Enhancers in higher eukaryotes and upstream activating sequences (UASs) in yeast have been shown to recruit components of the RNA polymerase II (Pol II) transcription machinery. At least a fraction of Pol II recruited to enhancers in higher eukaryotes initiates transcription and generates enhancer RNA (eRNA). In contrast, UASs in yeast do not recruit transcription factor TFIIH, which is required for transcription initiation. For both yeast and mammalian systems, it was shown that Pol II is transferred from enhancers/UASs to promoters. We propose that there are two modes of Pol II recruitment to enhancers in higher eukaryotes. Pol II complexes that generate eRNAs are recruited via TFIID, similar to mechanisms operating at promoters. This may involve the binding of TFIID to acetylated nucleosomes flanking the enhancer. The resulting eRNA, together with enhancer-bound transcription factors and co-regulators, contributes to the second mode of Pol II recruitment through the formation of a transcription initiation domain. Transient contacts with target genes, governed by proteins and RNA, lead to the transfer of Pol II from enhancers to TFIID-bound promoters. The first evidence of transcription at or through enhancers came from studies of Hox, globin, and B-cell-specific gene loci ,5,6,7,8.Pol II is recruited to basal promoter elements with the help of general transcription factors (GTFs) . Among tThe recruitment of Pol II and associated TFIIF to promoter-bound TFIID is mediated by TFIIB . Pol II The GTFs and Pol II are sufficient to initiate transcription of a TATA-box-containing promoter in the test tube. In the cellular context, DNA sequence-specific transcription factors and co-regulators are required for efficient transcription by Pol II. The co-regulators act by displacing nucleosomes, modifying histone tails, or facilitating recruitment of GTFs and transcription elongation factors. Of note is the acetylation and methylation of nucleosomes immediately downstream of the TSS. Acetylated nucleosomes are recognized by TAF1, and methylated nucleosomes (H3K4me3) interact with TAF3 . These iEnhancers are DNA sequences that positively regulate the transcription of target genes independent of distance, position, and orientation . These eActive enhancers, like promoters, are sensitive to digestion by DNaseI, referred to as hypersensitive sites (HSs) . The accSuper-enhancers (SEs) are extended genomic regions often containing multiple separated enhancer elements that function together to mediate extremely high-level expression of target genes ,25,26. TEnhancers activate transcription by different mechanisms, including modifying chromatin structure, relocating gene loci in the nucleus, or directly assisting in the recruitment of transcription complexes. Most current studies suggest that enhancers come in close proximity to target genes to modulate transcription efficiency . The proIt has been known for some time that enhancers recruit Pol II and other GTFs ,6. HowevA recent study demonstrated that Pol II, TFIIF, and TFIIE are recruited to upstream activating sequences (UASs) in yeast  Figure A. This sHow transcription complexes are recruited to enhancers in higher eukaryotes is not completely understood. It is possible that similar mechanisms operating at promoters, including the placement of a TFIID complex, also mediate the recruitment of Pol II to enhancers. This may involve the recruitment of TFIID, through TAF1, to H3K27 acetylated nucleosomes flanking enhancers and the subsequent recruitment of PolII, TFIIF, and TFIIE by TFIIB B. AssociIt is possible that most Pol II complexes recruited to enhancers/super-enhancers will not engage with DNA to initiate transcription. As a consequence, recruitment of Pol II via Mediator or other proteins may not be efficiently cross-linked to DNA in ChIP assays, thus potentially underestimating the amount of Pol II at SEs. In summary, there appear to be two modes of Pol II recruitment to enhancers and SEs. One mechanism involves the acetylation of H3K27 by CBP/p300, followed by the recruitment of TFIID via TAF1 and Pol II/TFIIF/TFIIE via TFIIB. This leads to the recruitment of TFIIH and initiation of eRNA transcription. The other mode involves the recruitment of Pol II/TFIIF/TFIIE via transcription factors and co-activators . The firTranscription at many genes occurs in bursts, which are characterized by burst size and frequency ,37,38. TTranscription at enhancers occurs in a cell-type-specific and mostly bidirectional manner . The eRNThe observation of bidirectional transcription at enhancers is restricted to the population of cells. At the single cell level, it appears that divergent eRNA transcripts rarely co-localize . MoreoveIt has been known for a while that the process of transcription alters chromatin structure. More recently, accumulating evidence suggests that eRNAs play functional roles in enhancer-mediated activation of transcription. The eRNA could function in a sequence-independent manner by participating in the establishment of a transcription initiation domain, or it could function in a sequence-dependent manner by interacting with proteins, RNA, or DNA. In the following, we discuss how enhancer transcription and/or the resulting eRNA may contribute to Pol II recruitment at enhancers and the transfer of Pol II from enhancers to promoters.The role of transcription initiated at enhancers/SEs: There are several mechanisms by which enhancer transcription could impact transcription activation. For example, the process of transcription at enhancers modifies flanking nucleosomes, and the modified nucleosomes recruit proteins important for enhancer function. Several histone marks are primarily found at enhancer regions, including H3K27ac (histone H3 lysine 27 acetylation) and H3K4me (H3K4 monomethylation) [ylation) . These mylation) .Other histone modifications are introduced during transcription and depend in part on the phosphorylation status of the CTD . For exaNoncoding transcription has also been associated with chromatin conformational changes, thereby facilitating promoter–enhancer interactions. Topoisomerases associate with the transcribing Pol II and change the topology and folding of DNA. Jha et al.  proposedThe role of eRNA in the formation of enhancer-associated transcription initiation domains: The nucleus contains membraneless organelles (MLOs) including the nucleolus where rRNA transcription and ribosome assembly take place; histone bodies, which are sites of histone gene transcription; and nuclear speckles, which are specialized for mRNA processing [ocessing . Severalocessing . Super-eocessing ,28. Moreocessing . Phase socessing ,63. A reocessing .There is still some debate about the physical nature of SE transcription initiation domains. A recent study demonstrated that interactions between transcription factors through intrinsically disordered activation domains enhanced transcription by increasing DNA residence time in the absence of phase separation . WhateveInteractions of eRNA with proteins: eRNAs have been shown to interact with transcription factors, co-regulators, and proteins involved in RNA metabolism [tabolism , and thetabolism . Furthertabolism ,67. Thistabolism .As mentioned previously, Pol II is paused by negative elongation factors DSIF and NELF . PhosphoHou and Kraus targeted eRNAs to estrogen receptor α (ERα) enhancers and found that specific eRNAs carrying a 40-nucleotide motif called FERM  enhanced binding of ERα and elevated levels of H3K27ac . ProteomInteraction of eRNA with RNA and DNA: There is increasing evidence that noncoding RNA interacts with DNA, either in the context of R-loops or in the context of RNA:DNA triple helices [cis or trans and often recruits co-regulators, including the PRC complex to repress or p300/CBP to activate transcription. For example, the SARRAH lncRNA acts in trans to recruit p300/CBP to several cardiac-specific genes, resulting in elevated H3K27ac levels [ helices ,72. For  helices . Furtherc levels . In addiRecently, several studies have shown interactions of eRNA with transcripts generated at TSSs in an antisense direction  . For exaIn summary, there is evidence that the transcription at enhancers changes the chromatin configuration and DNA topology, which may stimulate the binding of transcription factors and co-regulators and decrease the distance between enhancers and promoters. Moreover, there is evidence for a functional role of eRNA either through the formation of enhancer-centered transcription initiation domains, through the recruitment of positive and removal of negative regulators, or through interactions with RNA and DNA, thereby promoting transient enhancer–promoter contacts. Thus, increasing evidence suggests that transcription at enhancers and the resulting eRNAs participate in the formation of transcription initiation domains that concentrate Pol II and also contribute to the transfer of Pol II from the initiation domain to promoters by facilitating promoter–enhancer interactions.It is still unclear how enhancers select target genes in the genome. Transcription factors, like YY1, and cofactors, like Ldb1, have been shown to mediate proximity between specific enhancers and promoters via direct protein–protein interactions ,78. How In addition to the aforementioned mechanisms, CTCF (CCCTC-binding factor) and cohesin are involved in organizing the genome in a way that facilitates promoter–enhancer interactions . It is tEnhancers and promoters function in a specific chromosome environment and in a specific context with regard to distance and relative position. Often, genes are regulated by multiple enhancers that may drive expression in a cell-type- or developmental-stage-specific manner. Many highly expressed genes are regulated by SEs in which multiple enhancers act together to form a transcription initiation domain that concentrates eRNA, co-regulators, and Pol II. In the context of transcription initiation domains, Mediator and other co-regulators recruit a large pool of transcription-incompetent Pol II, which is transferred to TFIID-bound promoters during transient contacts. A small fraction of Pol II recruited to enhancers or SEs is used to generate eRNA . The recImproved new technologies for mapping RNA–RNA, RNA–DNA, RNA–protein, and DNA–protein interactions genome-wide, as well as for identifying enhancer and SE-specific protein complexes, will lead to further progress in our understanding of long-distance gene regulation. Furthermore, there has been enormous progress in high-resolution imaging, which will further illuminate the architecture of enhancers and promoters in the context of specific nuclear environments. Finally, continuous studies on mRNA-mediated feedback mechanisms that slow down transcription to allow ordered processing and efficient export of mRNA into the cytoplasm are anticipated to shed light on the function of enhancers and the interconnection of nuclear processes.Elucidating the mechanism(s) of enhancer function is not only relevant to understanding principles of gene regulation but also important for understanding the cause and progression of diseases. There are many diseases, including cancer, that are caused by mutations in noncoding regions that positively or negatively affect the function of enhancers/super-enhancers ,83. For


Endogenous retroviruses (ERVs), deriving from exogenous retroviral infections of germ line cells occurred millions of years ago, represent ~8% of human genome. Most ERVs are highly inactivated because of the accumulation of mutations, insertions, deletions, and/or truncations. However, it is becoming increasingly apparent that ERVs influence host biology through genetic and epigenetic mechanisms under particular physiological and pathological conditions, which provide both beneficial and deleterious effects for the host. For instance, certain ERVs expression is essential for human embryonic development. Whereas abnormal activation of ERVs was found to be involved in numbers of human diseases, such as cancer and neurodegenerative diseases. Therefore, understanding the mechanisms of regulation of ERVs would provide insights into the role of ERVs in health and diseases. Here, we provide an overview of mechanisms of transcriptional regulation of ERVs and their dysregulation in human diseases. Nearly two LTRs B. Among  protein . LTRs ar protein . However protein ,9. Basedus-like) . Howeverus-like) . In someus-like) . Recentlus-like) .ERVs have been considered as “junk DNA sequences” for a long time . HoweverCurrently, most discoveries about ERVs silencing and transcriptional regulation have been studied in mice, especially during embryonic development and in germ cells. Mouse embryonic stem cells (mESCs) are usually used as a cellular model to study the transcriptional regulation of ERVs, as the pluripotent state is capable of suppressing both exogenous and endogenous retroviruses ,14,15. MPro of provirus [Mus musculus [Trim28 is expressed in a variety of cell types with especially high levels during early embryonic development, in brain and mESCs [Trim28 results in embryonic lethal at E8.5, highlighting its essential role in early development [A common target for ERVs silencing is the PBS, an essential sequence used to prime reverse transcription by a host tRNA as primer. The first example for this mechanism is ZFP809, a member of the family of Krüppel-associated box-containing zinc-finger proteins (KRAB-ZFPs), which binds the PBSprovirus . Interesmusculus . ZFP809 musculus . Besidesmusculus ,21,22,23musculus ,24. TRIMmusculus   is a protein lysine methyltransferase methylating histone H3 at lysine 9 (H3K9) . Unlike The chromatin remodelers and the histone chaperones differing from well-known RNA chaperones or Janus chaperones have been considered as two important classes of factors involved in transcriptional regulation of ERVs, which are dependent on the KRAB-ZFPs/TRIM28 pathway. Recently, the SWI/SNF-like remodeler SMARCAD1 was identified as a key factor in the control of ERVs in mESCs . As key The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly, and genomic localization, which affects all chromosomal processes, including gene expression, chromosome segregation, and genome replication and repair . RecentlATRX is a chromatin remodeler and interacts with DAXX to form a histone chaperone complex, which deposits histone variant H3.3 into repetitive heterochromatin, including regions of retrotransposons, pericentric heterochromatin, and telomeres . A seriePost-translational modification with small ubiquitin-related modifier (SUMO) proteins is one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes, such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction, are subject to reversible sumoylation . Recent Trim28 or Setdb1 in MEFs does not lead to significant activation of ERVs [Trim28 or Setdb1 in neural progenitor cells or pro-B cells results in strong ERVs derepression with only a slight reduction in DNA methylation [Dnmt3l in mouse testis prevents the de novo methylation of both LTR and non-LTR retrotransposons, leading to the activation of IAPs and L1, as well as meiotic failure [In addition to histone-based silencing, ERVs exhibit distinctive DNA methylation patterns . Interes of ERVs ,29. Intehylation ,63,64,65hylation . Deletio failure . Notably failure . Young L failure .Xist, the master regulator of X chromosome inactivation in mammals [Xist-mediated silencing through directly recruited to Xist RNA [Xist may coopt ERVs RNA–protein interactions to repurpose powerful antiviral chromatin-silencing machinery [Drosophila that piRNA was first found to induce silencing through H3K9me3 formation [In addition to DNA-specific binding by co-repressors, histone chaperones, and chromatin remodelers, RNA-mediated targeting of ERVs also play a significant role for the silencing and transcriptional regulation of ERVs. It has been reported that siRNA- or antisense transcripts-based silencing pathways suppress IAPs and non-LTR retrotransposons such as L1 ,70. The  mammals . SPEN isXist RNA . A recenachinery . Anotherachinery . It is iormation . In miceormation ,76.It is worth noting that RNA epigenetic modifications play a significant role in regulation of ERVs. TET2, a member of the Ten-eleven translocation (TET) family, can be recruited to actively transcribed MuERVL RNAs by the RNA-binding protein PSPC1, then catalyzes 5hmC modification of MuERVL RNAs, resulting in their destabilization , which pgag RNA transcripts in lymphocytes and monocytic cells through transcription factors NF-κB or NF-AT, indicating that exogenous viral infection activates transcription factors, which also bind to ERVs LTR regions and induce their activation [Human ERVs activation can be triggered by infections of exogenous viruses such as HIV-1, hepatitis B virus (HBV), hepatitis C virus (HCV), human T-lymphotropic tumor virus-1 (HTLV-1), influenza A virus, and Kaposi’s Sarcoma-associated herpesvirus (KSHV) ,81. For tivation . An in-dKdm1a mutant mESCs exhibit increased methylation of histone H3K4, increased acetylation of H3K27, and decreased methylation of H3K9, indicating that chromatin modification mediated by KDM1A is part of the host’s defense against excessive ERVs activity [Additional factors also contribute to transcriptional regulation of ERVs. TIP60, a lysine acetyltransferase, was found to be involved in silencing of ERVs, through positively regulating the expression of SUV39H1 and SETDB1, and thereby establishing global H3K9me3 levels . KDM1A  that define the cleavage-specific transcriptional programs in humans and mice [As a DNA-binding protein that is specifically expressed in two cell-stage embryos during mouse development , ZSCAN4Cand mice . In addiand mice . Notablyand mice . cis-regulatory element activities or encoded proteins of ERVs can be beneficial to the host. For example, syncytin-1 and syncytin-2, which are specifically expressed in the placenta, are envelope proteins encoded by HERV-W and HERV-FRD, respectively, and with cell–cell fusogenic activities, contributing to the formation of placenta syncytiotrophoblast layer at the materno–fetal interface [Fv1 gene of mice, which is an endogenous gag gene related to ERV-L family [Arc mRNA into new target cells, where it can undergo activity-dependent translation, suggesting that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system [Several studies have suggested that TEs are domesticated for the benefit of the host. This process, in which the host makes use of TEs (including ERVs)-derived functions, are called exaptation, co-option, or repurposing . Either nterface ,93. TherL family ,95. Fv1 L family ,97. Fv1 L family ,99 and sL family , suggestL family . NotablyL family , is ableL family . The Arcs system .cis-regulatory element activities; (ii) insertion of ERVs sequences induces chromosomal rearrangements and genome instability; (iii) ERVs encode proteins, long non-coding RNAs (lncRNAs), and double-stranded RNAs (dsRNAs) to affect host physiology.In spite of the exaptations of ERVs by the host, the dysregulation of them is involved in numbers of pathological processes. Although there is no direct evidence for ERVs causing diseases, aberrant expression profiles of the ERVs transcripts and their regulatory activities on proximal host genes have been identified in different diseases, such as cancer and neurodegenerative diseases. Mechanistically, ERVs may participate in pathological processes through several pathways: (i) ERVs act as promoters or enhance cellular gene expression through LTR The transcriptional activation of HERVs is a common feature in human cancers, suggesting that ERVs are causative elements or cofactors contributing to the onset and progression of human cancer . So far,CSF1R transcription initiates at an LTR element of the MaLR THE1B family, rather than from its own promoter [TP63 and TNFRSF10B, which are regulated by upstream LTRs belonging to the ERV9 group of HERVs [Given the potential transposable ability of retrotransposon, it is the belief that the tumorigenicity of HERVs can depend on retroviral movement, thereby destabilizing the host genome . Indeed,promoter . Howeverof HERVs ,129.c-MYC or signaling pathways such as Notch, Wnt/β-catenin, Ras/ERK, and AKT [HERVs can take a direct action via their own proteins in cancer. The envelop proteins of HERVs, such as syncytin-1 and HERV-K (HML-2) ENV, have been reported to contribute to tumorigenesis by inducing cell–cell fusion in melanoma , endomet and AKT ,112,113.UCA1, a lncRNA consists of LTR7Y and HERV-H, has been shown to enhance proliferation, motility, invasion, and drug resistance of bladder cancer [SAMMSON and BANCR are involved in melanoma progression [lncRNAs play significant roles in various biological processes, including cancer progression. Strikingly, 75–83% of lncRNAs have been identified to contain TE sequences, especially ERVs . Severalr cancer . The HERgression ,135, andgression , pancreagression , and hepgression . A recengression . Higher gression . Anothergression . Interesgression . It has been reported that HERVs contribute to the modulation of innate immune response in different physiological and pathological conditions . For exaHERV deoxyuridine triphosphate nucleotidohydrolase (dUTPase) can trigger innate and adaptive immune responses . In pulm+ T cells are able to lyse melanoma tumor cells in an antigen-specific manner [Considering the activation of HERVs in many human cancers and that HERVs expression has been shown to be associated with proliferation, metastasis, TNM stage, and overall survival, HERVs can be used as biomarkers for tumor diagnosis and/or prognosis . For insc manner . Moreovec manner ,138. As mentioned above, ERVs are largely transcriptionally silenced through heterochromatic structures. However, there may be a net loss of heterochromatin with aging, leading to the abnormal activation of TEs, including ERVs, in aging individuals ,150. It MS is an autoimmune-mediated neurodegenerative disease of the central nervous system characterized by inflammatory demyelination with axonal transection . Althougpol transcripts are upregulated in patients with ALS but not detectable in Parkinson disease or in healthy controls [pol, env, and gag genes in brains of ALS patients [TDP-43 expression strongly correlates with HERV-K (HML-2) [Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive loss of cortical and spinal motor neurons, is another neurodegenerative disease associated with HERVs . Activatcontrols . A subsepatients . Moreovepatients . Severalpatients . As a mu (HML-2) , and has (HML-2) . Besides (HML-2) . NonetheRecent studies have revealed aberrant expression of HERVs in neurodevelopmental disorder ASD. HERV-H is more abundantly expressed while HERV-W shows lower expression levels in PBMCs from ASD patients compared to healthy controls . FurtherERVs are involved in various biological processes by encoding proteins, lncRNAs, dsRNAs, or acting as promoters/enhancers, thereby affecting human health and disease. Recent progress suggests that the implication of ERVs in cancer and neurodegenerative diseases provides an opportunity to develop novel therapeutic strategies. For example, nucleoside reverse transcriptase inhibitors (NRTIs) have shown promise in the treatment of neurodegenerative diseases . DNMTis


Vascular endothelial cells are a mesoderm-derived lineage with many essential functions, including angiogenesis and coagulation. The gene-regulatory mechanisms underpinning endothelial specialization are largely unknown, as are the roles of chromatin organization in regulating endothelial cell transcription. To investigate the relationships between chromatin organization and gene expression, we induced endothelial cell differentiation from human pluripotent stem cells and performed Hi-C and RNA-sequencing assays at specific time points. Long-range intrachromosomal contacts increase over the course of differentiation, accompanied by widespread heteroeuchromatic compartment transitions that are tightly associated with transcription. Dynamic topologically associating domain boundaries strengthen and converge on an endothelial cell state, and function to regulate gene expression. Chromatin pairwise point interactions (DNA loops) increase in frequency during differentiation and are linked to the expression of genes essential to vascular biology. Chromatin dynamics guide transcription in endothelial cell development and promote the divergence of endothelial cells from cardiomyocytes. •In endothelial differentiation, compartment transitions regulate gene expression•New TAD boundaries and chromatin loop anchors coordinate endothelial activation•Frequent, growing chromatin loops accompany essential endothelial gene expression•Cardiomyocytes evidence a markedly different topology versus endothelial cells cis contacts increase in endothelial cell differentiation, as do hetero-euchromatic compartment transitions, which are tightly associated with transcription. Specification occurs alongside new TAD boundaries and chromatin loop anchors, features that distinguish endothelial cells from cardiomyocytes, which evidence a dissimilar topology. Multiple forms of chromatin organization guide transcription in differentiation, driving endothelial divergence from cardiomyocytes.Alavattam, Mitzelfelt, and colleagues reveal that long-range  Endothelial cells, a mesoderm-derived cell population, line the entirety of the circulatory system. Their functions are complex and critical, including angiogenesis, blood clotting, barrier function, vasomotor function, and fluid/nutrient filtration. Endothelial cell dysfunction is a prominent feature of many pathological conditions, including nearly all cardiovascular diseases . Complexin situ DNase Hi-C  that spread outward with differentiation, increasing the numbers of “far” cis interactions. Next, we examined the cis chromatin contact probability P(s) for pairs of genomic loci stratified by distance s  for cis interactions . TADs comprise local “neighborhoods” of increased chromatin contact frequency and are often delimited by insulator sequences . Becausecis chromatin interactions are increasingly restricted within TAD boundaries  proximal to (within ±80 kb of) boundaries lost in differentiation (hPSC-specific boundaries), boundaries gained in differentiation (EC-specific), and shared boundaries (common to hPSC and EC). We found that DEGs are depleted from lost and gained boundaries and enriched at shared boundaries D. RelatiTo investigate this, we examined the interplay of TADs and compartments. TADs in A compartments are smaller than those in B compartments G, a likeThis led us to explore how TAD-compartment interrelationships influence gene expression. We examined the enrichment of up- and downregulated DEGs at TAD boundaries grouped by stable and dynamic compartments F. RegardOur analyses also revealed that shared TAD boundaries in stable A regions of the genome are slightly but significantly enriched in both up- and downregulated DEGs F, gray. CD31  in transcriptional regulation and suggests the existence of PPIs that function in specific aspects of development . These iSince PPI dynamics occur amid changes in genomic compartmentalization  and S3, MECOM, which encodes a transcription factor that promotes arterial EC identity  contacts for a single chromosome, chromosome 3 .Further information and requests should be directed to and will be fulfilled by co-lead contact Charles E. Murry  or various software packages . StrategK.G.A., K.A.M., W.S.N., and C.E.M. wrote the manuscript with edits provided by all other authors. K.G.A., K.A.M., G.B., W.S.N., and C.E.M. designed the experiments. K.A.M. conducted wet-lab experiments; K.G.A., G.B., P.A.F., and X.Y. conducted dry-lab experiments. K.G.A., K.A.M., X.Y., L.P., A.B., N.J.P., H.S.C., W.S.N., and C.E.M. interpreted the results. W.S.N. and C.E.M. obtained resources and were the overall supervisors of this work.


Insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs) family belongs to a highly conserved family of RNA-binding proteins (RBPs) and is responsible for regulating RNA processing including localization, translation and stability. Mammalian IMPs (IMP1-3) take part in development, metabolism and tumorigenesis, where they are believed to play a major role in cell growth, metabolism, migration and invasion. IMPs have been identified that are expressed in ovary, placenta and embryo. The up-to-date evidence suggest that IMPs are involved in folliculogenesis, oocyte maturation, embryogenesis, implantation, and placentation. The dysregulation of IMPs not only contributes to carcinogenesis but also disturbs the female reproduction, and may participate in the pathogenesis of reproductive diseases and obstetric syndromes, such as polycystic ovary syndrome (PCOS), pre-eclampsia (PE), gestational diabetes mellitus (GDM) and gynecological tumors. In this review, we summarize the role of IMPs in female reproductive pathophysiology, and hope to provide new insights into the identification of potential therapeutic targets. Vegetal-1 mRNA-binding protein (Vg1RBP/Vera) in Xenopus laevis; zipcode-binding protein 1 (ZBP1) in chickens; murine coding region determinant-binding protein (CRD-BP) in mice; and Drosophila IMP (dIMP) [IGF2BP2a and IGF2BP2b in Danio rerio. Moreover, the p62 protein, a 62 kDa isoform generated by IMP2, has been identified as an autoantigen in human hepatocellular carcinoma [Insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs or IGF2BPs) belong to a highly conserved family of RNA-binding proteins (RBPs), which function to regulate the localization, stability and translation of mRNA and fine-tune the physiological function of the proteins encoded . MembersP (dIMP) . IMP1 isP (dIMP) , 5. Vg1RP (dIMP) . Homologarcinoma . Mammaliarcinoma  and subsarcinoma –11 and marcinoma , due to arcinoma . Accumularcinoma , ovulatiarcinoma , implantarcinoma  and placarcinoma . Recent arcinoma , pre-eclarcinoma –21 and garcinoma , in addiarcinoma , 24. TheThe protein structures of the three identified human IMPs (IMP1-3) are highly similar in domain order, and share an overall amino acid sequence identity of 56% . Their cβ-actin mRNA to the leading edge of motile cells.The function of IMPs in the regulation of RNA metabolism has been widely studied, including RNA localization, stability and translation. IMPs are found to be predominantly expressed in the cytoplasm where they form messenger ribonucleoprotein (mRNP) granules around the nucleus which function to transiently “lock” target mRNAs. IMP1 granules have been shown to be particularly prevalent in neurons and oocytes. Compositional analysis has identified the exon-junction components, CBP80 and poly(A)-binding protein in IMP1-containing mRNP granules; the presence of which represents pre-translational status. IMP1 has also been shown to mediate transport of IGF2 gene generates numerous mRNAs with different 5’UTRs. The human IGF-2 gene is a complex transcription unit driven by four promoters. Each promoter directs transcription of a different RNA designated leader. Four designated leaders of IGF2 have different translational properties. IMP1-3 have been shown to regulate the translation of IGF2 through binding with the IGF2 mRNA designated leader 3 rather than IGF2 mRNA designated leader 4 in a rapamycin-sensitive manner. The co-translational phosphorylation of IMP1 is catalyzed by mTOR complex 2, which mediates initiation of IGF2-L3-luciferase mRNA translation by cap-independent internal ribosomal entry [IMPs were originally discovered as mRNA-binding proteins capable of binding to the 5’ untranslated regions (5’UTR) of IGF2 . The IGFal entry . IMP2 maal entry . Knockoual entry . The IMPal entry . In caseal entry . In chical entry , granuloal entry  and thecal entry , and plaal entry  and embral entry . IGF2 isal entry . IMPs maExpression of all three IMPs have been identified in adult ovarian and placental tissue. The physiological expression of IMP1–3 exhibits a biphasic pattern during development. IMP1–3 first appears in the oocyte and persists from pre-fertilization to the stages of zygote and blastocyst during early embryogenesis, and is then found to decline until murine embryonic day 10.5–12.5, when expression increases again . In the ♀+/♂+), but exerts no effect on IMP2−/−female-derived embryos (IMP2♀−/♂+). This effect of IGF2 has also been observed in human embryos [After fertilization, the early embryo undergoes maternal-to-zygotic transition (MTZ), which is the stage during which regulation of development, previously controlled by maternal factors, changes to be under the control of new genetic products from the zygotic genome. The transition includes two distinct processes: first, the maternal information (mRNAs and proteins) is gradually cleared; second, zygotic transcription is activated . Many ma embryos . The datEmbryo implantation is a dynamic and complex reproductive process regulated by a series of molecular and cellular events with its ultimate success based on the established uterine receptivity , 48. BotEarly placentation is critical to both perinatal fetal growth and postnatal fetal and maternal health. Transcriptome analysis performed during the implantation period revealed that there is overexpression of some placental growth factors, and IMP1 and IMP3 in human trophoblast ectoderm cells, as well as upregulation of corresponding receptors in the receptive endometrium , implyin6-methyladenosine (m6A) readers, IMPs recognize m6A modifications, which regulate splicing, translocation, stability, and translation. In mammals, the occurrence of abnormal m6A modifications during early development results in parthenogenetically activated oocytes that are not capable of developing to term [6A expression [In mouse parthenogenetic embryos, the expression of IMP1 and IMP2 is perturbed compared to that in embryos which have been normally fertilized. As N to term . Reducedpression . This efPolycystic ovary syndrome (PCOS) is a common disorder of the ovary in women of reproductive age, and is characterized by hyperandrogenism, ovulatory dysfunction (anovulation or oligo-ovulation) and a polycystic ovarian morphology . Most PCcyclin D2 (CCND2) and SERPINE1 mRNA binding protein 1 (SERBP1) mRNA [In polycystic ovaries, early follicular growth is excessive, resulting in massive ovarian preantral follicles, which become arrested and do not proceed to develop a dominant follicle. PCOS is also usually accompanied by high rates of GC proliferation and low rates of GC apoptosis. GC is an important source of anti-Müllerian hormone (AMH), IGF-1 and IGF-2 in the ovary. Elevated AMH has an antagonistic effect against the action of follicle stimulating hormone (FSH) on GCs, and represses the conversion of androgens to estrogens, therefore aggravating the androgen excess found in PCOS. Large numbers of GCs associated with excessive amounts of small ovarian follicles, which have become arrested in part, account for the characteristic polycystic morphology of the ovaries in PCOS. The expression of IMPs in PCOS patients is controversial and IMP2 may play a more important regulatory role in ovaries than IMP1 and IMP3. A recent microarray analysis identified that IGF1R, IGF2R, IMP2, IGFBP2 and IGFBP7 were all found to be down-regulated in the cumulus cells from patients with PCOS compared with patients without PCOS . In contP1) mRNA . In the P1) mRNA . Abnormacyclin B1 mRNA [Ovulatory dysfunction may be partially due to impaired oocyte maturation. In the zebrafish oocyte, IMP3 has been found to retard the progression of oocyte maturation by repressing translation of  B1 mRNA . Upregul B1 mRNA , howeverAs mentioned earlier, many patients with PCOS have other concurrent metabolic disorders, such as insulin resistance, hyperinsulinemia, dyslipidemia and activation of proinflammatory factors, and these patients also have a long term risk of developing T2DM and obesity , 73, 74.Abnormal proliferation and differentiation of human placental trophoblasts result in abnormal trophoblast invasion and dysfunctional syncytialization, which are involved in the pathogenesis of PE . As a seMeta-analyses of candidate gene studies and GWAS have identified multiple genes which are reproducibly associated with GDM, including TCF7L2, GCK, KCNJ11, KCNQ1, CDKAL1, IMP2, MTNR1B, and IRS1. These genes are also associated with T2DM . Among pGestational trophoblastic disease is caused by excess cellular proliferation of placental villous trophoblast cells, and includes a spectrum of cellular abnormalities including hydatidiform mole (HM), invasive mole, choriocarcinoma, and placental site trophoblast tumor. HMs can be divided into complete, partial and invasive HMs . AccordiOverexpression of IMP3 is associated with an unfavorable overall survival of patients with epithelial ovarian carcinoma (EOC) , such asUterine leiomyomas (ULM) is characterized by histological and molecular heterogeneity. The pathogenesis of ULM is partially a result of several driver gene mutations, such as MED12 mutation, HMGA2 overexpression, and biallelic FH inactivation , 105. ULEndometrial cancer is a common gynecologic malignancy, which originates from the endometrium and is composed of heterogeneous populations of cells. Traditionally, it has been classified into two types, type I (estrogen-dependent) and type II (estrogen-independent) based on clinical and epidemiological features . PatholoIn summary, IMPs are implicated in the physiology and pathology of the female reproductive system. As a biomarker, IMPs promise to be very useful in being able to monitor the healthy embryogenesis and oocyte maturation, and to predict the occurrence and progression of some female reproductive diseases Table . The exp


To evaluate the expression profile of MCM6 in HCC and the relationship between MCM6 level and clinicopathological parameters through bioinformatics analysis of several databases. MCM6 in HCC compared to normal liver tissues.  MCM expression level, clinical parameters, survival data, and gene set enrichment analysis were analyzed by bioinformatics database, including Oncomine™, UALCAN, HCCDB, TCGA, cBioPortal, and LinkedOmics. Real-time PCR, western blotting, and IHC staining were conducted to identify the expression of MCM6 was obviously increased in multiple cancer types, especially in HCC. MCM6 level was positively associated with multiple clinical parameters (stage 3 and grades 3 and 4) and negatively associated with patient outcomes . Moreover, enrichment of functions and signaling pathways analysis of MCM6 suggested that MCM6 might mediate DNA replication and cellular metabolism to promote the development and progression of HCC. Furthermore, IHC staining and western blotting indicated that the MCM6 was enhanced in HCC tissue, and MCM6 could promote HCC proliferation in activating Notch pathway via WB and bioinformatic analysis.  Bioinformatics analysis indicated that the mRNA of MCM6 in HCC. Furthermore, MCM6 is a carcinogenic role in activating Notch pathway to promote HCC cell proliferation, which may be a new prognostic biomarker and therapeutic target for HCC patients. This study actually revealed the expression and related functions of  Hepatocellular carcinoma (HCC) is the sixth most lethal human malignancy globally, with 841,080 new and 781,631 deaths cases estimated for 2018 [MCM2-7 forms a hexameric ring-shaped complex to promote DNA replication of chromatin by binding to a DNA replication starting point in the middle to late cell cycle to the early G1 phase [Minichromosome maintenance (MCM) proteins family, a classical family on the eukaryotic genome replication, in Saccharomyces cerevisiae, was found first, which were necessary for the maintenance of extrachromosomal DNA . During G1 phase . A biochMCM6, as an important member of the MCM family, could interact with Cdt1 at NTD to stabilize the coil structure [DDK at NTD terminus to activate pre-RC [MCM2-7 complex [MCM6, as a transcriptional regulation factor, could activate MEK/ERK signaling pathway to mediate EMT progression, resulting in metastasis of HCC [MCM6 and p53-binding protein 1 (53BP1) was involved in 53BP1 chromatin fraction and foci formation in HepG2 cells. However, little is known about the role and mechanism of MCM6 in hepatocarcinogenesis. Until now, the previous studies indicated that the expression of MCM6 was observed in 70 patients with HCC, and it may relate to poor prognosis [tructure , be phose pre-RC , and be  complex . It is rs of HCC . Chen ets of HCC  found thrognosis .MCM6 in HCC and the relationship between MCM6 level and clinicopathological parameters through bioinformatics analysis of several databases. In addition, the biological functions and genomic changes correlated with biological functions in HCC were examined. The present study may provide a novel and potential molecule target for the early diagnosis and targeted therapy of HCC.This study evaluated the expression profile of http://www.oncomine.org) is a web tool to extract data for the mRNA level and gene expression of MCM6, currently containing 715 datasets and 86,733 samples. A series of HCC datasets were utilized for this study, including Chen Liver, Roessler Liver, and Roessler Liver 2. HCCDB database (http://lifeome.net/database/hccdb/search.html) is an integrative molecular database of HCC based on 4000 HCC samples. We utilized the HCCDB database to confirm the level of MCM6. UALCAN database  is an interactive web portal to analyze the expression of MCM6 in HCC, which is based on gene expression analysis of TCGA (The Cancer Genome Atlas). The cBioPortal database  is an interactive web tool for elucidating the altered expression of MCM6, which is used to explore the multidimensional genomics datasets for multiple cancer types. LinkedOmics database (http://www.linkedomics.org/login.php), as an online tumor database, was utilized for elucidating multiomics data. Users can extract multiple cancer-type protein expression profiles, which are differential expression in specific cancer. We used it to analyze the GO and KEGG pathway of MCM6 coexpression genes in HCC.Oncomine database  were cultured in Dulbecco's Modified Eagle Medium  containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin, which were maintained at 37°C in a humidified atmosphere of containing 5% COMCM6 or shRNA negative control were constructed by RiboBio Co. Ltd. siRNA was transfected into cells using riboFECT transfection reagent  according to the manufacturer's protocol. The sequences of the siRNA were as follows: si-MCM6, GGCGCATAGTAGATTTGCA. The shRNAs were transfected by lip3000 for 6 hours. The effect of MCM6 knockdown was confirmed by western blot.The shRNA against MCM6 , NICD , Notch1 , Hey1 , Hes1 , and GAPDH .Western blotting was conducted according to our previous report . The expThe slices are dewaxed and then hydrated. After blocking endogenous peroxidase, the slices were treated with a hot citric acid buffer to reveal the epitope. The sections were then incubated with a primary antibody using an immunohistochemistry kit  according to the manufacturer's instructions.Immunohistochemical staining scores were assessed by staining percentage of tumor cells and staining intensity. The percentage of stained cancer cells was graded from 0 to 4: 0 (0% stained), 1 (0%-25% stained), 2 (26%-50% stained), 3 (51%-75% stained), and 4 (76%-100% stained). Similarity, stained tumor cell intensity was divided into 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The final semi-quantitative immunohistochemical fraction was obtained by multiplying the percentage of stained cancer cells by the intensity grade.3) and cultured for one, two, and three days. 20 μl MTT solution  was used to incubate for 2 hours. Next, 150 μl DMSO was added in each well. Last, the OD value was confirmed at 490 nm by a microplate reader. For the clone formation assay, cells were seeded into 24-well plates with 500 cells in each well and cultured for 14 days.For the MTT analysis, two HCC cancer cell lines were seeded in 96-well . Data are presented as the mean ± SD. All assays were tested in three independent experiments. Experimental data were analyzed using SPSS statistical software . The significance of the group difference was evaluated by one-way analysis of variance followed by Tukey's post hoc test. Multiple comparisons between the groups were performed using Student-Newman-Keuls method. MCM6 transcription levels from a variety of cancers from TCGA. The Oncomine database indicated significant increases in MCM6 mRNA expression in HCC , and MCM6 were increased in HCC compared to normal liver tissue (logFC = 0.34) and corresponding paracancer tissue (logFC = 0.91) . MCM6 profile was obviously increased in tumor grade, cancer stage, patient age, gender, race, and weight  . In addi Figures . In summMCM6 genetic alteration in HCC. Three HCC databases  analysis results indicated that the highest alteration frequency of MCM6 was only 1%  for biological process (BP), these DEGs were obviously enriched in biological regulation, metabolic process, response to stimulus, multicellular organismal process, localization, and cellular component organization  fornization ; (3) fornization . The signization .MCM6 in HCC, we detected the MCM6 expression in HCC tissues by IHC staining  foci formation and chromatin fraction in response to DNA damage, indicating that MCM6 was not just a DNA replication regulator [MCM6 could obviously promote migration and invasion ability via activating MEK/ERK signaling pathway to induce EMT cascades [MCM6 in the formation of HCC is still largely unknown. This study was based on the bioinformatics analysis of databases. After analysis of Oncomine, HCCDB, and TCGA databases, MCM6 transcription and posttranscriptional levels were significantly upregulated in HCC. In subgroup analyses based on disease stage, tumor grade, age, gender, ethnicity, and body weight, the MCM6 levels were significantly higher in HCC patients than in healthy people. Furthermore, the cBioPortal databases revealed that the frequencies of alterations of MCM6 might be one reason for its ectopic expression and induce a low survival rate in HCC patients with MCM6 alteration. The UALCAN databases also indicated that HCC patients with high expression of MCM6 had a poorer survival rate.l in HCC . MCM6 haegulator . Liu andcascades . HoweverMCM6 is mainly involved in primary bile acid biosynthesis, valine, leucine and isoleucine degradation, arginine biosynthesis, DNA replication, fatty acid degradation, and homologous recombination by mining-related functional networks through the KEGG pathway. As we know, MCM6 is a DNA replication license regulator that plays a key role in the cell cycle progression. It has been reported that MCM6 could interact with other MCM proteins  to assemble into MCM complex, regulating the DNA replication to accelerate cell cycle [MCM6 on cell metabolism, such as primary bile acid, amino acid, and fatty acid, remains unclear [MCM6, like a molecular switch, has a divergence in these critical nodes, with one branch forming MCM complex to directly accelerate DNA replication and another branch regulating cell metabolism to provide the necessary materials and energy in DNA replication. unclear . We specMCM6. The level of NDUFAF1, LDHD, and SERPING1 were negatively correlated with MCM6 expression levels. NDUFAF1, also known as NADH ubiquinone oxidoreductase complex assembly factor 1, is an essential assembly mitochondrial protein for the stability of complex I (NADH: ubiquinone oxidoreductase), which may play a key role in energy metabolism [LDHD, lactate dehydrogenase D, is responsible for human D-lactate metabolism [LDHD in HCC progression is still unclear. SERPING1, serpin family G member 1, plays a key role in hereditary angioneurotic edema by inhibiting the classical complement pathway activation [MCM6 might repress the tricarboxylic acid cycle by inhibiting the expression of NDUFAF1, inhibit aerobic glycolysis by inhibiting the expression of LDHD, and regulate the classical complement pathway activation by regulating the expression of SERPING1. DTL, ZWINT, and MCM2 were increased with MCM6 in HCC. DTL (denticleless E3 ubiquitin protein ligase homolog), also known as DNA replication factor 2, could mediate many cell cycle regulatory proteins levels to accelerate the cell cycle and maintain DNA repair and replication [ZWINT (ZW10 interacting protein) interacted with ZW10, a key factor for spindle checkpoint control and chromosome motility, inhibited the chromosome bridge phenotype from interacting with sister chromatids [ZWINT knockdown could attenuate mitotic arrest mediated by the microtubule inhibitor nocodazole, resulting in cell death [MCM2 could interact with MCM3-7 to form a hexameric ring-shaped complex around DNA, which could promote DNA replication to accelerate the cell cycle [MCM2 could be an important biomarker of multiple cancer types, such as oral squamous cell carcinoma [MCM6 not only interact with MCM2 to form a DNA replication license complex to directly activate DNA replication, but also interacts with DTL as well as ZWINT to accelerate the cell cycle. We also confirmed that MCM6 was significantly upregulated in HCC samples, which was in accordance with previous studies [MCM6 can interact with Notch pathway to enhance the proliferation level in HCC cells.Furthermore, we found several genes associated with tabolism . LDHD, ltabolism . The uprtabolism . Howevertivation , which dtivation , prostattivation , and luntivation . Therefolication . ZWINT (romatids . ZWINT kll death . MCM2 coll cycle . Severalarcinoma , cervicaarcinoma , and medarcinoma . Hence,  studies , 29. MorκB, integrin, and miRNAs, to significantly drive cell progression [Notch pathway is a classical and important signaling in multiple cancer types, such as breast cancer, HCC, lung cancer, ovarian cancer, cervical cancer, gastric cancer, and renal carcinoma . This sigression . Previougression . Becausegression . Our stuMCM6 is highly expressed in HCC compared to normal liver tissues and is correlated with multiple networks. MCM6 is an excellent biomarker for predicting the prognosis of HCC. However, current research focuses on the role of MCM6 in DNA replication. The role of MCM6 in tumor metabolism is required to confirm the effect of MCM6 on tumor metabolism and transcriptional regulation in HCC and other cancers. Moreover, MCM6 is a carcinogenic role in activating Notch pathway to promote HCC cell proliferation, which may be a new therapeutic target for HCC patients.This study indicates that


During cell division, drastic cellular changes characteristic of mitosis result in the inactivation of the transcriptional machinery, and global downregulation of transcription. Sequence-specific transcription factors (TFs) have thus been considered mere bystanders, devoid of any regulatory function during mitosis. This view changed significantly in recent years, upon the conclusion that many TFs associate with condensed chromosomes during cell division, even occupying a fraction of their genomic target sites in mitotic chromatin. This finding was at the origin of the concept of mitotic bookmarking by TFs, proposed as a mechanism to propagate gene regulatory information across cell divisions, by facilitating the reactivation of specific bookmarked genes. While the underlying mechanisms and biological significance of this model remain elusive, recent developments in this fast-moving field have cast new light into TF activity during mitosis, beyond a bookmarking role. Here, we start by reviewing the most recent findings on the complex nature of TF–chromatin interactions during mitosis, and on mechanisms that may regulate them. Next, and in light of recent reports describing how transcription is reinitiated in temporally distinct waves during mitosis-to-G1 transition, we explore how TFs may contribute to defining this hierarchical gene expression process. Finally, we discuss how TF activity during mitotic exit may impact the acquisition of cell identity upon cell division, and propose a model that integrates dynamic changes in TF–chromatin interactions during this cell-cycle period, with the execution of cell-fate decisions. Their activity is thus tightly controlled by a wide-variety of mechanisms. Binding of a TF to a gene regulatory region is to large extent dependent on its concentration . EukaryoMitosis is characterized by drastic cellular changes that altogether result in a global downregulation of transcription . In addiIn spite of the above, the finding that an increasing number of TFs is able to associate with mitotic chromosomes, sometimes even occupying part of their cognate binding sites in mitotic chromatin, is driving a paradigm change ,8. Altho. 2Distinct types of interactions mediate the association of TFs with DNA. Long-lasting sequence-specific interactions are established between residues in the DNA-binding domain (DBD) and DNA bases within consensus sequence recognition motifs. In addition, TFs engage in non-specific interactions with chromatin, mediated primarily by electrostatic forces that do not rely on specific DNA sequences –15. ThesWhile it was initially thought that complexation with positively charged histones neutralized the negative charges of DNA molecules, both computational and experimental work concluded nucleosomal DNA has similar net charges to naked DNA ,19. In s. 3in vitro assays), dramatically diminished binding to mitotic chromosomes [Live-cell imaging experiments using fluorescently tagged proteins have been a major drive in recent studies characterizing how TFs interact with mitotic chromosomes . Using tomosomes . In a reomosomes . On a diomosomes .Box 1.β, when fixed and visualized with the same protocol, cannot be observed at mitotic chromosomes, although significant mitotic binding can still be detected using ChIP-seq [β and Sox2 is preserved whereas Oct4 is not [in vitro [Understanding the impact of chemical fixation when assessing TF–chromatin interactions has helped to reconcile recent observations with old data. Formaldehyde fixation is widely used both in immunofluorescence and Chip-seq protocols. This fixator was found to artifactually exclude most TFs from mitotic chromosomes as observed by immuno-staining ,22, raisChIP-seq . Interes4 is not , in line4 is not  and low in vitro . Thus, iThe transient nature of interactions underlying MCB was revealed by studies using fluorescent recovery after photobleaching (FRAP) and single molecule tracking (SMT), showing decreased TF residence time on mitotic chromatin as compared to interphase ,25,30,31. 4β and CTCF bookmarked sites, counteracting large chromatin rearrangements characteristic of this stage [While growing evidence indicates MCB observed by live-cell imaging analysis reveals mostly electrostatic interactions, in some cases this phenomenon occurs concomitantly with sequence-specific binding. This is difficult to demonstrate using mutagenesis, given that mutations affecting sequence-specific binding may also disrupt electrostatic interactions ,16. The is stage ,34. Thisβ and CTCF [Altogether, available studies suggest that non-specific electrostatic interactions are the main determinants of the widespread binding of TFs in mitosis observed in live-cell imaging, and that in some cases such interactions contribute to sequence-specific binding to mitotic chromatin. As a consequence, only a subset of TFs that associate with mitotic chromosomes in live-cell imaging, are expected to display mitotic bookmarking activity as this was originally described . In lineand CTCF ,34.FiguOne possibility is that TFs that interact with mitotic chromosomes solely via electrostatic interactions may reside at the chromosome periphery, a domain covering the outer surface of mitotic chromosomes, which is formed by the accumulation of proteins and RNAs originated from the nucleolus ,36. Alth. 5The different behaviour of TFs with regard to the way they interact with mitotic chromatin  raises a still controversial question in the field: is MCB an intrinsic property imposed by the biochemical characteristics of each individual TF, or is this an activity modulated by specific regulatory mechanisms? Below, we first review what is known of the structural determinants required for MCB, before discussing two possible major layers of regulation.. 5.1Studies on MCB have initially associated this property with the ability of TFs to bind nucleosomal DNA (i.e. pioneer TF activity), a property known to rely on specific features of the DBD . AlthougBox 2.D. melanogaster [The TF FoxA1 is perhaps the most paradigmatic mitotic bookmarker. This master regulator of liver development can bind its target sites on ‘closed chromatin’ , a property referred to as ‘pioneer activity’. This precedent linked mitotic bookmarking (and to some extent MCB) with pioneer activity, a rather intuitive association whose molecular basis however, failed to materialize. Indeed, sequence-specific TFs without pioneer activity have been reported to bind mitotic chromosomes, one example being Brn2 ,38,39. Cnogaster . Furthernogaster . As a coIn addition to the DBD, several reports described the ability of a nuclear localization signal (NLS) to promote the association of proteins with mitotic chromosomes. The initial interpretation of these findings came with some controversy, as they have been seen as evidence for a role of the nuclear import machinery driving MCB . The proBox 3.β mutant to interact with mitotic chromosomes upon cold shock, further implicating an NLS-based mechanism [One of the first mechanisms proposed to regulate the association of TFs with mitotic chromosomes involved the nuclear import machinery, more specifically the Ran-GTP gradient which in interphase regulates nuclear import of proteins containing an NLS. Retention of the Ran-GTP promoting factor RCC1 on condensed chromosomes results in a gradient of Ran-GTP being maintained throughout mitosis, which is required for the activation and loading of Spindle Assembly Factors (SAFs) once these are at vicinity of mitotic chromosomes. It has been proposed that the Ran-GTP gradient could function as a ‘molecular GPS’ to direct specific sets of proteins such as SAFs to condensed chromosomes, a model that could be extended to other nuclear proteins such as TFs . In suppechanism . Althoug. 5.2β in such compacted chromosomes was reduced, as found by proteomics and immunofluorescence. Of note, no differences were observed at TF binding sites with ATAC-seq (investigated in case of ESRRβ), thus not supporting changes of chromatin accessibility as the underlying mechanism of decreased TF retention [The chromatin environment may provide one important level of regulation of MCB. For example, PTMs of core histones may influence the ability of a TF to bind mitotic chromatin by altering chromatin compaction, and/or the associated electrostatic field. A recent study tested the idea that repressive chromatin may hinder the association of TFs with mitotic chromosomes, by abolishing the mitotic levels of H3K9me3 in ES cells . Somehowetention . Moreoveetention ,20. AltoOther players involved in chromatin condensation that may also regulate MCB are divalent cations, particularly Ca2+ and Mg2+. These are sufficient to induce chromatin condensation, either directly via electrostatic neutralization or indirectly by promoting the activity of non-histone proteins such as TopoII –52. A tr. 5.3in vitro assays) [bona fide mitotic bookmarking activity. A striking example is that of Brn2, with a phospho-mimetic form devoid of sequence-specific binding (S362D) retaining to large extent its MCB ability in neural stem cells [One way the cellular context can influence TF interactions with DNA is via their targeting by PTMs. In line with the pivotal role of phosphorylation in cell-cycle regulation, high-resolution mass spectrometry-based proteomics has shown around 70% of proteins are phosphorylated along the cell-cycle, with nuclear proteins at the top of the list . Indeed, assays) . Importaem cells . In the em cells ,56. Alsoem cells .Altogether, growing evidence suggests phosphorylation plays a role in modulating how TFs interact with chromatin during mitosis, although we have only begun to fully explore its functional importance in this context. Future studies should address whether mitotic phosphorylation (for example under the direct control of cell-cycle regulators) may explain the reported exclusion of some TFs from mitotic chromosomes, and what are the mechanisms that revert this inactivation process upon mitotic exit .Another important possibility is that cell-type specific phosphorylation events may confer distinct MCB abilities to one TF across different cell types. One illustrative example is that of CTCF, which in ES cells was shown using ChIP-seq to function as a mitotic bookmarker and maintain nucleosome positioning . Bookmar. 6β, where sequence-specific binding during prometaphase is predictive of earlier reactivation on a genome-wide scale [Although the function of MCB and mitotic bookmarking of TFs remains largely unknown, one interesting possibility is that such interactions contribute to a temporal gradient of gene reactivation during mitotic exit. Initial studies had already proposed that sequence-specific binding to regulatory regions may result in early reactivation of bookmarked genes upon mitotic exit ,30. Howede scale . The linde scale ,11,62. Ide scale . By contde scale ,63. ThisOther studies on three-dimensional chromatin architecture provided further support to the hierarchical model of gene reactivation. In mitotically arrested erythroblasts, the reestablishment of enhancer-promoter contacts starts shortly after nocodazole release, reaching maximum contact in early G1 . Formatibona fide mitotic bookmarking activity of Brn2. Instead, this study proposed an alternative model, whereby increased TF concentration on chromatin (promoted by Brn2 MCB) fosters the early reactivation of its target transcripts. In line with this possibility, the late reactivation of Dll1 observed in neural stem cells correlates with increased nuclear concentration (upon nuclear-envelope reformation) of its main activator Ascl1, which remains excluded from mitotic chromatin during M-G1 [Altogether, accumulated evidence shows gene reactivation during M-G1 results from a highly coordinated succession of events, and suggests TFs may play an important regulatory role. This idea is supported by a recent study, which used single-molecule RNA-FISH (smRNA-FISH) as a method to image unprocessed transcripts at places of active transcription in single cells, in this way probing with high temporal resolution and sensitivity, the reactivation of specific transcripts in neural stem cells . In lineing M-G1 . Howeverin silico predictions of binding sites, and MCB data from a large number of TFs [With the aim of providing a large scale understanding of a regulatory role of TFs during M-G1, a recent study used mathematical modelling to integrate transcriptional profiling of gene reactivation, with r of TFs ,9,67 AltUnderstanding the role of TFs during mitotic exit will most likely require an integrative view of TFs and their cofactors during this time window, several of which have been shown to interact with mitotic chromatin . Given i. 7The large-scale changes in genome and chromatin organization characteristic of mitosis, associated with major downregulation of transcription , are thoRecently, an elegant study performed in ES cells characterized the genome-wide binding of Sox2/Oct4 along the cell-cycle, and how this correlates with chromatin accessibility, providing important mechanistic information. Here the authors report that recovering Oct4 levels after its short and acute depletion during M-G1 transition, results in some Oct4-dependent genomic regions becoming permanently inaccessible . SurprisDrosophila has shown retention of the neurogenic TF Prox1 on pericentromeric regions of mitotic chromosomes in dividing ganglion mother cells (GMCs), to be required for their terminal differentiation into post-mitotic neurons [de novo interaction of Prox1 with mitotic chromosomes in GMCs (which the authors refer to as ‘mitotic implantation’) can be explained by alterations in the cellular distribution of Prox1 (found tethered to the cell membrane in NBs), and does not necessarily entail changes to its ability to associate with mitotic chromosomes. Nevertheless, this is an important first example of dynamic regulation of MCB by a TF, in the context of a cell fate transition.A recent study in  neurons . Mitotic neurons ,80. StriHow can modulation of MCB and changes in the temporal pattern of gene reactivation impact the activity of TFs with a role in cell fate decisions? The mutually exclusive nature of alternative cell fates results often from antagonistic cross-regulatory interactions between their associated transcriptional programmes. Thus, the ability of one of such TFs to reactivate its target genes early during M-G1, may confer a significant advantage over others. In this context, one interesting possibility is that signalling pathways regulating cell fate decisions may operate at one level by governing the association of TFs with mitotic chromosomes, and consequently the timing of reactivation of their programmes . Investi


Transient receptor potential M3 (TRPM3) cation channels regulate numerous biological functions, including gene transcription. Stimulation of TRPM3 channels with pregnenolone sulfate activates stimulus-responsive transcription factors, which bind to short cognate sequences in the promoters of their target genes. In addition, coregulator proteins are involved that convert the chromatin into a configuration that is permissive for gene transcription. In this study, we determined whether TRPM3-induced gene transcription requires coactivators that change the acetylation pattern of histones. We used compound A485, a specific inhibitor of the histone acetyltransferases CBP and p300. In addition, the role of bromodomain proteins that bind to acetylated lysine residues of histones was analyzed. We used JQ1, an inhibitor of bromodomain and extra terminal domain (BET) family proteins. The results show that both compounds attenuated the activation of AP-1 and CREB-regulated gene transcription following stimulation of TRPM3 channels. Inhibition of CBP/p300 and BET proteins additionally reduced the transcriptional activation potential of the transcription factors c-Fos and Elk-1. Transcriptional upregulation of the interleukin-8 gene was attenuated by A485 and JQ1, indicating that proinflammatory cytokine expression is controlled by CBP/p300 and bromodomain proteins. We conclude that TRPM3-induced signaling involves transcriptional coactivators and acetyl-lysine-bound bromodomain proteins for activating gene transcription. TRPM3  channels are nonselective cation channels that can be activated with the neurosteroid pregnenolone sulfate ,2. NumerThe signaling cascade triggered by stimulation of TRPM3 channels leads to binding of stimulus-activated transcription factors to short DNA sequences in the promoters of their target genes. In addition, transcription factors interact with numerous coregulator proteins to decompact repressive chromatin structures. Chromatin comprises DNA complexed with histone proteins. The DNA packaging in chromatin is essential for gene transcription because the chromatin can be compact so that transcriptional regulators cannot bind to the DNA, or it can have an open configuration that allows interactions between DNA and transcription factors and recruitment of RNA polymerase II to DNA. Transcriptional activators bind to various coregulator proteins that either function as chromatin-modifying enzymes or are part of ATP-dependent protein complexes that modulate the chromatin status. The architecture of the chromatin is modified by enzymes that transfer or remove post-translational modifications of histones. One important modification is acetylation ,9. HistoHere, we investigated whether TRPM3-induced gene transcription depends on the recruitment and biological activity of coactivators that change the acetylation pattern of histones. We used a pharmacological approach to inhibit the histone acetyltransferase activities of the paralogs CBP (CREB-binding protein) and p300. Both proteins are master regulators of gene transcription that interact with numerous transcription factors and modify their activities ,14,15. IPharmacological and genetic experiments have shown that stimulation of TRPM3 induces transcription mediated by the transcription factor activator protein AP-1 ,3,6. We 2+ channels -2,3-dihydro-5-[[(methylamino)carbonyl]amino]-2′,4′-dioxo-N-spiro -3′-acetamide) was purchased from Tocris , dissolved in DMSO and used at a final concentration of 3 μM. The BET bromodomain inhibitor JQ1 (PubChem ID 46907787),(6S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno triazolo diazepine-6-acetic acid 1,1-dimethylethyl ester) was purchased from Adooq Bioscience , dissolved in DMSO and used at a final concentration of 0.5 μM. The chemical structures of A485 and JQ1 are depicted in ((1The lentiviral transfer vectors pFUW-C2/CREB, pFUW-GAL4-c-Fos, and pFUW-GAL4-Elk-1 have been described elsewhere ,42. The 5Sp12.luc, and pFWc-FosCRE4.luc have been described elsewhere [The lentiviral transfer vectors pFWColl.luc, pFWIL-8.luc, pFWUASlsewhere ,16,42. Tt-test. Statistical probability is expressed as * p < 0.05, ** p < 0.01, and *** p < 0.001. Values were considered significant when p < 0.05.Statistical analyses were done by using the two-tailed Student’s 2+ channel stimulation. The studies have shown that Ca2+ ions, protein kinases, and calcineurin link TRPM3 channel activation to transcription factor stimulation. The present study provides evidence that epigenetic modulators are required in addition to the sequence-specific binding of transcription factors to DNA. CBP/p300 are histone acetylases that acetylate lysine residues of histones and in this way induce an open chromatin structure. Our pharmacological analysis revealed that the coactivators CBP and p300 are essential for connecting TRPM3 stimulation with gene transcription. Acetylated lysine residues function as recognition motives for epigenetic BET proteins that further regulate epigenetic-controlled transcription via the recruitment of transcription factors, co-activators, and chromatin-organizing proteins. The fact that inhibition of BET protein interaction with acetyl-lysine residues attenuates TRPM3-mediated gene transcription indicates that BET proteins are important epigenetic regulatory proteins of TRPM3-mediated transcription. Based on these data, we propose a chromatin-based signaling cascade, involving DNA-bound stimulus-responsive transcription factors, recruitment of CBP/p300, acetylation of histones, and subsequent recruitment of the BET proteins to the transcription unit (In recent years, we have described in detail the intracellular signaling cascade triggered by TRPM3 Caion unit . Future Transcription factors such as CREB and AP-1 bind to specific DNA binding sites and recruit the coactivator and histone acetyltransferase proteins CBP and p300. These proteins catalyze the acetylation of histones, and by this means generate an open chromatin architecture. CBP/p300 also acts as a bridge to the basal transcriptional apparatus to support transcriptional activation. BET proteins bind to acetylated lysine residues in histone proteins and recruit coactivators and epigenetic regulators required for transcription.


Among the AP2/ERF transcription factor family, group VII ERF (ERF-VII) genes, such as RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, and AtERF71/HRE2, are known to be involved in the response to hypoxia in Arabidopsis. Notably, HRE2 has been reported to be involved in responses to hypoxia and osmotic stress. In this study, we dissected HRE2 promoter to identify hypoxia- and salt stress-responsive region(s). The analysis of the promoter deletion series of HRE2 using firefly luciferase and GUS as reporter genes indicated that the −116 to −2 region is responsible for both hypoxia and salt stress responses. Using yeast one-hybrid screening, we isolated HAT22/ABIG1, a member of the HD-Zip II subfamily, which binds to the −116 to −2 region of HRE2 promoter. Interestingly, HAT22/ABIG1 repressed the transcription of HRE2 via the EAR motif located in the N-terminal region of HAT22/ABIG1. HAT22/ABIG1 bound to the 5′-AATGATA-3′ sequence, HD-Zip II-binding-like cis-regulatory element, in the −116 to −2 region of HRE2 promoter. Our findings demonstrate that the −116 to −2 region of HRE2 promoter contains both positive and negative cis-regulatory elements, which may regulate the expression of HRE2 in responses to hypoxia and salt stress and that HAT22/ABIG1 negatively regulates HRE2 transcription by binding to the HD-Zip II-binding-like element in the promoter region.In the signal transduction network, from the perception of stress signals to stress-responsive gene expression, various transcription factors and  Transcription factors can control the expression of many target genes via the specific binding to the cis-regulatory element in the promoters of the respective target genes [Arabidopsis thaliana), such as AP2/ERF, bZIP, zinc finger, WRKY, MYB, bHLH, and NAC families [Abiotic stresses have been shown to regulate the expression of genes with various functions in a variety of plants . In the et genes . Severalfamilies ,4.RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, and AtERF71/HRE2, in response to hypoxia has been demonstrated [cis-regulatory element, HRPE, and activate downstream genes such as LBD41 and PCO1 under hypoxic conditions, whereas HRE1 and HRE2 bind to the GCC box [The AP2/ERF family is a large group of plant-specific transcription factors with 145 members in Arabidopsis, and these 145 genes are classified into the following four subfamilies: AP2, ERF, DREB/CBF, and RAV subfamilies [nstrated . In resp GCC box ,9,10.RAP2.2 is transactivated by WRKY33 and WRKY12 under hypoxic conditions via the W-box, 5′-AGTCAA-3′, present in its promoter, while WRKY33 and WRKY12 do not regulate the other four ERF-VII genes [cis-regulatory element(s) in the promoters of ERF-VII genes, except RAP2.2, have not yet been studied.The expression of ERF-VII genes is regulated at transcriptional and/or post-transcriptional levels. Post-transcriptional regulation of ERF-VII is mediated by N-degron pathway-targeted sequences in their N-terminal regions . In termII genes . In tranAtHB7 and AtHB12 function as negative regulators of abscisic acid (ABA) response in Arabidopsis [AtHB13 is positively regulated by low temperature, drought, and salinity and overexpression of AtHB13 confers cold, drought, and broad-spectrum disease resistance [AtHB2, HAT1, HAT2, HAT3, and AtHB4 are rapidly induced by changes in the red/far-red ratio light and promote shade avoidance, a process regulated at multiple levels by auxin [Homeodomain-leucine zipper (HD-Zip) family is unique to plants, and is characterized by the presence of a homeodomain closely linked to a leucine zipper motif . A totalbidopsis . AtHB13 sistance ,16,17. Aby auxin ,19. In aby auxin ,22,23,24AtHB2, HAT2, HAT3, and AtHB4, and is involved in the shade avoidance response [HAT22/ABIG1, a member of the HD-Zip II subfamily, is required for ABA-mediated growth inhibition under drought conditions [HD-Zip proteins are transcription factors that function as positive or negative regulators of gene expression . Among tresponse ,29. Recenditions . Howevernditions , its funHRE2 has been reported to be involved in both the hypoxia and osmotic stress responses [HRE2 transcription increases under hypoxic, salt, and drought stress conditions, and HRE2-overexpressing transgenic plants (OXs) are more tolerant to flooding, salt, and drought stresses. In addition, the promoter activity of HRE2 is increased by hypoxia and salt stress [HRE2 to identify the abiotic stress-responsive region(s). Promoter analysis using two reporter genes has indicated that −116 to −2 region of HRE2 promoter is responsible for hypoxia and osmotic stress responses and contains both positive and negative cis-regulatory elements. In addition, HAT22/ABIG1, a member of HD-Zip II, binds to the −116 to −2 region via a 7-bp negative cis-regulatory element to repress the transcription of HRE2.Previously, esponses ,35. HRE2HRE2 responds to hypoxia and salt treatment in Arabidopsis transgenic plants [HRE2. To this end, we generated constructs of firefly luciferase genes controlled by the −180 to +212, −116 to +212, −2 to +212, and +52 to +212 regions from the transcriptional start site of HRE2 promoter  involved in the response to hypoxia.We further confirmed this result by measuring GUS activity controlled by the same promoter deletion series of er assay c. For ther assay d. These HRE2 promoter in Arabidopsis plants. To this end, we generated Arabidopsis transgenic plants harboring firefly luciferase gene controlled by the −180 to +212 region from the transcriptional start site of HRE2 promoter  responsible for hypoxia response.To reconfirm the positive response of the −116 to −2 region of toplasts a. The lo control a. Tandempromoter b, demonsHRE2 promoter, we performed yeast one-hybrid screening using a cDNA library of Arabidopsis seedlings subjected to hypoxia, in which cDNAs were fused to the GAL4 activation domain (AD). As a result of the screening, a total of 25 positive colonies were obtained from 8.8 × 105 yeast transformants by growth assay using HIS3 and ADE2 as reporter genes (To isolate the transcription factor(s) that bind to the −116 to −2 region of er genes . Plasmider genes . Intereser genes .AUR1-C or lacZ reporter genes controlled by the tandem repeats of the −116 to −2 region of HRE2 promoter. Based on the yeast growth and β-galactosidase orthonitrophenyl-β-D-galactopyranoside (ONPG) assays, At4g37790 transactivated the reporter genes most strongly , in which 52 aa of the N-terminus of HAT22/ABIG1, including the EAR motif, were deleted. We then analyzed the effect of ΔN52 HAT22/ABIG1 on the firefly luciferase activity controlled by the tandem repeats of the −116 to −2 region of HRE2 promoter  (data not shown), suggesting that ΔN52 HAT22/ABIG1 is translocated to the nucleus and binds to the −116 to −2 region of HRE2 promoter. Indeed, GFP signal of the sGFP-ΔN52 HAT22/ABIG1 construct was observed in the nucleus  and transformed into yeast. As expected, HAT22/ABIG1 did not show transactivation activity in the yeast growth and β-galactosidase ONPG assays .HRE2 promoter contains 5′-AATGATA-3′ (−69 to −63 region) in its hypoxia- and salt-responsive −116 to −2 region ATT-3′ . Our res2 region . This obTGAGG-3′ a. The coTGAGG-3′ b. In theTGAGG-3′ . These rluciferase gene under the control of four tandem repeats of the 16 bp HRE2 promoter region and co-transformed it with HAT22/ABIG OX construct into Arabidopsis protoplasts  results showed that the expression of HAT22/ABIG1 increased at 1 h after being subjected to hypoxia and then gradually decreased until 8 h after hypoxia treatment . In addition, we isolated HAT22/ABIG1 as a transcriptional repressor of HRE2 transcription in responses to hypoxia and salt stress, and identified a negative cis-regulatory element bound by HAT22/ABIG1 in HRE2 promoter.HRE2 is a member of the ERF-VII transcription factor group in Arabidopsis, and the ERF-VII group is well known to be involved in the hypoxia response in plants . The ERFHRE2 is involved in responses to both hypoxia and salt stress and that the 180 bp promoter of HRE2 includes positive cis-regulatory element(s) responsible for these responses [HRE2, we first analyzed the region of HRE2 promoter responsible for hypoxia and salt stress responses. The analysis using firefly luciferase and GUS as reporter genes controlled by deletion series of the 180 bp HRE2 promoter showed that the −116 to −2 region of HRE2 promoter includes positive cis-regulatory element(s) responsible for both hypoxia and salt stress responses , software for the analysis of plant cis-regulatory element(s) in the promoter. However, we could not find candidate(s) for hypoxia-responsive positive cis-regulatory element(s) (data not shown).We have previously reported that esponses . To elucesponses . We analHRE2 promoter  that activate HRE2 transcription remain unidentified in this study, further studies using the −116 to −2 region of HRE2 promoter are needed to isolate and characterize the transcriptional activators.Using yeast one-hybrid screening, we isolated HAT22/ABIG1, a member of the HD-Zip II subfamily, which binds to the −116 to −2 region of promoter . It has  element . The yeant  and repressor(s).Gene expression is tightly regulated by transcriptional activators and repressors. Regulation of the balance between activators and repressors is important for proper gene expression and responses to abiotic stresses . DREB1/Celopment . NAC016 ignaling ,39. Our RAP2.2 is transactivated by WRKY33 and WRKY12 in the hypoxia response via the W-box, 5′-AGTCAA-3′, in RAP2.2 promoter. However, HRE2 and HRE1 are not regulated by WRKY33 and WRKY12 [HRE2 promoter does not contain the W-box (data not shown). On the other hand, RAP2.12 and RAP2.2 transactivate downstream genes via HRPE, a hypoxia-responsive cis-regulatory element, whereas HRE1 and HRE2 transactivate downstream genes via the GCC box [The ERF-VII group of the AP2/ERF family can be divided into two types, namely, the RAP-type, which includes RAP2.12, RAP2.2, and RAP2.3, and the HRE-type, which includes HRE1 and HRE2 . Recentld WRKY12  and our  GCC box ,8,9,10. HRE2 promoter contains both positive and negative cis-regulatory elements involved in hypoxia and salt stress responses and that HAT22/ABIG1 transcriptionally represses HRE2 via 5′-AATGATA-3′ sequence, which is a negative cis-regulatory element present in the −116 to −2 region.Taken together, our results demonstrate that the −116 to −2 region of Arabidopsis thaliana plants used in this study were of the Columbia (Col-0) ecotype. Arabidopsis seeds preparation, germination, and growth were performed according to previous study [All us study .HRE2 promoter, −180 to +212, −116 to +212, −2 to +212, and +52 to +212 regions from the transcriptional start site of HRE2 were amplified by PCR and cloned into pFGL1495 or pFGL539 fused with firefly luciferase or GUS, respectively. Two tandem repeats of −116 to −2 region of HRE2 promoter were cloned into pFGL1437 fused with firefly luciferase.To generate deletion series of HRE2 were amplified by PCR and cloned into pAbAi or pLacZi fused with AUR1-C or lacZ, respectively. The full-length ORF of HAT22/ABIG1 was amplified by PCR and cloned into pGADT7 in-frame with GAL4 AD.To construct plasmids for the yeast one-hybrid assay, the promoter regions of HRE2 were amplified by PCR and cloned into pFGL1437 fused with firefly luciferase.To generate plasmids for the transrepression assay in Arabidopsis protoplasts, the promoter regions of The primers for cloning are listed in Agrobacterium tumefaciens strain GV3101 (pMP90) using the freeze–thaw method [The constructs for expression in Arabidopsis were transformed into w method  and thenw method . Transge2 gas under dark conditions for 0, 1, 2, 4, and 8 h.For the hypoxia treatment, 10-day-old WT seedlings grown on MS plates were transferred to MS medium-saturated filter paper and then were treated with 99.99% NFor NaCl and mannitol treatments, 10-day-old WT seedlings grown on MS plates were transferred to filter papers saturated with MS medium containing 150 mM NaCl or 300 mM mannitol and kept for 0, 1, 2, and 4 h.GUS activity was detected histochemically following a previously described protocol .Arabidopsis protoplast isolation and transformation were conducted according to Yoo et al. .® Dual-Luciferase® Reporter Assay System  and the GloMax®-Multi+ Detection System , in accordance with the manufacturer’s instructions.Luciferase activity was quantified using the Nano-Glo7. Yeast one-hybrid screening was performed using Matchmaker® Gold Yeast One-Hybrid Library Screening System. pADE2i harboring two tandem repeats of the −116 to −2 region of HRE2 promoter was used as the bait in yeast one-hybrid screening. cDNA library generation and yeast one-hybrid screening were performed by PanBioNet .To generate a hypoxia cDNA library, 7- and 14-day-old seedlings grown under short-day conditions were subjected to hypoxia for 1 and 3 h. Total RNA was isolated using RNAqueous Kit  and Plant RNA Isolation Aid . Subsequently, cDNA library was generated using Make Your Own “Mate & Plate” Library System . The cfu value of the cDNA library was 1.43 × 10TM Kit , in accordance with the manufacturer’s instructions. A quantitative β-galactosidase assay was performed using ONPG as a substrate. The unit of β-galactosidase activity was calculated using the formula 1000 × OD420/(OD600 × assay time in min × assay volume in mL). Transformants were analyzed using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as a substrate for the β-galactosidase filter assay. The reaction was carried out for 6 h. For the yeast growth assay, transformants were streaked onto synthetic minimal media lacking leucine and uracil containing 150 ng/mL Aureobasidin A (AbA) and incubated for 3–5 days at 30 °C.The constructs for the yeast one-hybrid assay were transformed into Y1HGOLD or YM4271. Yeast transformation was performed by the Frozen-EZ Yeast Transformation IITM 3 real-time PCR system , and QuantStudioTM Design and Analysis software v.1.4.3  in accordance with the manufacturer’s manual. Three independent reactions were conducted for each technical replicate. Two technical replicates were conducted for each biological replicate. The primers for RT-qPCR are listed in Total RNA was isolated by RNAqueous Kit  and Plant RNA Isolation Aid  in accordance with the manufacturer’s protocol. Two micrograms of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase . RT-qPCR was performed and analyzed using Power SYBR Green PCR Master mix , QuantStudioStatistical analysis was performed by IBM SPSS Statistics software version 23  with one-way ANOVA using Tukey’s multiple comparison test.


Nature Communications 10.1038/s41467-020-20543-z (2021)1 explored the cause-and-effect relationship between histone acetylation and transcription and reached the conclusion that transcription is a prerequisite for histone acetyl transferase (HAT) activity at both promoters and gene bodies. This thought-provoking proposal is particularly surprising with respect to gene promoters since it contradicts the widely accepted model in which histone acetylation occurs upstream of, and typically stimulates, RNA polymerase II (RNAPII) recruitment and transcription initiation. The conclusions in this paper are based upon the assumption that 1,10 phenanthroline , the primary agent used in this study to inhibit RNAPII, has no effect on the transcription factors (TFs) that recruit HAT and histone deacetylase (HDAC) complexes to promoters. We show here that this is not the case since 1,10-pt treatment rapidly alters major signaling pathways that have profound genome-wide effects on the localization and activity of TFs, as well as HAT and HDAC complexes. The causal relationship between promoter nucleosome acetylation and RNAPII transcription thus cannot be inferred using this experimental approach.A recent study by Martin et al.4, and a rapid (<10 min) decrease of both transcription and promoter histone acetylation at these genes5. The concomitancy of these events makes it difficult to infer a cause-effect relationship between RNAPII activity and acetylation.Establishment of histone acetylation patterns at gene promoters probably involves a complex balance between the opposing activities of HAT and HDAC complexes, modulated by condition-dependent binding or activity of TFs, which recruit these complexes to promoters. For example, two major growth-promoting TFs in yeast, Ifh1 and Sfp1, are rapidly displaced from ribosomal protein (RP) and ribosomal biogenesis (RiBi) gene promoters, respectively, following inhibition of TORC1 kinase. This is associated with release of the essential NuA4 HAT complex, the simultaneous recruitment of the Rpd3L HDAC6 used a thermosensitive allele of the large subunit of RNAPII (rpb1-1) to inhibit mRNA synthesis and concluded that promoter histone acetylation is not affected by transcription arrest. However, Martin et al.1 noted that subsequent studies raised concerns regarding the efficiency of Rpb1 inactivation at the non-permissive temperature and they thus chose to use the transcription inhibitor 1,10-pt to test the dependence of acetylation on transcription.One possible experimental approach towards untangling this relationship would be to rapidly block transcription initiation without affecting the activity of major growth or stress signaling pathways, which are known to modulate HAT and HDAC targeting at promoters. A previous study1 Rpb3 ChIP-seq data. For example, peaks of RNAPII binding are induced by 1,10-pt at the promoters of all Heat Shock Factor 1 (Hsf1) target genes (defined in ref. 7) and at plasma membrane transporter genes that are specifically up-regulated in response to environmental stress or drug treatment . Interestingly, in addition to Hsf1 activation, RASTR causes strong down-regulation of nearly all RP genes14, an effect also observed in the Martin et al.1 Rpb3 ChIP-seq data.None of the above should come as a surprise since two previous studies indicated that 1,10-pt induces a heat shock-like response11. Consistent with this view, Eshelman et al.11 demonstrated a rapid (<10 min) decrease in nuclear Sfp1 following 1,10-pt addition, concomitant with an increase in the nuclear import of Dot6, which recruits the Rpd3L HDAC to many RiBi and RiBi-like genes2. It would therefore appear that 1,10-pt causes widespread changes in activator and repressor binding, which in turn drives a major genome-wide redistribution of HDAC and HAT complexes. Consistent with this notion, Epl1 ChIP-seq analysis of the NuA4/Piccolo HAT complex by Martin et al.1 reveals important changes following 1,10-pt treatment  due to TORC1 inhibition by 1,10-pt1 analysis of their Epl1 ChIP-seq data, which they use to argue that 1,10-pt treatment has no significant effect on promoter binding of the essential NuA4 HAT complex. They focus on a set of 562 genes that display peaks of Epl1 promoter binding and claim, showing only average plots, that binding remains high following 1,10-pt treatment. However, their average plots hide the fact that this group of genes responds in a very heterogenous manner, with a subgroup displaying a dramatic binding increase in response to 1,10-pt treatment , Hsf1 target (n = 34), and Epl1 binding increase genes (n = 123) from all other genes (n = 307). This shows that Epl1 ChIP-seq signal significantly decreases at most of these 562 genes following 1,10-pt treatment  contend that if one looks globally at the ~5000 promoters where they report Epl1 binding one finds little of no effect of 1,10-pt treatment. However, Epl1 binding is at background levels at most promoters (the reader is invited to examine the data on a genome browser), and the same is true for other NuA4 subunits mapped by ChIP-exo (see http://yeastepigenome.org/). It is thus not possible to know from these data whether Epl1 (or NuA4) binding is altered by 1,10-pt treatment genome-wide. Nevertheless, at those (562) genes where Epl1 promoter binding is detectable by ChIP-seq above background levels, 1,10-pt treatment mostly leads to a significant decrease in binding  whose binding and activity they modulate. The most promising approaches may be rapid and specific depletion of either RNAPII or the TATA binding protein (Tbp1), using the anchor-away or auxin-induced degron (AID) methods. Martin et al.1 have in fact performed an AID experiment targeting the Rpb2 subunit of RNAPII, but they report only a few histone western blots on this strain which, interestingly, show an overall weaker decrease in acetylation compared to 1,10-pt treatment, even though the depletion would appear to be complete. Further characterization of the Rpb2-AID strain, or readily available anchor-away strains targeting Rpb1 or Tbp1, are likely to clarify the causal relationship between histone acetylation at both promoters and gene bodies and RNAPII promoter recruitment and transcription.In summary, we argue that the claim made by Martin et al.1. ChIP-seq signal was quantified for each promoter defined as the 400 bp region upstream of the transcription start site (TSS), as obtained from ref. 15. To calculate ChIP-seq signals fold change, a ratio between the total number of reads for each promoter from 1,10-pt treated cells and the number of reads from untreated cells was made. RP gene list was defined in ref. 14 and Hsf1 gene list in ref. 7.ChIP-seq datasets (Replicate 1) from 1,10-pt-treated or untreated cells were normalized to silent regions by ChIP Normalization Factors calculated in ref. https://yeastmine.yeastgenome.org/yeastmine/begin.do) was used to determine GO-terms and MEME16 was used for motif enrichment analysis, with the following settings: anr -nmotifs 5 -minw 6 -maxw 20 -objfun classic -revcomp -markov_order 0.Yeast Mine .In all of the box plots, the box shows the 25th–75th percentile, whiskers show the 10th–90th percentile, and dots show the 5th and 95th percentiles. Asterisks show significant difference according to the Wilcoxon test (*Further information on research design is available in the Description of additional Supplementary FileSupplementary data 1Supplementary data 2Reporting SummaryPeer Review File