reference genome GRCh38.hg38 was also searched with the alignment program BLASTN version 2.3.1+ using as the query a dimer of human alphoid 171 bp repeat consensus sequence derived from 293 cloned monomers of wide-ranging chromosomal origins . The anaAlu, L1, or LTR and for some alpha repeats flanking repetitive sequence prevented construction of single locus-specific primers. Analysis of insertion polymorphism was finally performed for all 10 alpha satellite elements located within introns as well as for 10 elements in intergenic regions (zinc finger protein 675 gene (ZNF675) and element No 10 located within intron of nuclear receptor subfamily 3 group C member 1 gene (NR3C1)  . Element (NR3C1) . Alpha e (NR3C1) , while ip < 0.05) relative to the controls (no HS) immediately after 2 h of HS. The level of alpha RNA is decreased during the recovery period of 30 min at 37 °C, while after 1 h of recovery it drops to the level of control (To investigate if heat stress (HS) affects transcription of alpha satellite DNA we followed its transcription dynamics in human cell lines by RT-qPCR under standard and heat stress conditions. Primers used for transcription analysis were constructed based on consensus sequence derived from cloned alpha satellite monomers of wide-ranging chromosomal origins  and were control . T. castaneum [t-test. In addition, we followed the dynamics of binding of IgG to dispersed alpha satellite repeats and tandemly repeated satellite arrays and the amount of bound IgG was very low resulting in a signal below the qPCR threshold. The enrichment of repressive histone marks at dispersed repeats of a major satellite DNA and their neighboring regions after HS was previously demonstrated in the beetle astaneum . Here wep = 0.011) increase of H3K9me3 was detected immediately after HS while no significant change in H3K9me3 level was detected at regions located 1.8 kb 5′, 1.9 kb 3′, and 6 kb 5′ to AR 10  immediately after HS, while at more distant regions positioned 1.8 kb 5′ and 1.9 kb 3′ to AR a statistically insignificant increase of H3K9me3 level was observed as well as no change at 6 kb distance , 19 (2.4 monomers), and 29 (0.6 monomer) in MJ90hTERT cells using specific primers. The primers used amplified regions encompassing ARs as well as several regions, approximately 200 bp long, positioned 0.5–6 kb from the corresponding AR  immediately after HS while at 30 min of recovery the level decreases and drops to the level of the control at 1h of recovery (p < 0.05) after HS on all ARs while no significant change was observed at the regions 2–6 kb distant to ARs which served as negative controls  were expressed at a very low level while the seven remaining genes were suitable for expression study: NR3C1, ZNF675, VAV1, SLC30A6, PRIM, STAM, and MYO1E. Finally, we also tested expression of 14 genes closest to the intergenic alpha repeats, which are up to 50 kb away from the intergenic ARs . Among genes associated with 20 dispersed alpha satellite repeats previously tested for insertion polymorphism , we seleenic ARs , and eigZNF675 and NR3C1 genes. The dynamics of expression of the ZNF675 gene in MJ90 hTERT fibroblasts and in HepG2 cells is characterized by a downregulation of 1.7× relative to the control immediately after 2 h of HS in both cell lines, while in Cal 27 and OV-90 cell lines where ZNF675 is not associated with an alpha satellite repeat, the expression was not affected by HS (NR3C1 expression relative to the control was observed immediately after 2 h of HS in MJ90 hTERT (3.5×), Cal 27 (3.7×), and OV-90 (2.7×) cells and is followed by an increase in expression after recovery periods of 30 min and 1 h, respectively .The expression analysis of genes ell line D. The exw levels . HS was w levels . The levDrosophila melanogaster and of its human ortholog Suv39H1 [To study the possible role of silent histone modification H3K9me3 on suppression of alpha repeat-associated genes we used chaetocin, a specific inhibitor of the histone methyltransferase SU(VAR)3-9 of  Suv39H1 . Suv39H1p < 0.05). No significant change in the H3K9me3 level was detected at regions 2–6 kb distant to ARs (p > 0.05). In the same way we tested the effect of chaetocin on H3K9me3 level at ARs 10 and 29 in Cal 27, OV-90, and HepG2 cells, respectively, where these ARs exhibit insertion polymorphism. In the absence of the corresponding ARs  no change of H3K9me3 level was observed  and eight genes associated with intergenic ARs  was measured by qRT-PCR after HS in the presence and in the absence of chaetocin  adjacent to intronic ARs and to most of intergenic ARs prevented elimination of alpha repeats using CRISPR/Cas9 and creation of modified cell lines suitable for further studies. For intergenic alpha repeats we assumed their association with the closest 5′ and 3′ positioned genes, however because of the 3-dimensional structure of the genome it might be possible that some intergenic repeats are associated with more distant genes and this can be assigned only based on high resolution chromatin interaction maps. The number of genes associated with dispersed alpha repeats is low (46) and gene ontology analysis revealed no significantly enriched pathways or molecular functions within this gene set. It is however interesting that some of the alpha repeat-associated genes are stress or disease-related: NR3C1 codes for the glucocorticoid receptor which upon binding of stress hormones participates in gene expression regulation [HTRA3 shows drastically downregulated expression in some cancers and it is considered to be a tumor suppressor gene [VAV1 and PHF20L1 have oncogenic potential [ZNF675 gene codes for a KRAB zinc-finger protein which specifically targets a subfamily of LTR retrotransposons and is responsible for their silencing [Considering the influence of dispersed, euchromatic alpha repeats on the expression of neighboring genes, the observed downregulation of all ARs-associated genes upon heat stress as well as differences in the expression of two genes associated with polymorphic alpha satellite repeats suggest a possible contribution of H3K9me3 enriched alpha repeats in gene silencing. It is known that the genome-wide transcriptional response to heat shock in mammals is modulated at promoter-proximal pause, either by increasing or reducing RNA polymerase release, resulting in up or downregulation of many genes . We can gulation , HTRA3 ssor gene ,46 whileotential ,48. In ailencing . Alu repetitive elements which is responsible for human genetic diseases including cancer [The dispersion of satellite DNA repeats throughout the genome is most probably a consequence of the molecular mechanisms of satellite DNA evolution which still need deeper investigation to be fully elucidated ,50,51. Wg cancer ,54. In ag cancer  or as a g cancer . A relevg cancer ,57,58, b


Thus, we have here identified that Nfe2l1Nrf1 and α-PalNRF1 exert synergistic and antagonistic roles in integrative regulation of the nuclear-to-mitochondrial respiratory and antioxidant transcription profiles. In mouse embryonic fibroblasts (MEFs), knockout of −/−Nfe2l1 leads to substantial decreases in expression levels of α-PalNRF1 and Nfe2l2, together with TFAM  and other target genes. Similar inhibitory results were determined in −/−Nfe2l2 MEFs but with an exception that both GSTa1 and Aldh1a1 were distinguishably upregulated in −/−Nfe2l1 MEFs. Such synergistic contributions of Nfe2l1 and Nfe2l2 to the positive regulation of α-PalNRF1 and TFAM were validated in −/−Keap1 MEFs. However, human α-PalNRF1 expression was unaltered by −/−hNfe2l1α, -/-ΔTAhNfe2l2, or even −/−+siNrf2hNfe2l1α, albeit TFAM was activated by Nfe2l1 but inhibited by Nfe2l2; such an antagonism occurred in HepG2 cells. Conversely, almost all of mouse Nfe2l1, Nfe2l2, and cotarget genes were downexpressed in NRF1+/-α-Pal MEFs. On the contrary, upregulation of human Nfe2l1, Nfe2l2, and relevant reporter genes took place after silencing of α-PalNRF1, but their downregulation occurred upon ectopic expression of α-PalNRF1. Furtherly, Pitx2 (pituitary homeobox 2) was also identified as a direct upstream regulator of Nfe2l1 and TFAM, besides α-PalNRF1. Overall, these across-talks amongst Nfe2l1, Nfe2l2, and α-PalNRF1, along with Pitx2, are integrated from the endoplasmic reticulum towards the nuclear-to-mitochondrial communication for targeting TFAM, in order to finely tune the robust balance of distinct cellular oxidative respiratory and antioxidant gene transcription networks, albeit they differ between the mouse and the human. In addition, it is of crucial importance to note that, in view of such mutual interregulation of these transcription factors, much cautions should be severely taken for us to interpret those relevant experimental results obtained from knockout of Nfe2l1, Nfe2l2, α-Pal or Pitx2, or their gain-of-functional mutants.There is hitherto no literature available for explaining two distinct, but confused, Nrf1 transcription factors, because they shared the same abbreviations from nuclear factor erythroid 2-related factor 1  and nuclear respiratory factor (originally designated  In all life forms, distinct types of cells are the most basic unit of their biological structures and functions, due to a fact that they are bounded by the membrane lipid bilayers and hence protected from extracellular oxidizing environments to which they have adapted. Of note, eukaryotic cells are evolutionarily compartmentalized by endomembrane systems, as a unique trait that distinguishes from prokaryotic cells , insofarα-proteobacterium, which was firstly surmised to be engulfed by a primordial anaerobic eukaryote  that is essential for the transcription of eIF2α gene [α-PalNRF1) and NRF-2  binding to distinct consensus sites within these nuclear-encoding respiratory gene promoters [Of particular concern is the mitochondrial origin from aerobic pothesis ) or origpothesis , 7). Thepothesis , 5. In tpothesis , 9. Suchpothesis , 11. Howpothesis , e.g., mpothesis –17. Tranpothesis –20. HereF2α gene –23, whicF2α gene , 25. Notromoters , 27.2+ storage and the disulfide relay system required for redox signaling; (ii) the cooperative contributions of the ER and mitochondria to eukaryotic lipid synthesis, as opposed to occurring at the plasma membrane as in all prokaryotes; (iii) the distinction in lipid compositions of between the endomembrane system and plasma membrane, which originated from the fact that bacterial-like lipids replaced the archaeal lipids from the inside in the first place; and that (iv) the ER-continuous nuclear envelope is not homologous to the plasma membrane of putative archaeal host, which is just due to the formation of newly enveloped nucleus from the ER in eukaryotic cells with open mitosis [Apart from mitochondria, the eukaryotic endomembrane system including the ER and nucleus has been posited to originate from the bacterial-like out membrane vesicles (OMVs) released by the endosymbiont mitochondrial ancestor within the cytosol of its archaeal host at eukaryote origin , 6. The  mitosis . These p mitosis , such th mitosis , 30. Howper se proteins [α-PalNRF1 and GABPNRF2 [α-PalNRF1-targeted eIF2α [Aside from only 13 mitochondrially encoded proteins , all othproteins , 28. Of GABPNRF2 , 27 and ed eIF2α –23. Parted eIF2α , 32, befed eIF2α –35. The ed eIF2α , in whiced eIF2α . Howeverα-PalNRF1), leading to the confused interpretation of relevant works, of which the most typical was published by L'Honore et al.  is significantly distinctive from α-PalNRF1, because the former Nfe2l1Nrf1 belongs to the family of cap'n'collar (CNC) basic region-lucine zipper (bZIP) transcription factors that regulate distinct subsets of antioxidant response element -driven cytoprotective genes [Nrf2 that is negatively regulated by Kelch-like ECH-associated protein 1 (Keap1), which is an adaptor subunit of Cullin 3-based E3 ubiquitin ligase and also a key sensor for oxidative and electrophilic stresses [Nrf1 and α-PalNRF1 exert synergistic and antagonistic roles in integral regulation of the nuclear to mitochondrial respiratory and antioxidant transcription profiles. Such synergistic contributions of Nfe2l1 and Nfe2l2 to positive regulation of α-PalNRF1 and TFAM were validated in −/−Keap1 mouse embryonic fibroblasts (MEFs). By contrast, human α-PalNRF1 expression was almost unaltered by −/−hNfe2l1α or -/-ΔTAhNfe2l2, albeit its target TFAM was activated by Nfe2l1, along with antagonism against Nfe2l2 in HepG2 cells. Conversely, almost all of mouse Nfe2l1, Nfe2l2, and cotarget genes were downexpressed to certain extents in NRF1+/-α-Pal MEFs. Further investigation revealed that human Nfe2l1, Nfe2l2, and relevant reporter genes were downregulated by an ectopic α-PalNRF1 expression, but this inhibitory effect was reversed by silencing α-PalNRF1. Moreover, Pitx2 (pituitary homeobox 2) was identified as a direct upstream regulator of Nfe2l1 and TFAM, besides α-PalNRF1. Collectively, these across-talks between Nfe2l1, Nfe2l2, and α-PalNRF1, together with Pitx2, are integrated through multiple extranuclear  signaling to the nuclear-to-mitochondrial communication for their mitochondrial target TFAM, so as to finely tune distinct cellular oxidative respiratory and antioxidant gene transcription networks responsible for maintaining the robust redox homeostasis and organelle integrity, albeit they differ between the mouse and human.Amongst the putative ER-derived redox signaling machineries, the most critical antioxidant arm is mediated by the membrane-bound nuclear factor erythroid-2 p45-related factor 1  . The abbtal Cell ). As a mve genes . This costresses . HoweverNfe2l1 has an effect on the constitutive expression of Nfe2l2 and target genes in MEFs was firstly examined by real-time quantitative PCR (RT-qPCR) and Western blotting. As anticipated, loss of Nfe2l1 in mice resulted in substantial decreases in both mRNA and protein levels of Nfe2l2 expressed constitutively in −/−Nfe2l1 MEFs , GCLM (glutamate-cysteine ligase modifier subunit), MT-1 , GSTp (glutathione S-transferase pi 1), and SOD1 (superoxide dismutase 1) were determined in −/−Nfe2l1 MEFs ) and Aldh1a1  were also obtained from −/−Nfe2l1 in MEFs , Ndufv1 (NADH:ubiquinone oxidoreductase core subunit V1), and Ndufb6 (NADH:ubiquinone oxidoreductase subunit B6), besides SOD1  is required for expression of α-PalNRF1 and its downstream targets, including TFAM  cells. The latter notion is also corroborated by evidence showing significant decreases in the constitutive protein expression of HO-1, GCLM, SOD1, and Aldh1a1  are also likely subjected to putative dominant negative regulation by, at least, a not-yet-identified transcription factor competitively against the positive effects of Nfe2l2 and Nfe2l1 in −/−Keap1 MEFs.Knockout of the Nfe2l2-inhibitor Keap1 caused significant increases in protein abundances of Nfe2l2 and its downstream targets HO-1, GCLM, Aldh1a1, and SOD1 in −/− MEFs , as cons−/− MEFs , albeit s  were only slightly promoted, whilst the expression of COX5a was roughly unaltered, in −/−Keap1 MEFs . This cell line was further confirmed to be true by its DNA sequencing and RT-qPCR  to constructive expression of Nfe2l1, Nfe2l2, and cognate ARE-driven target genes  MEFs , but rather significantly upregulated in -/-ΔTAhNfe2l2 cells, by comparison with their wild-type controls , when cobserved . However Figures . These o Figures . Collect−/−hNfe2l1α caused a marked increase in human Nfe2l2 protein abundances but not in its mRNA expression . RT-qPCR analysis unraveled that the basal mRNA expression of HO-1 was substantially upregulated in −/−hNfe2l1α cells but also significantly downregulated in -/-ΔTAhNfe2l2 cells  was almost completely abolished in −/−hNfe2l1α or −/−+siNfe2l2hNfe2l1α cells but also enormously augmented in -/-ΔTAhNfe2l2 cells . However, similar increases of α-PalNRF1, TFAM, and SOD1 were not observed in −/−hNfe2l1α cells, although hyperactive Nfe2l2 was further incremented by tBHQ (cf. E2 with E5-E7). Subsequently, RT-qPCR analysis revealed that mRNA expression levels of NRF1α-Pal, TFAM, COX5a, and SOD1 were induced, to greater or less extents . Similarly, activity of hNfe2l1-luc reporter was substantially activated by tBHQ (b2) but significantly suppressed by ectopic expression of α-PalNRF1-V5 (c1 and c3). Such overexpression of ectopic α-PalNRF1-V5 also led to similar transcriptional repression of GSTa2-ARE×6-luc reporter (c2 and c3). These data indicate a possible contribution of α-PalNRF1 to transrepression of Nfe2l1-luc and ARE×6-luc reporter genes.To clarify putative contributions of reporter , albeit reporter , b1. Sim by tBHQ , b2 but α-PalNRF1-expressing cells revealed that its targets TFAM and SOD1 were evidently enhanced, whilst abundances of Nfe2l1 and Nfe2l2 proteins were markedly diminished by ectopic α-PalNRF1-V5 (d1-d5). Accordingly, HO-1 and GCLM abundances were also partially inhibited by α-PalNRF1-V5 (d6 and d7). Conversely, knockdown of α-PalNRF1 by its specific siRNA . By contrast, both mRNA and protein levels of SOD1 were modestly promoted by silencing of α-PalNRF  were coincidently accompanied by marginal enhancements of their mRNA expression levels, particularly following transfection of HepG2 cells with 50 nM siRNA against α-PalNRF1 . Moreover, α-PalNRF1 also exerts disparate effects on GCLM, possibly depending on whether Nfe2l1, Nfe2l2, and other unidentified transcription factors were also stimulated by α-PalNRF1 overexpression or its RNA-silencing.Further examinations revealed that modestly increased proteins of Nfe2l1, Nfe2l1, and HO-1 , M4–6 we Figures . In addi-PalNRF1  and M7. and HO-1 . Howeverα-PalNRF1 was identified as a direct target of Pitx2/3  [Nfe2l1. As shown in mNfe2l1-luc reporter was significantly transactivated by the ectopic Pitx2 expression, when compared with the basal activity measured from its cotransfection with an empty pGL3-Basic vector. Similar results were also obtained from those hNfe2l1-luc reporters . The hNfe2l1-luc activity was also strikingly induced by tBHQ to the extent of hNfe2l1-luc1 transactivation mediated by Pitx2 (cf. C1 with C3). In addition, four distinct consensus Pitx-response elements (PitxREs), which are situated at the upstream of the indicated promoter region to have yielded hNfe2l1-luc1 .Clearly, ain 2/3) . Here, w Figures . Of noteeporters , C2-C4. 2l1-luc1 , were alby Pitx2 . TogetheNfe2l1 and HO-1 was increased by Pitx2 overexpression . These demonstrate that Pitx2 may also serve as an upstream regulator to mediate transcriptional expression of Nfe2l1 (and Nfe2l2), besides α-PalNRF1. This notion is further supported by the evidence obtained from silencing of Pitx2 by its three distinct siRNA sequences Nfe2l1, NRF1α-Pal, and their target genes , two similar α-helical structures were wheeled, respectively, by the successive mitochondria-targeting sequences (MTS1 and MTS2) in each nuclearly encoded TFAM. Alignment of three amino acid sequences from human and mouse showed that MTS1 (aa 1-18) and MTS2 (aa 21-38) are located within the N-terminal region of this MHG- (high mobility group-) box-binding transcription factor TFAM (lower panel). Further bioinformatic analysis revealed several putative consensus cis-regulatory elements, such as AREs, PitxRE, and α-Pal-binding site within the promoter region of human TFAM , albeitll lines .ATP5B (ATP synthase F1 subunit β), COX4I1 (cytochrome c oxidase subunit 4I1), OXA1L (oxidase cytochrome c assembly 1-like), CCND1, or RPA1 (C10orf2 (twinkle mtDNA helicase), CS (citrate synthase), CYCS , POLG2 (DNA polymerase γ2), RRM2B (ribonucleotide reductase subunit M2B), SSBP1 (single stranded DNA binding protein 1), eIF2S1 (eIF2α), eIF2S2 (eIF2β), eIF5, GABPβ1, GABPβ2, MEF2A (myocyte enhancer factor 2A), PPP1R15B, RUNX1, SIRT1, SUPT16H, UCP2, and YAF2 (YY1-associated factor 2). Reversely, changes in these gene expression levels were also determined by RNA-sequencing of −/−Nfe2l1α, −/−+siNfe2l2Nfe2l1α, and -/-ΔTANfe2l2 cell lines . Those differentially expressed genes (DEGs) as key players in this network were further analyzed by RNA-sequencing of Nfe2l1α-induced cells Nfe2l2-induced cells (n–p), when compared with their WT controls.An overview of multilayer interaction networks with the aforementioned key molecular nodes is illustrated in Figures BRIX1 (for biogenesis of ribosomes), eIF2α, GNL3 (G protein nucleolar 3), MKI67IP (a nucleolar protein interacting with the FHA domain of MKI67), RPF1 (ribosome production factor 1), and RPF2 were promoted by Nfe2l1, but as accompanied by downexpression of 7 genes NRF1α-Pal, COX4I1, CTNNB1 (catenin-β1), NIP7 (a nucleolar pre-rRNA processing protein), RRS1 (ribosome biogenesis regulator 1), SPC24 (a component of NDC80 kinetochore complex), and ZWINT (ZW10 interacting kinetochore protein). Further analysis revealed that Nfe2l2 contributed to 12 of DEGs, besides HMOX1 , DDIT3 (Chop), Jun, MafG, and SSBP1 were activated by Nfe2l2, while AKR7A2 , CS, LEF1, RPA1, SUPT16H, and UCP2 (uncoupling protein 2) were depressed by this CNC-bZIP factor.The result validated that HO-1 (encoded by HMOX1) is a direct cotarget of Nfe2l1 and Nfe2l1, because similar increased mRNA expression levels were attributed to induction of either CNC-bZIP factor Figures . Particu Figures . Amongstα/β and PRC1 are accepted as key transcription cofactors involved in the nuclear control of mitochondrial biogenesis and function, here we established an interaction network, with distinct families of 21 transcriptional factors and 6 cofactors, by using the STRING tool  and NR2C2, but as companied by its another negative relationship with PGC-1β, PPARγ, RARA (retinoic acid receptor α), and STAT6 , MLXIPL (MLX interacting protein like), RARA, and SREBF1 (sterol regulatory element binding protein 1) was also accompanied by its another negative relationship with CREB1, CREBBP , YY1, P300, PGC-1β, PPARα, PPARγ, FOXO1, HNF4A, MEF2C, NR2C1, NR2C2, and THRB (thyroid hormone receptor β). Together, these indicate that PGC-1α and STAT6 are Nfe2l1α-specific, while 9 of Nfe2l2-specific genes are ESRRA, MLXIPL, SREBF1, PPARA, CBP, FOXO1, HNF4A, MEF2C, and THRB. Rather, Nfe2l1α and Nfe2l2 have an overlapping effect to downregulate PGC-1β and PPARγ, apart from their opposite effects of both CNC-bZIP factors to bi-directionally regulate CREB1, YY1, EP300, NR2C1, NR2C2 and RARA.Since PGC-1ING tool . Differecontrols  and alsocontrols . FurtherP factor . There end STAT6 . In the ll lines . Among tNrf1 and α-PalNRF1, Figures Nrf2 and GABPαNRF2, respectively. Of note, the antioxidant master Nfe2l2Nrf2 is highly conserved with Nfe2l1Nrf1, but neither have evolutionary conservation with those nuclear respiratory factors α-PalNRF1 and GABPαNRF2, of which the latter two factors have also no any homology with each other  in −/−:Nfe2l2−/−Nfe2l1 MEFs, when compared with their individual knockout of −/−Nfe2l1 or −/−Nfe2l2. Hence, it is inferred that Nrf2 is also allowed for a partial compensation for the loss of Nrf1's function in synergistically regulating critical genes for the intracellular redox homeostatic setting at the robust threshold state during embryogenesis. Such partially overlapping functions of both Nfe2l1 and Nfe2l2 are determined by coexpression patterns of the two CNC-bZIP factors [ARE-containing gene promoters , although this was accompanied by an increased Nfe2l1β. Collectively, these results demonstrate that Nfe2l1 acts as a dominant positive regulator to monitor the basal constitutive expression of Nfe2l2, at least in MEFs  with either unctions , 49, 62.−/−hNfe2l1 or −/−hNfe2l2 on mRNA and protein levels of human α-PalNRF1 in this experimental setting, albeit both CNC-ZIP genes contain at least one consensus α-Pal binding site within their promoters  beyond α-PalNRF1. In this study, we also found that TFAM was downregulated in −/−hNfe2l1 cells  and hence upregulated in −/−hNfe2l2 cells. Our further evidence reveals that TFAM along with α-PalNRF1 is significantly induced by the redox inducer tBHQ, but such inducible expression levels of both factors are almost completely abolished by −/−hNfe2l1. Collectively, these indicate that Nfe2l1 and Nfe2l2 contribute, respectively, to the putative positive and negative regulation of TFAM, even though both CNC-ZIP factors are inhibited by α-PalNRF1 through an as-yet-unidentified mechanism  signaling to the nuclear-to-mitochondrial controls of distinct cellular respiratory and antioxidant transcription networks, albeit they differ between the mouse and human . Furtheevidence , 55, demn Figure B.α-PalNRF1 to certain extents in so much as that the intracellular redox homeostasis is maintained at a robust steady-state. Altogether, interaction between these transcription factors comprises a complex feedback circuit with hierarchical regulatory networks served to maintain robust redox homeostasis by balancing a distinct cellular oxidative respiratory system and an antioxidant cytoprotective response.However, the nucleus-controlled mitochondrial respiratory and oxidative phosphorylation are also a primary source of endogenous ROS byproducts in eukaryotic cells. This facilitates to trigger physiological activation of Nfe2l1, Nfe2l2, and Aldh1a1 and GSTa1). But, the inter-regulatory synergism of Nfe2l1 and Nfe2l2 confers both factors to exert their overlapping roles for cotarget genes  in MEFs. By sharp contrast, apparent synergistic and antagonistic relationships of between human Nfe2l1 and Nfe2l2 are also demonstrated. Nfe2l1 is a dominant repressor, whilst Nfe2l2 is thus negatively regulated by Nfe2l1 in HepG2 cells. Such function of Nfe2l1 is considered as a brake to avoid Nfe2l2 overshoot. For this simplification to be valid, there should always be a correlation between Nfe2l1 and Nfe2l2 so that both CNC-bZIP factors along with cognate target genes are finely tuned in order to meet the changing needs of cells under all conditions. In fact, Nfe2l1 and Nfe2l2 cannot be reduced as a simple module factor alone logical responses at different levels are determined by a range of regulatory mechanisms from various signaling pathways toward multiple transcription factor-mediated gene networks. Such being the case, these transcription factors and other regulatory molecules could be simplified as key modules which cross-talk amongst them, in order to form a hierarchical network. Nfe2l1 and Nfe2l2 are embedded in such a complex molecular interaction network, responsible for antioxidant, detoxification, and cytoprotective adaptation to distinct physio-pathological stresses during life process. Of note, Nfe2l1 is endowed with unique indispensable biological functions. This fact confers Nfe2l1 to be distinguished from Nfe2l2 at regulating distinct subsets of ARE-driven cognate genes  was from Sangon Biotech . Specific antibodies against Nfe2l1 were made in our own laboratory [α-PalNRF1 or V5 ectope were from Abcam and Invitrogen, respectively. Besides, both β-actin and secondary antibodies were from ZSGB-BIO . Of note, the detailed information about other antibodies, as well as all other key reagents and resources used in this study, was all shown in Table All chemicals were of the highest quality commercially available. The boratory , whilst α-palNRF1, were here made by cloning each of those full-length cDNA sequences into the pcDNA3.1 vector. Particularly, the CRISPR/Cas9 plasmid constructs containing guide RNAs specifically targeting mouse α-palNRF1 were created to generate a heterogeneous  knockout cell line from MEFs. Such sgRNAs  was amplified by PCR from its genomic loci and then inserted into the pGL3-Basic vector. Another group of the cis-regulatory consensus, e.g., ARE and PitxRE- (Pitx2 response elements-) adjoining sequences were cloned from the indicated gene promoter regions of, such as Nfe2l1, TFAM, and NRF1α-pal and then inserted into the pGL3-Promoter vector. In addition to those intact reporter genes, such as PitxRE-Luc and ARE-Luc, their relevant point-mutant reporters were also engineered herein. The fidelity of the above-described constructs was all confirmed to be true by sequencing. All these primers in the above gene manufacture, and other oligos for siRNA-mediated knockdown of the indicated genes, were listed in Table Furthermore, several specific −/−Nfe2l1, −/−Nfe2l2, and −/−Keap1, were also obtained from the groups of Profs. Kobayashi and Hayes. Of note, all of these cell lines were originally prepared from Prof. Yamamoto's laboratory. But, another heterogeneous knockout line of NRF1+/-α-pal in the mouse was established by its gene-editing in our own laboratory as described above. Besides, the human wild-type  hepatocellular carcinoma (HepG2) cells were originally from the American Type Culture Collection . The fidelity was conformed to be true by its authentication profiling and STR (short tandem repeat) typing map  [−/−hNfe2l1α and -/-ΔTAhNfe2l2 were previously established and characterized in our own laboratory [v/v) fetal bovine serum (FBS), and 100 units/mL penicillin-streptomycin, in the 37°C incubator with 5% CO2. Subsequently, the indicated cell lines were subjected to transfection for 8 h, which was performed by using Lipofectamine 3000  containing different combinations of indicated plasmids, and then allowed for 24 h recovery from transfection in a fresh medium before relevant experimentation. In addition, two Nfe2l1α-inducible and Nfe2l2-inducible cell lines, along with their control cells, were described by Wang et al. [Wild-type mouse embryonic fibroblasts (MEFs) were given as a gift from Akira Kobayashi. Their relevant knockout MEF lines, e.g., o., Ltd) . On thisboratory . These eg et al. .β-actin served here as an optimal internal standard control. All the primers used for qPCR (Table ) were synthesized by Tsingke .Equal amounts of experimental cells were subjected to isolation of total RNAs by using the RNA simple Kit . Then, 500 ng of total RNAs was added in a reverse-transcriptase reaction to generate the first strand of cDNA . The synthesized cDNA fragments were served as the template for qPCR, in the GoTaq®qPCR Master Mix (from Promega), before being deactivated at 95°C for 10 min, and then amplified by 40 reaction cycles of the annealing at 95°C for 15 s and then extending at 60°C for 30 s. The final melting curve was validated to examine the amplification quality, whereas the mRNA expression level of CR Table  were synβ-actin served as an internal control to verify equal loading of proteins in each of electrophoretic wells.Experimental cells were harvested in a lysis buffer , which was supplemented with the protease inhibitor cOmplete Tablets EASYpack. The lysates were then denatured immediately at 100°C for 10 min, sonicated sufficiently, and diluted in 3 × loading buffer  at 100°C for 5 min. Thereafter, equal amounts of protein extracts were subjected to separation by SDS-PAGE containing 5–12% polyacrylamide and subsequent visualization by immunoblotting with distinct primary antibodies as indicated Table . On some5) of indicated experimental cells were plated into 12-well plates. When allowed for growth to the density reaching 70–80% cell confluence, they were then transfected with Lipofectamine 3000 reagent (Invitrogen). For distinct lengths of the gene promoter studies, the pGL3-Basic vector containing 5 kb (−2248 to +2777), 2.7 kb (−2248 to +483), or 3 kb (−259 to +2777) of human Nfe2l1 promoter, or 3.2 kb (−2047 to +1210) of mouse Nfe2l1 promoter, or an empty pGL3-Basic vector as a blank control was cotransfected for 8 h with an indicated expression construct or an empty pcDNA3.1 as a control, plus another internal control pRL-TK reporter. Subsequently, the cells were allowed for a recovery from transfection in a fresh complete medium for 24 h, before they were lysed in a passive lysis buffer  for dual-luciferase assays. About 20 μL of the supernatant of cells was assayed for the luciferase activity. Both firefly and renilla luciferase activities in each of the same samples were measured by the dual-luciferase reporter assay system (Promega). For the short consensus sequences of the indicated transcription factor-binding sites, the specific driven luciferase plasmids or corresponding paired point-mutants were cotransfected with the internal control pRL-TK, together with an expression construct for Nrf1, Nrf2, Pitx2, or an empty pcDNA3.1. All the resultant data were normalized and calculated as a fold change (mean ± S.D) relative to the activity of the control group .Equal numbers . All the data presented in this study are shown as a fold change (mean ± S.D), each of which represents at least 3 independent experiments undertaken on separate occasions that were each performed in triplicate. In addition, statistical analysis of RNA-sequencing data was carried out as described by Wang et al. [The statistical significance of changes was determined using the g et al. .