To understand in detail the transcriptional and functional overlap of IFN-I- and IFN-II-activated responses, we used an integrative RNAseq-ChIPseq approach in Huh7.5 cells and characterized the genome-wide role of pSTAT1, pSTAT2, IRF9 and IRF1 in time-dependent ISG expression. For the first time, our results provide detailed insight in the timely steps of IFNα- and IFNγ-induced transcription, in which pSTAT1- and pSTAT2-containing ISGF3 and GAF-like complexes and IRF1 are recruited to individual or combined ISRE and GAS composite sites in a phosphorylation- and time-dependent manner. Interestingly, composite genes displayed a more heterogeneous expression pattern, as compared to GAS (early) and ISRE genes (late), with the time- and phosphorylation-dependent recruitment of GAF, ISGF3 and IRF1 after IFNα stimulation and GAF and IRF1 after IFNγ. Moreover, functional composite genes shared features of GAS and ISRE genes through transcription factor co-binding to closely located sites, and were able to sustain IFN responsiveness in STAT1-, STAT2-, IRF9-, IRF1- and IRF9/IRF1-mutant Huh7.5 cells compared to Wt cells. Thus, the ISRE + GAS composite site acted as a molecular switch, depending on the timely available components and transcription factor complexes. Consequently, STAT1, STAT2 and IRF9 were identified as functional composite genes that are part of a positive feedback loop controlling long-term IFNα and IFNγ responses. More important, in the absence of any one of the components, the positive feedback regulation of the ISGF3 and GAF components appeared to be preserved. Together, these findings provide further insight in the existence of a novel ISRE + GAS composite-dependent intracellular amplifier circuit prolonging ISG expression and controlling cellular responsiveness to different types of IFNs and subsequent antiviral activity. It also offers an explanation for the existing molecular and functional overlap between IFN-I- and IFN-II-activated ISG expression.The online version contains supplementary material available at 10.1007/s00018-023-04830-8. Interferons (IFN), initially discovered as antiviral proteins, can be divided into three subtypes IFN-I, IFN-II, and IFN-III. IFN-II or IFNγ, also named immune IFN, binds to the IFNγ receptor (IFNGR) and facilitates a more general immune response to pathogens. IFN-II is mainly produced by T lymphocytes and Natural Killer (NK) cells and modulates adaptive immunity as well as inflammation in response to foreign antigens or mitogens. Produced by many cell types, IFN-I is the main antiviral IFN subtype, mainly constituting IFNα and IFNβ subtypes. As part of a robust innate immune response, IFN-I collectively binds to the IFNα receptor (IFNAR) to potently inhibit viral infection. IFN-III consists of IFNλ1 to -4 subtypes, which largely are produced in a tissue-specific manner and bind the heterodimeric IFN-λ receptor (IFNLR) expressed only at certain cell types and tissues. These include macrophages, dendritic cells, plasmacytoid dendritic cells and respiratory epithelial cells. As such, IFN-λ confers more localized antiviral responses at the site of infection .Although IFN-I and IFN-II bind different cell-surface receptors expressed on most cell types, they both induce IFN-stimulated gene (ISG) expression through Janus kinase (JAK)-dependent phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT2. STAT1 homodimers, known as γ-activated factor (GAF), directly activate transcription of ISGs containing the IFN-II activation site  in response to both types of IFN. STAT1-STAT2 heterodimers together with interferon regulatory factor (IRF)9 (known as ISGF3) in response to IFNα expands the range of regulatory elements that can be targeted by the JAK-STAT pathway to the IFN-stimulated response element  .II (heterodimer of pSTAT1 and unphosphorylated STAT2 together with IRF9), have been identified to regulate IFNγ-activated transcription of ISRE genes , with a distance varying between 0 and < 415 bp (Table S4). Analyzing recruitment of pSTAT1, pSTAT2, IRF9 and/or IRF1 to these 211 common genes revealed different binding characteristics . As compared to wt cells , knocking out STAT1 dramatically delayed and prolonged the phosphorylation pattern of STAT2 in response to IFNα, which correlated with the prolonged expression pattern of STAT2, IRF1 and IRF9 . The response to IFNγ in these cells was completely abrogated, as marked by the absence of phosphorylated STAT2 and expression of IRF1 and IRF9 . Knocking out STAT2 resulted in a significant decrease in STAT1 phosphorylation levels, displaying a bi-phasic character in response to IFNα and a clear transient pattern after IFNγ treatment. IRF1 expression clearly correlated with this phosphorylation pattern of STAT1, with a more prolonged expression after IFNα treatment as compared to early and transient in response to IFNγ. In contrast, the expression of STAT1 and IRF9 under these conditions exhibited a prolonged character and increased even after long-term treatment, similar to wt cells . Knocking out IRF9 predominantly effected the IFNα response, with a weaker and more transient phosphorylation pattern of STAT1 and STAT2 slowly decreasing over time until 72 h. IRF1 expression clearly correlated with this phosphorylation pattern of STAT1 and STAT2, being transient but more prolonged as compared to wt cells. In contrast, the expression of STAT1 and STAT2 under these conditions exhibited a prolonged character and increased even after long-term treatment, similar to wt cells. The response to IFNγ in these cells was similar to wt cells, displaying a transient phosphorylation of STAT1 reflecting the increase in IRF1 expression. Expression of STAT1 and STAT2 exhibited a prolonged character and increased even after long-term treatment . IRF1KO cells responded opposite to IRF9KO cells, with a transient phosphorylation pattern for STAT1 and STAT2 after IFNα treatment (that mirrored wt cells), and prolonged phosphorylation of STAT1 after IFNγ stimulation. In these cells, both types of IFN induced a similar expression pattern of STAT1, STAT2 and IRF9, however being more prolonged as observed in IRF9KO cells . Finally, in IRF1/IRF9 double KO cells, phosphorylation of STAT1 and STAT2 in response to IFNα and STAT1 in response to IFNγ exhibited an extremely prolonged pattern. More important, in IRF1/IRF9 double KO cells, the IFNα- and IFNγ-mediated increase in STAT1 and STAT2 proteins was still observed .Together, this clearly shows that in the absence of any one of the components, IFNα- and IFNγ-activated responses still occur, except in IFNγ-treated STAT1 mutant cells, and that the positive feedback regulation of the ISGF3 and GAF components is preserved.A striking observation after comparing IFNα and IFNγ commonly up-regulated GAS genes revealed the differential recruitment of GAF and GAF-like complexes depending on the type of IFN Fig. B. Close To further characterize the dependence of IRF1 and ICAM1 on STAT1 and STAT2, we compared their expression in wt and STAT1-, STAT2-, IRF9-, IRF1- and IRF9/IRF1-mutant Huh7.5 cells Fig. C. As expIn contrast to GAS genes, binding characteristics of ISRE genes clearly pointed to the regulatory role of ISGF3 together with IRF1. Close examination of the known ISRE-containing genes ISG15, MX1, IFIT12 and OAS2, by ChIP-seq Fig. D and of Site-directed mutagenesis confirmed the functionality of the proximal ISRE sites in the promoters of the ISG15 and IFIT2 genes Fig. F, displaFurther characterization of the expression of ISG15 and IFIT2 in wt and STAT1-, STAT2-, IRF9-, IRF1- and IRF9/IRF1-mutant Huh7.5 cells Fig. E, revealThese results were further verified by quantitative ChIP-PCR, and demonstrated to correlate with binding of all ISGF3 components and IRF1 to ISRE motifs in ISG15 and IFIT2 in response to IFNα Fig. E, and maAs mentioned above, composite genes exhibited combined features of GAS and ISRE-containing genes, with the collective recruitment of pSTAT1, pSTAT2, IRF9 and IRF1 after IFNα stimulation and pSTAT1 + IRF1 and less frequently IRF9 after IFNγ  and the sustained expression and TF binding in mutant cell lines Fig. C, this pAs shown in Fig. STAT1 and STAT2 in a time-dependent manner (even after 72 h) in IFNα-treated cells and pSTAT1 and IRF1 and weak IRF9 to these genes in response to IFNγ   or without (IRF9) IRF1 in WT cells after IFNα stimulation. Likewise, collective pSTAT1 + IRF1 and weak IRF9 binding could be observed after IFNγ  was detected. As expected, for all three genes no pSTAT2 binding could be detected upon IFNγ treatment.Remarkably, IFN-induced expression of S7) Fig. E, which S7) Fig. E, with cFNγ Fig. E, exceptSTAT1, STAT2 and IRF9 in STAT1KO cells was marked by a delayed response and shift in maximum gene expression towards 72 h  and IRF1 binding, pointed to a shift from an ISGF3/ISRE dependent to a pSTAT1 + IRF1/composite site-dependent mechanism. For IRF9, the absence of IRF1 binding under these conditions pointed to a shift from an ISGF3/ISRE-dependent to a pSTAT1 + pSTAT2/GAS (in case of IFNα) or pSTAT1/GAS (in case of IFNγ) site-dependent mechanism. In the IRF1KO cells, intact expression of all three genes in response to IFNα correlated with recruitment of all ISGF3 components to the ISRE site. The IFNγ response of STAT1, STAT2 and IRF9 in these cells was marked by strong pSTAT1 and weak IRF9 recruitment , could recognize the binding to typical ISRE or GAS-containing genes, but also to ISRE and GAS composite sites. Together with the results from our integrative analysis this offers clear evidence for the existence of a GAS + ISRE composite gene group, which together with GAS and ISRE genes are commonly involved in IFNα- and IFNγ-mediated transcriptional responses through recruitment of STAT1- and STAT2-containing ISGF3 and GAF-like complexes and IRF1 in a time- and phosphorylation-dependent manner.Historically, IFNα signaling was perceived as a pre-dominant ISRE-dependent ISGF3-mediated response  and IRF1Accordingly, early expression of GAS genes was supported by early and transient binding of pSTAT1 alone (GAF) or pSTAT1 together with pSTAT2 (GAF-like) in response to IFNα. This coincided with individual or combined roles of GAF and GAF-like complexes and corresponded with the presence of a single functional GAS element and no ISRE in their proximal promoter. Upon IFNγ treatment only pSTAT1 was recruited, which was stronger and more prolonged and pointed to the sole role of GAF. This also correlated with the prolonged expression of GAS genes in response to IFNγ. In contrast, later expression of ISRE genes in response to both types of IFN correlated with collective recruitment of all ISGF3 components  with or without IRF1, or only IRF1 in response to IFNα. IFNγ predominantly directed IRF1 binding to these genes, but less frequently and with lower affinity also IRF9 and pSTAT1. This corresponded with the presence of a functional ISRE element and no GAS in their proximal promoter. In addition, it further highlighted the exclusive IFN-dependent binding of ISGF3  as opposed to IRF1 which exhibited binding to these ISRE genes also under basal conditions. Obviously, IFN-dependent binding of ISGF3 components and/or IRF1 to these ISRE genes appeared later as compared to GAF and GAF-like complexes to GAS genes. Moreover this binding to ISRE genes was still clearly detectable after 72 h and correlated with long-term ISG expression. This pointed to the later recruitment of ISGF3 and IRF1 to ISRE sites and correlated with the time-dependent transcriptional differences of GAS (early) and ISRE (later) genes. Stronger binding of ISGF3 components to ISRE sites as compared to GAS, reflected the potency of ISGF3 and the higher induction of ISRE genes as compared to GAS genes.Finally, the newly identified group of composite genes displayed a more heterogenous expression profile in response to both types of IFN, resembling that of GAS (Early) and ISRE (Later) genes. The combined recruitment of pSTAT1, pSTAT2, IRF9 and IRF1 after IFNα stimulation and pSTAT1 + IRF1 and less frequently IRF9 after IFNγ correlated with the close proximity of GAS and ISRE sites, and revealed combined binding features of GAS and ISRE-containing genes. IFNα-dependent binding of ISGF3 components to these composite genes appeared earlier (and stronger) as compared to ISRE genes, and also correlated with the more dominant role of the ISRE site as compared to the GAS site under these conditions. Our data are, therefore, suggestive of a minor role of GAF or GAF-like complexes in early activation followed by a major role of ISGF3 and IRF1 in maintaining long-term IFNα-activated expression of composite genes. On the other hand, IFNγ-activated long-term transcriptional responses of composite genes clearly depended on both GAS and ISRE sites, directed by time-dependent recruitment of GAF and IRF1. Together, this could explain the more heterogeneous nature of IFN-dependent composite gene expression, being later (and more prolonged) than GAS genes upon IFNα treatment and earlier than ISRE genes in IFNγ-treated cells.More important, our results provide further proof that ISRE and GAS sites in composite genes are functionally important, either alone or in a collaborative fashion, in response to both types of IFN through co-binding of GAF, GAF-like, ISGF3 and IRF1 complexes. Technical limitations of our ChIPseq experiments, however, can’t rule out the existence of gene fractions that are occupied by only one TF and not the other. The mechanism of STAT and IRF co-binding in transcriptional regulation of ISRE and GAS-containing ISGs is a known phenomenon. For example, both STAT1 and IRF1 were shown to control the IFNγ-induced expression of CIITA, GBP1, and gp19 , 13, 53.Our integrative analysis also resulted in the identification of a selective group of IFNα- and IFNγ-specific genes, which in general displayed lower expression and weaker binding characteristics as compared to the common genes. Interestingly, the main function of these IFNα- and IFNγ-specific genes were connected to the defence response and immune system process, and in case of IFNγ-specific genes also to RNA biosynthetic process. Based on the presence of the binding site and the binding pattern of pSTAT1, pSTAT2, IRF9 and/or IRF1, it is tempting to speculate that IFNα-specific gene regulation involves both ISRE- and GAS-dependent mechanisms, whereas IFNγ-specific transcription tends to be more GAS-GAF-driven. In this respect, we cannot rule out the involvement of other STATs and/or IRFs. On the other hand, STAT- and IRF-independent mechanisms or IFNα- and IFNγ-specific changes in chromatin remodeling/histone modifications  can alsoClose examination of gene expression in combination with chromatin binding characteristics, in wt and STAT1-, STAT2-, IRF9-, IRF1- and IRF9/IRF1-mutant Huh7.5 cells, further supported a number of additional conclusions connected to the transcriptional regulation of GAS, ISRE and composite genes. First, in addition to the accepted role of GAF (pSTAT1 homodimers), our results are in line with a novel role of a pSTAT1–pSTAT2 GAF-like complex in the transcriptional response of GAS genes to IFNα , 57 confSecond, in IFNα-treated STAT1KO cells, delayed transcription of ISRE and composite genes correlated with a functional role of the STAT2/IRF9 complex under these conditions as a replacement of ISGF3 activity in wt cells . Thus, SThird, in contrast to GAS and ISRE genes, composite genes were able to sustain IFN responsiveness in all mutant cell lines as compared to Wt cells. Collectively, it shows that the ISRE + GAS composite site shares features of GAS and ISRE genes and is able to act as a molecular switch, which depends on the combinatorial action of canonical and non-canonical ISGF3 and GAF complexes and IRF1. The involvement of other STATs and/or IRFs as compensatory mechanisms cannot be ruled out, however. Nevertheless, this highlights the flexibility of the composite site in the transcriptional response to both IFNα and IFNγ as compared to individual GAS and ISRE genes, which correlates with their conserved role in the immune response . More imFourth, comparing the proximal promoters of the common composite genes identified in our study, revealed a random organization (ISRE-GAS or GAS-ISRE), with a distance varying between 0 and 415 bp (Table S4)  and epigDetailed assessment of the STAT1, STAT2 and IRF9 genes by ChIP-seq, recognized a similar binding pattern as seen for composite genes and revealed novel insights into the autoregulatory mechanisms of IFN-dependent transcription of these genes. The binding characteristics for the STAT1, STAT2 and IRF9 genes corresponded with the presence of a functional ISRE and a GAS element in close proximity in the promoters of STAT2 and IRF9, and an ISRE in the STAT1 gene. Recently, a novel distal regulatory element was described positioned 5.5-kb upstream of the mouse STAT1 gene  with a sAs potential IRF1 target genes, the feedback regulation of ISGF3 and GAF components also depends on IRF1 . TherefoII) in IFNγ-stimulated human A549 cells with a role in antiviral activity [IFNγ-mediated tyrosine phosphorylation of STAT2 was reported in a study using IFNγ-treated mouse primary embryonic fibroblasts that resulted in the formation of ISGF3 , 72. Moractivity .Literature describes the involvement of unphosphorylated ISGF3 (U-ISGF3) or U-GAF in long-term IFN responses, where increased levels of ISGF3 components are no longer phosphorylated , 75. In An additional role of U-ISGF3  and STATFigure S1. Global distribution of pSTAT1, pSTAT2, IRF9 and IRF1 binding sites in response to IFNα and IFNγ stimulation.  Global distribution of peaks annotated for pSTAT1, pSTAT2, IRF9 and IRF1 after IFNα (A) and pSTAT1, IRF9 and IRF1 after IFNγ (B) stimulation. The number assigned to each bar on the right side presents the total number of identified binding regions. Binding sites annotation to the categories of genomic localizations were performed on the lists of all non-redundant peaks combined from all time-points (0-72h) for each antibody.  Genomic localization of occupied binding sites with identified motifs GAS and/or ISRE in response to IFNα (C) or IFNγ (D) stimulation. The number on the right side represents the total number of identified binding regions with the specific motif. The number in bold indicate the most abundant motif identified in the regions occupied by the given signaling protein. (PDF 1366 KB)Figure S2. GAS matrices used for binding sites annotation in the peak regions from ChIP-seq experiments. 10 matrices representing GAS binding site, 6 retrieved from HOMER database and 4 calculated deNovo using HOMER on pSTAT1 ChIP-seq data, were used in the first binding site annotation in the peak regions from the ChIP-seq experiment with pSTAT1 antibody. The UpSetR plot presents the number of motifs recognized with the intersections between matrices. Matrices selected for the final TF binding sites annotation based on the number of motifs recognized commonly with other matrices, as well uniquely by each matrix separately. (PDF 1612 KB)Figure S3. ISRE matrices used for binding sites annotation in the peak regions from ChIP-seq experiments. 8 matrices representing ISRE binding site, 7 retrieved from HOMER database and 1 calculated deNovo using HOMER on pSTAT1 ChIP-seq data, were used in the first binding site annotation in the peak regions from the ChIP-seq experiment with pSTAT1 antibody. The UpSetR plot presents the number of motifs recognized with the intersections between matrices. Matrices selected for the final TF binding sites annotation based on the number of motifs recognized commonly with other matrices, as well uniquely by each matrix separately. (PDF 1551 KB)Figure S4. Data integration and filtering pipeline. An integrative analysis of our RNAseq-ChIPseq data performed, using the BETA tool . By concentrating on the promoter/5’UTR regions with annotated GAS and/or ISRE motifs, our multi-omics data integration approach identified IFNa and IFNg commonly induced genes that bound any of the 4 components individually or in a random combination in their promoter region. (PDF 1359 KB)Figure S5. Characterization of IFNα- and IFNγ-stimulated responses in WT, STAT1-, STAT2-, IRF9-, IRF1KO and IRF9/IRF1 dKO Huh7.5 cell lines. (A) WT, (B) STAT1-, (C) STAT2-, (D) IRF9-, (E) IRF1KO and (F) IRF9/IRF1 dKO Huh7.5 cells were treated with IFNα (1000 U/ml) or IFNγ (10 ng/ml) for the indicated time. The expression levels of STAT1, STAT2, IRF9 and IRF1 were evaluated by immunoblotting; p-phosphorylated, t- total. (PDF 2335 KB)Figure S6. pSTAT1 and STAT2 recruitment to GAS, ISRE and composite genes in IFNα and IFNγ treated Hu 7.5 cells. Views of the ChIP-seq peaks detected in the promoter regions of pre-selected GAS, ISRE and composite genes in untreated or IFNα and IFNγ treated Huh7.5 cells. All peaks were mapped onto human reference genome hg38 and visualized using the IGV genome browser; scale for pSTAT1 and STAT2 0-1000. (PDF 128 KB)Figure S7. Expression of GAS, ISRE and composite-containing representative genes in STAT1KO Huh7.5 cells after IFNγ treatment. GAS , ISRE  and composite  gene expression was analyzed using qPCR. Mean +/- SEM, n=2. (PDF 32 KB)Table S1. Primer sequences for RT-PCR and cloning. (DOCX 19 KB)Table S2. List of IFNα- and IFNγ-specific integrated genes. (DOCX 24 KB)Table S3. List of IFNα- and IFNγ-common integrated GAS, ISRE and composite genes. (DOCX 19 KB)Table S4. List of IFNα- and IFNγ-common integrated composite genes with spacing between and orientation of GAS and ISRE sites. (DOCX 19 KB)Below is the link to the electronic supplementary material.


G‐quadruplexes (G4s) are a kind of non‐canonical nucleic acid secondary structures, which involve in various biological processes in living cells. The relationships between G4s and human diseases, such as tumors, neurodegenerative diseases, and viral infections, have attracted great attention in the last decade. G4s are considered as a promising new target for disease treatment. For instance, G4 ligands are reported to be potentially effective in SARS‐COV‐2 treatment. However, because of the lack of analytical methods with high performance for the identification of intracellular G4s, the detailed mechanisms of the biofunctions of G4s remain elusive. Meanwhile, through demonstrating the principles of how the G4s systematically modulate the cellular processes with advanced detection methods, biochemical targeting of G4s in living cells can be realized by chemical and biological tools and becomes useful in biomedicine. This review highlights recent methodological advances about intracellular G4s and provides an outlook on the improvement of the bioanalysis and biochemical targeting tools of G4s. G‐quadruplexes involve in various biological processes in living cells, and are considered as a promising new target for disease therapeutics, but the detailed mechanism of G4 biofunction remains elusive. This review highlights recent methodological advances about intracellular G‐quadruplexes and provides an outlook on the improvement of the bioanalysis and biochemical targeting tools of G‐quadruplexes. G4s undergo the formula G≥3NxG≥3NxG≥3NxG≥3 (x = 1–7), while there are also other forms of G4s, such as two‐quartet, long‐loop, and bulge G4s. The sequences between G‐tracts form three loop regions. The sequence component and length of the loops play an important role in the stability of G4 structures. In general, G4s are more stable than other competing nucleic acid structures. G4s can form in both DNA and RNA, and have diverse substructures. Dictated by the orientation of G‐tracts, DNA G4s can fold into parallel, anti‐parallel, and mixed topologies  are a class of non‐canonical nucleic acid secondary structures. Four guanine bases self‐associate via Hoogsteen hydrogen bonds into a square planar conformation, namely, G‐quartet Figure . Two or > > Li+).1, 2s Figure , while R and transcriptome of living cells. There are more than one million G4‐forming sequences in human and mouse cells. G4s participate in various cellular processes related to gene expression, such as transcription, translation, DNA replication, and so on. In addition, G4s have been reported to have critical regulatory functions in human diseases, including tumors, neurodegenerative diseases, and viral infections. In this way, G4s have been regarded as a potential therapeutic target. An important example of G4‐based therapeutics is that targeting the G4 in the SARS‐COV‐2 genomic RNA with a small molecular ligand is proved to be a potential treatment for COVID‐19. Therefore, there has been a recent surge in the study towards understanding G4s in living cells and organisms for the development of biomedicine applications based on intracellular G4s. However, limited by analytical methods, the detailed mechanisms of G4‐dependent biofunctions remain unclear. For instance, it is hard to specifically detect G4s in an individual gene, while it is necessary to characterize the G4 in every single gene, to clarify its detailed molecular mechanism in a certain gene pathway.G4s are widely present in the genome8 and  fluorescence resonance energy transfer, ultraviolet‐visible spectrophotometry, nuclear magnetic resonance, surface‐enhancedRaman scattering, and so on. These methods have greatly expanded our understanding of G4 structures and their biochemical properties. However, in vitro experiments cannot reflect the real properties of G4s in the cells. Therefore, it is necessary to study the function and regulation of intracellular G4s in situ. Identification and characterization of G4s in cellular context raise higher requirements on analytical methods. Firstly, the in situ intracellular physiological state needs to be maintained to the greatest extent. Secondly, the G4s in the cells may interact with other nucleic acids and proteins, resulting in a very complicated chemical environment, thus the probes of G4s are susceptible to be interfered from non‐specific binding. Thirdly, there are millions of G4s in the cells, which are very difficult to be differentiated. In the past ten years, methodological advances such as cell imaging, high‐throughput sequencing, and proteomic technologies have been developed to systematically identify and characterize G4s in the cells. Although there are other methods that can be useful in intracellular G4s, these three types of methods have the greatest power to advance our understanding of G4 biology. Moreover, based on the knowledge of G4 biology, several chemical and biological tools have been established to regulate the biofunction of G4s in vivo, which lay the foundation for G4‐based biomedical applications.The prerequisite for revealing the biofunction of G4s and developing novel G4 mediated biological applications is to establish a powerful toolkit of detection and characterization methods. In the last decade, various biophysical methods for characterizing G4‐forming oligonucleotides in vitro have been established, including circular dichroism (CD),20 fluAs early as 10 years ago, G4s were considered as targets for therapeutics. In the past decade, some progress has been made in the development and testing of G4‐based therapeutic methods, such as G4 ligands for anti‐tumor treatment and the detailed mechanism underlying G4‐related tumors. However, G4‐based biomedical applications are still in their infancy. Methodological advances are under demand to promote this field. On the one hand, improved bioanalytical methods can drive the discovery of the overall mechanism of G4‐mediated intracellular processes. On the other hand, it is essential to expand the biochemical toolkits for targeting G4. Herein, we believe that it is necessary to illustrate the currently applicable methods and highlight the recent advances about intracellular G4s. Importantly, discussing their principles, application scenarios, advantages and disadvantages can provide a guidance of the future improvement of intracellular G4 bioanalysis and biochemical targeting. In this review, we first briefly introduce the biological functions of G4s. Then, we overview G4 probes as the key molecular tools required for G4 detection and biochemical targeting, and focus on the screening methods of these probes. Next, the bioanalytical technologies of intracellular G4s are discussed from three categories: imaging, high‐throughput sequencing, and proteomic methods. Meanwhile, the biochemical targeting tools of G4s in living cells are summarized. Finally, we point out the currently unresolved questions in G4 bioanalysis and biochemical targeting.2 Secondly, many endogenous proteins have been proved to bind with DNA and/or RNA G4s, in other words, G4s function as an assembly element to recruit additional regulatory proteins. The complex and diverse biofunctions of G4s have already been reviewed elsewhere. However, what G4s do in the cells remains a mainly unanswered question. Herein, we briefly introduce the major biofunction of G4s to facilitate the understanding of the scientific meaning of G4 bioanalysis and biochemical targeting.G4s are abundantly present in the genome and transcriptome, and the ever‐increasing number of discoveries has revealed that G4s participate in various biological processes related to DNA and RNA. G4s function in two major ways. Firstly, due to its high stability, DNA polymerase, RNA polymerase, and ribosome can be stalled by downstream G4s.27 Sec2.1 which is the most important biofunction of DNA G4s  to form 8‐oxo‐2′‐deoxyguanosine (8‐oxo‐dG). Some proteins related to DNA damage repair and transcription, such as APE1 and OGG1/2, can be recruited to the oxidation sites. In this way, promoter G4s are related to transcriptional activation in response to oxidative stress.Finally, G4s are also related to DNA oxidative damage. The last G of the NGG sequence has the lowest redox potential,42 whi2.2 , long noncoding RNAs, mitochondrial RNAs, microRNAs, telomere‐associated RNAs. Increasing evidence shows that G4s are widely involved in various RNA bioprocesses, which further confirms the complexity of G4 biology. In cellular mRNA, G4s are present in 5′‐UTR (untranslated region), 3′UTR, and CDS (coding sequence) regions. Similar to other stable secondary structures, G4s in mRNAs inhibit translation45 .The detection and biochemical targeting of intracellular G4s depend on the chemical tools for probing G4s with high specificity. Small molecule ligands and antibodies are the major types of widely used G4 probes. At present, a series of G4 ligands have been developed. These ligands have the following three characteristics in common Figure : (1) hav60 Cur Thus, PDS has been widely applicated in biophysical characterization and biochemical targeting of G4s. CarboxyPDS has higher binding affinity to RNA G4s than DNA G4s, which can be very useful in the detection of RNA G4s. Another special G4 fluorescent probe derived from PDS is SiR‐PyPDS, which cannot induce unfolded G4‐forming sequences to fold into G4 structures at low concentration (20 nM). This feature is very necessary in the detection of intracellular G4s with its intact folding state.The most widely used G4 ligands are pyridostatin (PDS) and its derivatives Figure . PDS has62 Thu 1H6, and D1. These antibodies have played an important role in G4 imaging and sequencing due to its high binding affinity and selectivity to the G4 structures.In addition to the small molecule ligands, G4 antibodies are also a powerful tool for G4 probing, including BG4,66 1H63.2 In contrast, the methods for screening small molecule ligands are more complicated.Given the importance of G4 probes, it is worth to introduce the methods for the design and optimization of G4 probes, which are critical in the future development of G4 bioanalysis and biochemical targeting toolkits. The G4 antibodies are screened with the common phase display method.72 In   has been used to screen ligands of the MYC promoter G469  for screening the ligands of MYC G4. ALIS utilized mass spectrometry to identify the G4 molecules, the ligands, as well as the G4‐ligand complexes. The binding affinity of ligands can be calculated by the ratio between the signal intensity of G4‐ligand complexes and that of the ligands. ALIS has the advantages of being fast and easy to operate. ALIS does not need to immobilize small molecules on the substrate and label G4 molecules, and only needs a small amount of reagents. In this way, as many as 500,000 molecules can be screened with an ALIS system in only one day.Alternatively, Flusberg f MYC G4.73 ALI 70 . Immunofluorescence imaging of DNA and RNA G4s has been realized in fixed mammal cells. This method enables the measurement of the overall G4 folding state in dynamic cellular processes, such as the transformation of chromosomes during mitosis. However, whether this method can detect a single G4 has not been effectively confirmed. The number of fluorescent scatters in the nucleus is relatively small, far lower than the theoretical number of G4s contained in various genes. In other words, immunofluorescence may only visualize G4 structures that are clustered together in space. Moreover, G4 antibodies may not be able to bind to all possible G4 configurations, which restricts the detection efficiency.An alternative G4 imaging method is immunofluorescence based on G4 antibodies. BG4 is the most widely used G4 antibody Figure , which c4.2 let alone differentiate the G4 of an individual gene among the entire genome. Therefore, how to achieve the specific detection of the G4 on a single gene is the ultimate goal for this field.The above‐mentioned imaging methods have promoted the understanding of the biological roles of G4s, but still have some shortcomings. The major problem of the imaging methods is that the entirety of intracellular G4s always been probed at the same time. Regardless of small molecule ligands or antibodies, they can only distinguish G4s from other nucleic acid types, such as double strands, single strands, hairpins, and so on, but cannot distinguish the G4s with different sequence contexts in various genes. In actual, all G4s have the consensus feature of stacked G‐quartets, while the differences only lie on the loop regions. The impact of these differences on the overall global similarity of most G4s is very weak, resulting in difficulty in detecting the single G4 motif. It was unsuccessful to screen specific probes from a small pool of three different G4s,85 letet al. reported the G4‐triggered fluorogenic hybridization (GTFH) probe, ISCH‐nras1. This probe consists of two parts, one is a hybridization probe targeted to the proximity sequence near the G4 motif of NRAS mRNA, and the other is a G4 light‐up probe, of which the fluorescence can be enhanced by adjacent G4s  for visualizing monogenetic G4s in fixed cells86 . A sequencing buffer with Na+ ions was used in the read 1 process, in which the G4s cannot fold and the accuracy of DNA sequence recognition can be ensured. Then, a sequencing buffer containing K+ was used in the read 2 process, generating drastically reduced sequencing quality scores on the same clusters. In this way, the sequence capable of folding into G4s can be accurately identified.Investigating the sequences that can fold into G4 structures has been achieved by a polymerase stalling method.87 As ] Figure . During  which has provided important insights for the formation and distribution of genomic G4s.G4‐seq has been applied to identify the observable G4s in the human genome. Only 60–70% of the predicted G4s in the human genome can be observed experimentally to be folded. There are in total 716,310 different observable G4s discovered by G4‐seq, of which more than 70% are non‐canonical G4 structures that cannot be predicted by computational algorithm. The non‐canonical G4s include long loops with more than 7 bases, bulges with discontinuous G‐tracts regions, and two‐tetrad G4s. These results indicate that the changes in the local sequence context in the genome have very complex affection on the formation and stability of the G4 structures. Therefore, computational algorithms always over‐predict or under‐predict many G4 structures, making it difficult to accurately identify the formation of G4s in the genome. The G4‐seq method has been further optimized and applied to the genomic G4 sequencing of 12 species,91 whi In the standard whole‐genome sequencing process of Illumina sequencing, the reaction buffer without K+ cannot lead to the formation of stable G4s, but the existing unstable G4s can still cause abnormal fluctuations of the Phred quality scores. Through capturing the subtle changes in the sequencing quality of these sites with computational algorithm, the G4 structures can be directly predicted. G4‐Miner detected 736,689 G4 sites in the human genome, 89% of which were consistent with the results obtained by G4‐seq. Although G4‐Miner is simple and accurate, its positive detection rate is still somewhat low, compared to the improved G4‐seq method. Therefore, it is necessary to develop more genomic G4 sequencing methods to promote the advancement of DNA G4 biology.However, the G4‐seq method identifies G4s based on the polymerase stalling site, resulting in insufficient spatial resolution. The G4s in proximity may not be effectively distinguished. Moreover, G4‐seq relies on customized Illumina sequencing reagents with the flexibility of adding different monovalent ions , which limited the usage of this method by the other groups. Recently, the G4‐Miner method was reported to be used to detect genomic G4s through a novel principle.95 In 5.2 with the similar principle of G4‐seq  and 2‐methylnicotinic acid imidazolide (NAI) are widely used to identify the secondary structures of RNA. After treatment by these reagents in the living cells, the unpaired and unconstrained nucleotides are kinetically more susceptible to be reacted and cannot form any secondary structures later. In this way, if the G4‐forming sequences are unfolded in the living cells, the associated G bases are methylated or acylated, and subsequently the RNA does not form G4 structures and cause polymerase stalling. However, this method may also cause some bias due to the unexpected reaction on the G bases of folded G4s, which has been discussed in detail elsewhere. In addition, G4RP‐seq is a more accurate method to detect transcriptomic G4s in the physiological state  is a recently invented method for efficiently profiling chromatin DNA binding maps.100 Co] Figure . However G4s have been proved to shape the cell specific transcriptome. Furthermore, G4s have also been confirmed to mold DNA methylomes and inhibit the activity of DNA methyltransferase DNMT1. However, ChIP‐seq and Cut&Tag all depend on G4 antibodies, which are expected to have high binding affinity and high specificity. Among the known G4 antibodies, BG4 has the best performance, but is likely to have some bias in the detection of chromatin G4s. Recently, a new G4‐binding peptide G4P has been reported to detect more G4 peaks at high resolution with ChIP‐seq. A greater proportion of peaks are perfectly matched with the observed genomic G4s. G4P is expected to become a better chemical tool for improving our understanding of chromatin G4s.These techniques for mapping the chromatin G4s, have promoted the exploration of the functions of intracellular G4s. The chromatin G4s have been demonstrated to be a transcription factor hub, which plays a key role in the recruitment of transcription factors and the assembly of RNA Pol II.102 G46http://bsbe.iiti.ac.in/bsbe/ipdb/index.php). It has been demonstrated that many G4‐associated biological processes are mediated by G4BPs. G4BPs participate in G4‐associated processes with the following three mechanisms: (1) G4BPs work directly after binding with G4s without unfolding G4 structures; (2) G4BPs bind to and unfold G4 structures to reduce the hindrance of G4s in biological processes such as transcription and translation; (3) G4BPs recruit other proteins after binding with G4s, regarding G4 structures as scaffolds. In order to generate a robust molecular understanding of intracellular G4s, it is necessary to uncover which proteins can bind to G4 structures with high affinity and specificity, what is the interaction network between G4BPs and G4s in living cells, and how these G4BPs participate in biological processes. Therefore, a series of methods have been developed to analyze G4BPs http://bs These methods can help to understand the binding affinity between G4BPs and G4s in vitro. However, these methods have low throughput, and cannot reflect the in situ G4‐protein binding state in living cells. Therefore, it is necessary to develop high‐throughput methods to characterize G4BPs.In the early stage of research on G4BPs, in vitro methods, such as electrophoretic mobility shift assay, surface plasmon resonance, and DMS footprinting, were used to study the interaction between one single G4BP and one single G4‐forming sequence.105 Th6.1et al. used quantitative mass spectrometry based on SILAC (stable isotope labeling by amino acids in cell culture) to identify G4BPs  to realize multiplexing relative quantification and obtain the binding curves. Similar to the DNA G4 pull‐down method, Herdy et al. developed the RNA G4 pull‐down method to study the G4BPs located at the G4 sites of the proto‐oncogene NRAS 5′UTR region. Through this method, 80 proteins were identified to specifically bind to NRAS RNA G4, and five new G4BPs were discovered, including DDX3X, DDX5, GRSF1, and NSU5.Mass spectrometer is widely used to identify proteins, which is called high‐throughput proteomics. Proteomic technologies have been applicated to discover and characterize G4BPs. Williams ify G4BPs106  107  molecule, GTC365, can cause the hTERT promoter G4 to misfold and induce death of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) has a variety of tumor‐related genes in which G4s are enriched in promoter regions. Targeting these genes through G4 ligands can produce anti‐tumor effects. CX‐5461 can block replication forks and induce DNA damage after stabilizing the DNA G4 structures. Patient‐derived xenograft model has shown that BRCA1/2‐deficient tumors were very sensitive to this G4 ligand. Similarly, CX‐3543 has a good therapeutic effect on ATRX‐deficient tumors. Currently, CX‐5461 has been put into clinical trials, which shows a great promising of G4 targeted therapeutics.It is worthy to detailly introduce the exploration of using G4 ligands in tumor therapy. It was firstly discovered about 10 years ago that PDS can inhibit the growth of tumor cells.115 In Therefore, the ligand for regulating transcription may also interfere translation, resulting in complex and unpredictable final regulatory results. How to specifically regulate the function of G4s is the key goal for this field. Recently, Dickerhoff et al. have reported a new drug‐like small molecule PEQ, which can bind MYC promoter G4s in a sequence‐specific way. This result provides the first example of single‐target regulation of G4s and will promote the rational design of small molecule ligands targeting individual G4s.Small molecule ligands can regulate G4 biofunctions via many different pathways. These ligands bind to the G4 structures at multiple sites in the genome and transcriptome, which has been confirmed by cell imaging experiments.75 The7.2 ASO has been used to unwind the G4 structures on a specific promoter region and down‐regulate the expression of the corresponding gene. Besides, the modulation of G4s can also be precisely induced through the regulation of G4BPs. For example, the small molecule inhibitor of G4BP NME2 can inhibit the expression of the tumor gene hTERT. However, although this method targets a single protein, G4BPs such as NME2 can bind to multiple G4 sites throughout the genome, and its regulatory outcomes may still be diverse. Another strategy to target the G4 structure by protein is to bind the G4‐forming regions through the dCas9/sgRNA protein complex. dCas9 has a strong binding affinity to nucleic acids and can unwind the G4 structures, thus affecting G4‐dependent biological functions. This strategy is expected to become an effective tool to target and regulate gene‐specific G4 structures. However, dCas9 is a large exogenous protein, which is not easy to be introduced into living cells. Therefore, biochemical tools that target individual G4s are always under demand.In addition to G4 ligands, there have been other kinds of G4 regulators, which have enhanced specificity. In other words, these G4 regulators can target G4 in a specific gene, giving a specific regulatory result. Antisense oligonucleotides (ASOs) are short‐stranded nucleic acids that can be designed to hybridize to specific sequences of DNA or RNA in living cells, thereby mediating RNA degradation or DNA transcription inhibition.121 AS8 has been screened and tested in vivo. Moreover, a small peptide G4 probe has been reported to detect more chromatin G4s with a high signal‐noise ratio. Secondly, in situ imaging of G4 in the cells has reached monogenic and single‐cell level. mRNA G4s of a specific gene can be detected in many genes and many cell lines. Thirdly, the omics methods have been able to profile the G4 landscape in an intact physiological state, which can give more exact data of intracellular G4s. Although further improvement is needed, G4 ChIP‐seq and G4 CUT&Tag have achieved the profiling of G4 in cellular chromatin context. In addition, in situ recognition and labeling methods for characterizing G4BPs have been realized by CMPP and G4‐LIM‐CAP. Taking advantage of these imaging and omics methods, the formation, distribution, localization, dynamics, and interactions have been discovered, providing a systematic landscape of intracellular G4s.In the last decade, the methodological advances in G4 detection and biochemical targeting have greatly expanded our understanding of the function and mechanism of intracellular G4s. There are three aspects of advances that are worth highlighting. Firstly, the recognition and targeting tools of G4s have been developed to be more powerful. The first small molecule ligand that can selectively bind to G4 structures at sequence‐specific level, PEQ,120 haOverall, these genome and transcriptome scale studies provide future directions to unravel the G4‐associated biological processes. There are still unanswered and arising questions in fundamental biology. Future research is expected to advance our understanding of the detailed mechanisms of the intracellular G4s and unravel the underlying biological consequences, and their connections to human diseases. However, the currently applicable methods remain in their infancy for solving these questions. Firstly, it remains possible that existing sequencing methods cannot detect the full entirety of intracellular G4s. Secondly, transient and dynamic G4 structurome and interactome in living cells may not be accurately detected with current technologies. Thirdly, there are only few tools that can be used to target and regulate intracellular G4s at single‐motif resolution. Lastly, the detection and regulation methods for G4s have rarely been tested with clinic samples for further development of diagnostic and therapeutic applications. The development of novel methodologies with superior resolution, throughput, and sensitivity will provide groundbreaking discoveries and applications that would be useful in the clinic in the future.The authors declare no competing financial interests.


G-quadruplexes (G4s) have long been implicated in the regulation of chromatin packaging and gene expression. These processes require or are accelerated by the separation of related proteins into liquid condensates on DNA/RNA matrices. While cytoplasmic G4s are acknowledged scaffolds of potentially pathogenic condensates, the possible contribution of G4s to phase transitions in the nucleus has only recently come to light. In this review, we summarize the growing evidence for the G4-dependent assembly of biomolecular condensates at telomeres and transcription initiation sites, as well as nucleoli, speckles, and paraspeckles. The limitations of the underlying assays and the remaining open questions are outlined. We also discuss the molecular basis for the apparent permissive role of G4s in the in vitro condensate assembly based on the interactome data. To highlight the prospects and risks of G4-targeting therapies with respect to the phase transitions, we also touch upon the reported effects of G4-stabilizing small molecules on nuclear biomolecular condensates. G-quadruplexes G4s) are planar arrangements of Hoogsteen bonded guanine tetrads. They can be formed through the association of several nucleic acid strands that harbor G2+ tracts or an intramolecular tetrahelical folding of a single strand . Within s are plaBiomacromolecular condensates are assembled spontaneously through the liquid–liquid phase separation (LLPS) of biopolymers, typically nucleic acids and proteins with low-complexity domains (LCDs) or intrinsically disordered regions (IDRs) ,21. SuchRNA G4s have recently emerged as prospective drivers for various phase transitions in the cytoplasm . The eviA substantial number of acknowledged G4 interactors have been identified using non-high-throughput methods and assays that do not fully recapitulate the intracellular conditions . Early sThe established or predicted LLPS drivers can be found in each category of the G4 binders. Those related to infection sensing and innate immune signaling include the DEAD box helicases, such as DDX1 and DDX24 , DDX5 4, Dbp1 4, and DbpThe other G4-recognizing LLPS drivers from the RNA binding category include the plasminogen activator inhibitor 1 mRNA binding protein (SERBP1) , nucleopSERBP1 assists in transcription, chromatin remodeling, and the assembly of promyelocytic leukemia (PML) bodies in the nucleus, while in the cytoplasm, it stabilizes mRNA and controls translation . This prThe FUS protein, one of the few TFs enriched at the endogenous DNA G4s and displaced efficiently by the G4-specific ligand PDS , also biAdditional examples of the known G4-binding LLPS drivers are provided in It can be concluded that LLPS drivers are abundant, if not enriched, in the G4 interactome. Nucleoplasm–cytosol shuttling RNA-binding LLPS drivers with a remarkable affinity for G4s typically contain Arg-rich domains that are capable of weak electrostatic/cation–pi interactions, and/or aromatic fragments that are capable of weak pi–pi interactions, which are commonly observed in cellular condensates ,93 Figub. The diSynthetic DNA G4s were typically used in interactome studies ,35, GMV-Nucleoli, the condensates in which rRNA is synthesized, are assembled around the so-called nucleolar organizer regions of genomic DNA that contain clusters of G4-harboring ribosomal gene repeats (rDNA) . The traIt should be emphasized once again that rDNA G4s are typically mentioned as possible targets of the ligands because most in vitro data were obtained using synthetic DNA. However, nucleoli staining using a G4-specific benzothiazole-based light-up probe thioflavin T (ThT) revealed a decreased foci number after the RNAse but not the DNAse treatment, pointing to the prevalence of RNA rather than DNA G4s . In the To summarize, the G4s in noncoding RNA and rDNA are capable of recruiting established and presumed LLPS drivers, presumably increasing their local concentrations to a critical point needed for a spontaneous phase transition. Since Arg-rich motifs (RM) are present in the majority of those proteins, G4s might contribute to LLPS through weak cation-π interactions c. In theThe G4s appear to play context-dependent permissive/repressive roles in the gene expression, thus contributing to the cell-type-specific transcription control ,13. In pThe in vitro assays with polynucleosomes evidenced that H1 is the key separation driver . In the HP1α co-separates with H1 in vitro and plays an important accessory role in the heterochromatinization in vivo . In soluImportantly, the heterochromatin establishment at the sub-telomeric regions is dependent on the HP1α recruitment to the telomeric repeat-containing RNA (TERRA), rather than the DNA, and appears to be interlinked with the shelterin assembly ,118 Figc. ShelteA recent semi-comprehensive immunocytochemistry-based investigation of the G4 location relative to various nuclear condensates  revealedThe permissive role of the promoter/enhancer G4s in the gene expression can be attributed to the accumulation of TFs, including the LLPS drivers TAF15 , SP1 7878, and, Another possibility is the transcription activation through the LLPS-driving bridging interactions . The G4sAll the above hypotheses imply that G4s can be prerequisites rather than mere consequences of chromatin opening upon active transcription . The samInterestingly, despite the accumulation in the S phase, evident from both small-molecule-based  and antiThe mechanisms of the G4 oxidation, base excision repair, and the possible consequences for transcription factor loading have been reviewed elsewhere . The recIn this review, we outlined the current understanding of the G4 contributions to the assembly and function of nuclear macromolecular condensates. This understanding is grounded by G4 interactome studies, in vitro LLPS assays, and intracellular imaging assays using antibodies for the G4s and condensate markers, as well as small-molecule light-up probes. The limitations of such studies should be taken into account.First, short oligodeoxyribonucleotides prone to intramolecular folding were used as the model G4s in most of the in vitro experiments. However, the key role of the intermolecular structures, RNA G4s, or RNA/DNA hybrids in the nuclear condensates could not be excluded. Second, the in vitro LLPS assays rarely followed the complete protocols for a bona fide verification of the condensate liquid state. The intracellular LLPS verification was even more challenging, and the requirements were never fully met in the G4 studies. Classical immunofluorescence staining and the usage of light-up probes hardly provided sufficient spatiotemporal resolution, while optogenetic tools are not yet commonly accessible.Despite the technical limitations, several lines of research have converged to support the importance of G4s in nuclear condensates. Some of the major results and interpretations are summarized below.G4s promote the LLPS of heterochromatin-associated proteins in artificial systems, but the biological relevance of these findings awaits verification.G4s promote the LLPS of RNA-binding proteins in the pseudo-cellular environment. These findings are in line with the studies of cytoplasmic condensates and may be relevant to the assembly of nuclear RNA processing factor-rich condensates, namely nucleoli, speckles, and paraspeckles.The integrity and/or functions of speckles/paraspeckles are disrupted by G4 mutations and G4-stabilizing ligands. The shelterin integrity and function are also disrupted by G4 ligands. The effects of these ligands are attributed to their interference with G4 protein interactions.The colocalization with Pol II clusters, TFs, and chromatin loop boundaries supports the idea that G4s assist in the transcription initiation. However, conclusive evidence is lacking. A comparison of transcription burst rates at G4-rich and non-G4 SEs could probably clarify this matter.The proposed mechanisms for the G4-mediated LLPS can be classified as follows.The nucleobase exposure in the G4 outer tetrads and the adjoining ssDNA regions for transient π–π interactions with aromatic amino acid-rich proteins and cation–π interactions with Arg-rich ones .The exposure of a protein IDR/LCD for transient interactions with other macromolecules following a G4-binding-induced conformational transition .The accumulation of multiple IDR/LCD-containing proteins at the G4 repeats through the G4 recognition by the structured domains of these proteins or their partners b–d.The assembly of a transient nucleic acid “net” through the formation of G4–G4 kissing complexes, intermolecular G4 folding, or chromatin looping mediated by G4-binding proteins b–d.Most G4-stabilizing small molecules are planar polyaromatic structures with positively charged  substituents tailored to form stacking contacts with an outer G4 tetrad. They may interfere with transient π–π/cation–π interactions and outcompete the G4-binding proteins, including those involved in chromatin looping. This makes the typical G4 ligands prospective remodelers of the G4-containing nuclear condensates. The consequences of such a remodeling would be systemic at a cellular level and may outweigh the desired specific effects on G4 targets from oncogene promoters, telomeres, etc. This thesis undermines the predictability of G4 targeting with small molecules to some extent, but also points to exciting new possibilities for manipulating nuclear condensates.


Nlrp6 promoter region (−400–−391 bp and −33–−24 bp) and activate the transcription of Nlrp6. Altogether, our study demonstrated that NPAS4, as a transcription factor, can exacerbate pyroptosis and transcriptionally activate NLRP6 in the acute phase of intracerebral hemorrhage in mice.Intracerebral hemorrhage (ICH) is a severe cerebrovascular disease with a high disability rate and high mortality, and pyroptosis is a type of programmed cell death in the acute phase of ICH. Neuronal Per-Arnt-Sim domain protein 4 (Npas4) is a specific transcription factor highly expressed in the nervous system, yet the role of NPAS4 in ICH-induced pyroptosis is not fully understood. NLR family Pyrin-domain-containing 6 (NLRP6), a new member of the Nod-like receptor family, aggravates pyroptosis via activating cysteine protease-1 (Caspase-1) and Caspase-11. In this study, we found that NPAS4 was upregulated in human and mouse peri-hematoma brain tissues and peaked at approximately 24 h after ICH modeling. Additionally, NPAS4 knockdown improved neurologic dysfunction and brain damage induced by ICH in mice after 24 h. Meanwhile, inhibiting NPAS4 expression reduced the levels of myeloperoxidase (MPO)-positive cells and Caspase-1/TUNEL-double-positive cells and decreased cleaved Caspase-1, cleaved Caspase-11, and N-terminal GSDMD levels. Consistently, NPAS4 overexpression reversed the above alternations after ICH in the mice. Moreover, NPAS4 could interact with the  Intracerebral hemorrhage (ICH) is a severe cerebrovascular disease with a poor prognosis, a high disability rate, and high mortality, resulting in a heavy burden on patients’ families and society ,2. The pVarious forms of cell death occurring after intracerebral hemorrhage have been identified, including apoptosis, necrosis, and ferroptosis in humans and experimental animals ,9,10,11.NLR family pyrin domain protein 6  is a new member of the human NLR protein family ,19. LikeNlrp6 promotor region using bioinformatics prediction approach. Hence, we hypothesized that NPAS4 may modulate pyroptosis by regulating the transcriptional expression of Nlrp6 and the activation of the NLRP6 inflammasome after ICH. In summary, this study investigated the role and potential mechanism of NPAS4 in a brain injury after intracerebral hemorrhage in mice.Neuronal Per-Arnt-Sim domain protein 4 (NPAS4), a member of the basic helix–loop–helix (BHLH)-PAS protein family, is a specific transcription factor highly expressed in the nervous system ,25. BHLHp < 0.01). To investigate the spatiotemporal expression of NPAS4 in the peri-hematoma tissue, Western blotting was performed to detect the protein levels of NPAS4 at 6, 12, 24, 48, and 72 h after ICH compared with a sham control. The results indicated that the NPAS4 expression level started increasing at 6 h and reached a peak at approximately 24 h after ICH , which revealed that NAPS4 deficiency could reduce the infiltration degree of neutrophils. Altogether, these results indicated that the interference of NPAS4 expression attenuated brain injuries, neurological function damage, and neutrophil infiltration in the ICH model mice, which suggests that NPAS4 played a pernicious role in the ICH model mice.Firstly, we injected NPAS4-siRNA fragments  into the right lateral ventricles of wild-type mice; then, we screened out the optimum interference fragments , but the treatment of NAPS4-siRNA injection before ICH presented a distinctly reversible alternation in comparison with simply ICH modeling (p < 0.01). Besides, the protein levels of GSDMD-FL, pro-Caspase-1 and pro-Caspase-11 were showed none significant differences among these groups (p > 0.05). Immunofluorescence staining together with TUNEL assays were conducted to further explore the impact of NAPS4 on pyroptosis. The representative pictures (p < 0.0001). Consistently, the numbers of these counted positive cells were decreased in the ICH + NPAS4-siRNA group in contrast with the ICH group (p < 0.01). All these data reveal that inhibiting NAPS4 expression might offer a protective function against ICH mice via attenuating cell pyroptosis.To specifically explore the function of NPAS4 toward ICH, Western blotting, immunofluorescence staining, and TUNEL assays were performed to investigate the effect of inhibiting NPAS4 on ICH-induced pyroptosis. Western-blotting analysis A,B reveae groups p > 0.05pictures C,D exhibNlrp6 transcription levels, and Western blotting was carried out to determine the protein levels of NLRP6, cleaved-Caspase-1, and cleaved-Caspase-11 in each group. As shown in Nlrp6 was greatly upregulated in the ICH and ICH + NC groups compared with the sham group (p < 0.0001), whereas no differences were found between the ICH and ICH + NC groups. However, this alternation was remarkably reversed after NPAS4-siRNA injection before modeling in contrast with the ICH group (p < 0.001), which revealed that NPAS4 is a positive regulator of the Nlrp6 gene. As presented in p < 0.01), while the treatment of NPAS4-siRNA greatly decreased their expression in comparison with the ICH group (p < 0.01). Briefly, these results support the notion that inhibiting NPAS4 downregulates the expression of NLRP6, cleaved-Caspase-1, and cleaved-Caspase-11, indicating that NPAS4 deficiency is likely to attenuate inflammasome activation via regulating NLRP6 expression after ICH.In our previous studies, we found that NLRP6 could aggravate brain injuries in an ICH-induced rat model and exaggerate cell pyroptosis in an oxygen glucose deprivation (OGD) model. To explore the regulation of NPAS4 on NLRP6 in vivo, quantitative real-time PCR was conducted to detect Nlrp6 promotor sequence. Therefore, a dual luciferase reporter assay was carried out to preliminarily explore the interaction of NPAS4 and the Nlrp6 promotor. Firstly, N2A cells from murine neuroblastoma were divided into two groups and then co-transfected with pGL4.10-Nlrp6 promotor(fLuc) and pGL4.74(rLuc), respectively, together with NPAS4 overexpression plasmids (pIRES2-Npas4-Flag) or empty vector plasmids (EV-Ctrl). Forty-eight hours later, firefly and renilla luciferase activity was detected and analyzed to estimate the Nlrp6 transcription level. As presented in Npas4-Flag) compared with the empty vector (p < 0.001), which demonstrated that the NPAS4 protein upregulated the transcriptional activity of the Nlrp6 promotor (−1902–+496 bp). Secondly, N-terminally truncated Nlrp6 promotor sequences  were constructed to investigate the possible NPAS4 action sites according to a schematic diagram  were transfected with plasmids (fLuc) containing an intact promotor sequence (−1902–+496 bp) or two designed truncations; then, luciferase activity in each group was tested using dual luciferase reporter assay kits. From Nlrp6 promotor truncations showed no obvious alternations in relative luciferase activity when compared to the intact Nlrp6 promotors (p > 0.05). Moreover, there was no significant difference between P1 and P2 truncations (p > 0.05). The results indicated that NPAS4 protein might interact with any one of the three binding sites predicted above in the Nlrp6 promotor. To further explore the specific NPAS4-Nlrp6 interaction sites, Nlrp6 promotor mutants  were constructed by respectively mutating ten bases of the predicted binding-sites  were co-transfected with either pGL4.10-Nlrp6 promotor mutants  or the original control vector, and luciferase activity in every group was detected to evaluate the effect of NPAS4 on Nlrp6 transcriptional activation level. The results (p < 0.0001), while no difference was observed in mut1 (p > 0.05) compared with the original control. Consequently, it was surmised that NPAS4 is more likely to activate the transcription of Nlrp6 through interacting with site2 (−400–−391 bp: CACACGTGCC) and site3 (−33–−24 bp: AGCACATATA). Additionally, a chromatin immunoprecipitation (Ch-IP) assay involving N2A cells was conducted to confirm whether NPAS4 protein interacts with the Nlrp6 promotor sequence. PCR primers for site2 were designed to testify the Ch-IP products. As shown in Nlrp6 promotor (site2) fragments was significantly higher in the anti-NPAS4 group than the IgG group that act as the negative control (p < 0.0001), which illustrated that NPAS4 protein can interact with the Nlrp6 promotor sequence at site2.To determine how NPAS4 regulates the expression of NLRP6, we predicted and analyzed these cis-acting elements interacted with the transcriptional factor NPAS4 using the Jasper2020 online tool and found that there were three possible NPAS4-binding sites F in the  diagram G. Subseqng-sites H. Then,  results H showed Taken together, the NPAS4 transcription factor may promote inflammasome activation in the acute phase of ICH by interacting with the Nlrp6 promotor.Npas4-AAV injection significantly increased NPAS4 expression after ICH (p < 0.01), and NPAS4 overexpression exacerbated a series of ICH-induced injuries. A forelimb-placing test  had more severe muscle weakness than the ICH  and ICH + NC  groups. Nissl staining (Npas4-AAV injected mice in contrast to the ICH control (p < 0.05). And the protein levels of GSDMD-FL, pro-Caspase-1 and pro-Caspase-11 were showed no obvious differences among the three groups (p > 0.05). The data above clearly uncovered that NPAS4 overexpression aggravated neurologic function deficit, brain injuries, and cell pyroptosis. In summary, all the results implied that NPAS4 tends to participate in the development of ICH through promoting pyroptosis induced by NLRP6 inflammasome activation.To verify the detrimental function of NPAS4 in ICH, we injected NPAS4-overexpressing AAV or mock-AAV (no vector) into the right basal ganglia of wild-type mice one month before ICH modeling. As ing test B,C reveastaining A,J demonstaining D–I reveae groups p > 0.05Nlrp6 promoter region using a bioinformatics prediction approach. Therefore, this study was designed to explore whether NPAS4 affects intracerebral hemorrhage and interacts with the Nlrp6 promoter.After ICH occurs, both primary and secondary brain injuries can contribute to cell death, which includes pyroptosis, necroptosis, ferroptosis, and autophagy ,32,33. A−/− mice following cerebral ischemia modeling were reported, thus confirming the neuroprotective role of NPAS4 in ischemic stroke [NPAS4 is a specific neuronal transcription factor that is highly expressed in the nervous system . An incrc stroke . Alternac stroke . ConsistNlrp6 promoter region (−400–−391 bp and −33–−24 bp) and subsequently upregulate the mRNA expression of Nlrp6.NLRP6 can recruit apoptosis-associated speck-like proteins containing a CARD (ASC) and promote the activation of Caspase-1 . The porNlrp6 transcriptional levels, but did not intervene in NLRP6 expression simultaneously so as to further clarify the regulatory function of NPAS4 in an NLRP6-induced brain injury. In follow-up experiments, Nlrp6−/− mice should be raised to further explore the interaction of NPAS4 with the NLRP6 inflammasome in order to determine the potential mechanism of NPAS4 after intracerebral hemorrhage. Moreover, NPAS4 plays a crucial role in neurogenesis and memory formation [Remarkably, there are still some limitations to our current study. We investigated the effects of NPAS4 on mice subjected to experimental ICH and ormation . We focuormation ,54. FurtNlrp6 promoter −400–−391 bp and −33–−24 bp regions to activate Nlrp6 transcription . Control tissues were provided by the Department of Forensic Medicine, Chongqing Medical University (n = 3). The details are shown in This study was approved by the Ethics Committee of Children’s Hospital of Chongqing Medical University (File No. 2021295) and conducted in compliance with the ethical standards outlined in the Declaration of Helsinki. Brain tissues were obtained from spontaneous intracerebral hemorrhage patients , which were provided by the Laboratory Animal Center of Chongqing Medical University , were maintained in a standardized environment with controlled temperature (25 °C) and humidity (relative humidity 55%) and provided ad libitum access to food and water under a 12 h light/dark cycle. All animal procedures and protocols were approved by the Institutional Animal Ethics Committee of Chongqing Medical University and were conducted in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines .Modeling intracerebral hemorrhage in mice: firstly, mice were anesthetized with sodium pentobarbital via intraperitoneal injection and then positioned on a stereotaxic apparatus . After having their hair cut and disinfected by 75% ethanol, mice were made a small sagittal incision and their skulls were exposed extensively with hydrogen peroxide in order to drill a burr hole on the right side of the skull (anteroposterior (AP), −0.2 mm; mediolateral (ML), −2.0 mm). Second, autologous blood (30 µL) from the tail-tips of the mice obtained via a Hamilton micro-syringe rinsed with heparin was automatically infused into the right basal ganglia , −3.5 mm) at a rate of 1.5 µL/min with micro-infusion pump. Subsequently, micro-syringe was maintained in situ for an additional 10 min to minimize blood reflux and was then withdrawn slowly. Lastly, the burr hole was covered with bone wax, and the wounded scalp was sutured and sterilized again with 75% ethanol. Additionally, the sham group was injected with an equal volume of sterile saline solution.NPAS4 siRNA ; si-848 ; si-2253 ) and scramble siRNA as a negative control were designed and synthesized by Sangon Biotech Company . NPAS4 siRNA (0.05 OD/µL) was pumped at a rate of 1.0 µL/min within 10 min into the right lateral ventricle (anteroposterior (AP), −0.3 mm; mediolateral (ML), −1.2 mm; dorsoventral (DV), −2.5 mm) and transfected 24 h before ICH modeling.Npas4 gene was packaged into the Vector (pcAAV-CMV-EGFP-P2A-Npas4-3xFLAG-WPRE) for overexpression (AAV-Npas4). A plasmid (pcAAV-CMV-EGFP-P2A-MCS-3xFLAG-WPRE) not encoding NPAS4 was used as a control AAV vehicle (AAV-NC). Both were purchased from Obio Technology Corporation . Adeno-associated viruses, namely, AAV-Npas4  or AAV-NC , were administered in the right basal ganglia (anteroposterior (AP), −0.2 mm; mediolateral (ML), −2.0 mm; dorsoventral (DV), −3.5 mm) over 10 min one month before the establishment of ICH model.To evaluate the neurological function of the murine models, a grip strength test was conducted using a grip strength meter  that recorded the maximum and average force. The forces were measured in Newtons. During the test, the grip strength meter was positioned horizontally, and the mice were held by the tail and lowered towards the apparatus. For the four-limb placing test, mice were allowed to grasp the metal grid with four limbs; for the forelimb placing test, mice were allowed to hold the metal grid with their forelimbs, while their hindlimbs were not allowed to make contact with the grid. Then, mice were gently pulled backwards by the tail until they could no longer hold the grid. Grip strength test was carried out in triplicate for each mouse.H&E staining was used to assess brain edema and neuronal injury after ICH. After sequential perfusion of normal saline and 4% paraformaldehyde from apex of the heart into the whole body, the brains were extracted and soaked in 4% paraformaldehyde for 24 h fixation and then dehydrated with gradient ethanol. After being transparentized in dimethylbenzene and immersed in paraffin wax, the brains were sliced into 5 µm thick, paraffin-embedded coronal sections. Following deparaffinization and benzene removal, the brain sections were stained with hematoxylin–eosin and, in a gradient, dehydrated again so that they could be observed under a microscope.Nissl staining was performed to measure neuronal loss after ICH. The brain sections were collected through the above-mentioned approach. After being debenzolized and deparaffinized, the brain slices were soaked in a tar purple solution  for 5–15 min and then decolorized in gradient ethanol for 2 min each. Finally, the slices were transparentized, sealed, and observed under a microscope.Brain sections were collected through the above-mentioned approach. After being deparaffinized in xylene and dehydrated with gradient ethanol, brain slices were submerged in antigen retrieval solution and then heated in a high-pressure cooker for 3 min for antigen retrieval. Slices were allowed to cool to room temperature before proceeding. Brain slices were washed three times with phosphate-buffered saline (PBS) and permeabilized with 0.4% Triton X-100 for 30 min. After being blocked with 5% goat serum  for 30 min at room temperature (RT), slices were incubated with primary antibodies overnight at 4 °C and, subsequently, corresponding secondary antibodies  in the dark for 1 h at RT after being washed three times with PBS. Lastly, the slices were counterstained with 4′,6 diamidino-2-phenylindole . TUNEL assay was conducted using a TUNEL Apoptosis Detection Kit . The primary antibodies used included rabbit anti-Iba-1 , rabbit anti-NeuN , rabbit anti-GFAP , goat anti-NPAS4 , rabbit anti-MPO , and rabbit anti-Caspas-1 . Images were captured with a fluorescence microscope  and analyzed using ImageJ software (1.48v).Immunohistochemical staining was conducted according to the universal two-step test kit . Briefly, 5 µm sections of the brain tissues were baked at 70 °C for 2 h. Then, the sections were de-paraffinized in xylene, rehydrated using a gradient of ethanol concentrations, boiled in citrate antigen retrieval solution in a high-pressure cooker for 3 min for antigen retrieval, blocked with 3% hydrogen peroxide for 15 min to inhibit endogenous peroxidase activity, and incubated with goat serum for 30 min to reduce background non-specific staining. Subsequently, sections were incubated with goat anti-NPAS4  overnight at 4 °C and then a corresponding secondary antibody at room temperature for 15 min. Subsequently, color development was determined using a DAB Substrate kit . Lastly, the sections were counterstained with hematoxylin, dehydrated, transparentized, and sealed. The intensity of immunostaining was scored as 0 (no immunostaining), 1 (weak immunostaining), 2 (moderate immunostaining), or 3 (strong immunostaining). The percentage of positive cells was scored as 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). Finally, the IHC score for each sample was calculated as the intensity of the immunostaining score multiplied by the percentage-of-positive-cells score.Brain tissue around hematoma at different timepoints after ICH was collected for protein homogenate extraction. Protein concentration was quantified using the BCA method with a commercial kit . A total of 30 µg of protein from each group was electrophoresed on 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane. The membranes were incubated at 4 °C overnight with primary antibodies, which was followed by incubation with corresponding anti-goat  or anti-rabbit  secondary antibodies for 2 h at room temperature. Enhanced chemiluminescence reagent kits  were used to visualize the bands. Integrated optical density (IOD) values were measured for the quantification of target protein using ImageJ software (1.48v) and expressed as a relative value normalized to β-actin levels. The following primary antibodies were used: goat anti-NPAS4 , rabbit anti-β-actin , rabbit anti-IL-6 , rabbit anti-TNF-α , rabbit anti-IL-1β , rabbit anti-N-GSDMD , rabbit anti-GSDMD , rabbit anti-cleaved-Caspase-1, rabbit anti-pro-Caspase-11 , and rabbit anti-NLRP6 .−ΔΔCT method. Primers used for the analysis are listed as follows: NLRP6—sense (CGGGACGAGAGGAAGGAGAG), antisense (CACACGATCCAGCACACGAGG); NLRP6(ChIP-PCR)—sense (GCTTGGCTTTGTATTTCTTCCCTT), antisense (TGGTTTCCTGATCCCACATGAAG); GAPDH—sense (GATGCAGGGATGATGTTCTG), antisense (GTGAAGGTCGGTAACGG).Total mRNA was extracted from isolated brain tissues using trizol reagent. qRT-PCR was performed in duplicate using the SYBR Green method, and the transcriptional level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. mRNA expression levels of candidate genes were calculated using 2E. coli strain was used for plasmid construction. Liquid medium used for bacterial cultures in this study was Luria–Bertani–Miller (LB) broth. Premixed antibiotic solution of penicillin and streptomycin was purchased from Gibco . T4 DNA ligase was acquired from Takara Biomedical Technology . Phusion High-Fidelity DNA Polymerase and all restriction enzymes were purchased from New England BioLabs . Genomic DNA templates of a murine origin were obtained from Qi, H. at Basic Medical College, Chongqing Medical University. All plasmids were constructed using basic molecular cloning methods, including standard steps such as PCR, restriction digestion, ligation, and transformation. In brief, to generate NPAS4 overexpression plasmids (H340-Npas4-Flag), oligonucleotide primers (Nlrp6 promotor(fFluc) and pGL4.10-Nlrp6 promotor mutant (fFluc), first, Nlrp6 gene promotor regions  were obtained from the NCBI online database and then corresponding NPAS4 binding sites  were predicted using the Jaspar2020 website. Particularly, mutations in the predicted binding sites were performed using base mutation from A/T to G/C and vice versa, and then the primers  and pGL4.74(rLuc) plasmids. After transfection, cells were harvested, and lysates were used to detect firefly and renilla luciferase activity using dual luciferase reporter gene assay kits  (used according to the manufacturer’s protocol). Normalized values (firefly/renilla activity) were used for analysis.® Enzymatic Chromatin IP Kit , which was used according to the manufacturer’s instructions. In brief, N2A cells were fixed with 1% formaldehyde for 15 min at room temperature; then, MNase digestion was carried out. Cell lysate was sonicated for 5 min at 0 °C. Subsequently, protein lysate was incubated with anti-NPAS4 , anti-histone H3, or normal IgG for immunoprecipitation overnight at 4 °C. After releasing crosslink, purified DNA was subjected to PCR (using the primers 5′-GCTTGGCTTTGTATTTCTTCCCTT-3′ and 5′-TGGTTTCCTGATCCCACATGAAG-3′) to amplify the −400 to −391 region of the mouse Nlrp6 promoter (site 2).ChIP assay was performed by employing the SimpleChIPt test was used between two groups. Normal one-way ANOVA followed by Tukey’s test were conducted for multiple comparisons. The data are presented as means ± SD. A p value < 0.05 was considered significant.GraphPad Prism software (version 8.0.2) was used for statistical analysis. Student’s In conclusion, our study demonstrated for the first time that NPAS4 can interact with the Nlrp6 promoter region (−400–−391 bp and −33–−24 bp) and subsequently upregulate Nlrp6 transcription levels. Additionally, it also confirmed that NPAS4 might aggravate pyroptosis and brain injury via activating the NLRP6 inflammasome after intracerebral hemorrhage in mice.


IbMYB1-4. In this study, a yeast one-hybrid screening aimed at identifying transcription factors upstream of the promoter IbMYB1-4 was performed in the storage roots of the purple-fleshed sweet potato, and IbPDC, IbERF1, and IbPGP19 were identified as upstream binding proteins for the promoter IbMYB1-4. A dual luciferase reporter assay, and yeast one-hybrid assays, were employed to confirm the interaction of these binding proteins with promoters. IbERF1 was found to be an upstream transcription factor for the promoter IbMYB1, and is implicated in the biosynthesis of anthocyanin in the purple-fleshed sweet potato. IbERF1 plays a major role in the biosynthesis of anthocyanin in the purple-fleshed sweet potato.IbMYB1 is a transcription factor involved in the biosynthesis of anthocyanin in the purple-fleshed sweet potato. So far, few studies have investigated transcription factors that are upstream of the promoter  CHI, CHS, DFR, UF3GT, ANS, and F3H. Transcription factors (TFs) regulate the expression of these structural genes. These trans-acting factors comprise a group of DNA-binding proteins that bind specifically to cis-acting elements of eukaryotic genes, and regulate the activation or inhibition of gene transcription [IbMYB1 gene expression was reported to correlate positively with that of related structural genes in anthocyanin biosynthesis. The IbMYB1 promoter (length 2183 bp) was cut into four segments . A previous study investigated the IbMYB1-1 promoter for upstream TFs [IbMYB1-2 and the promoter IbMYB1-3, with 400 ng/mL being the minimum inhibitory AbA concentration for the PAbAi-PIbMYB1-4 strains. However, little is known regarding TFs that are upstream of PIbMYB1-4 (the promoter of IbMYB1-4) in the anthocyanin synthesis pathway of the PFSP storage roots. In this study, a yeast one-hybrid screening aimed at identifying transcription factors upstream of the promoter IbMYB1-4 was performed in the storage roots of the purple-fleshed sweet potato. IbPDC, IbERF1, and IbPGP19 were identified as upstream binding proteins for the promoter IbMYB1-4. IbERF1 was found to be an upstream transcription factor for the promoter IbMYB1, and is implicated in the biosynthesis of anthocyanin in the purple-fleshed sweet potato.The sweet potato is a major food crop in China, and is mainly grown in tropical and subtropical regions. High levels of anthocyanin with specific physiological activity and functions are present in the purple-fleshed sweet potato (PFSP). Anthocyanin has anticancer and antioxidation functions, and plays a role in lowering blood pressure and preventing arteriosclerosis . The acccription . The biocription . The IbMream TFs . It was IbMYB1-4 promoter.The AP2/ERF families are ethylene response factors (ERFs) involved in multiple physiological processes during the growth of plants, and in their response to biological, as well as nonbiological, stress ,7. The eArabidopsis thaliana) was used for dual luciferase and subcellular localization assays. It was grown at 20 ± 2 °C in a chamber, using a day/night cycle (16 h/8 h), and a constant light intensity (100 μmol/m2/s).The PFSP cv. A5 and the white-fleshed sweet potato (WFSP) cv. Yubeibai were cultivated in a garden at South China Normal University, Guangdong, China. Arabidopsis  was ground into a powder with liquid nitrogen using a mortar and pestle. The DNA extraction was then carried out using a plant DNA kit , and RNA extraction was conducted using a Hipure plant RNA kit . To eliminate the possibility of DNA contamination, the RNA underwent DNase I digestion using an RNase-free kit . A BioPhotometer Plus  was used to estimate the DNA concentrations and purity by measuring the absorbance at 230, 260, and 280 nm. The DNA samples showed 1.8 ≤ ODTM Reverse Transcription System  was used for cDNA synthesis, with the promoter and genomic fragments subsequently cloned and sequenced. Agarose gels (1.2%) were used to analyze the PCR products. The fragments were ligated into plasmid, transformed into Escherichia coli DH5α competent cells, then sent to be sequenced by Sangon Biotech, Shanghai, China.A GoScriptIbMYB1-4 promoter was inserted into pAbAi, resulting in pAbAi-bait. The plasmids containing pAbAi-bait were subsequently linearized, and transformed into Y1HGold cells. Colonies growing in a synthetic dextrose medium lacking uracil were then selected. After the minimal inhibitory concentration of aureobasidin A (AbA) for bait strains was identified, the linear pGADT7-Rec vector was co-transformed into bait yeast strains, and then selected using synthetic dextrose (SD)/-Leu/AbA plates. The primers used in the Y1H screening assay are shown in Y1H screening was performed using Matchmaker Gold Y1H Library Screening, as described previously . The RNAIbMYB1-4 promoter. In brief, fragments of the promoter were ligated into the pAbAi vector, while upstream regulators were cloned into pGADT7. The pAbAi was linearized and transformed into Y1HGold cells. The IbMYB1-4 promoter was inserted into the pAbAi, to create pAbAi-bait. The complete CDSs for IbERF1, IbPGP19, and IbPDC were inserted separately into the pGADT7 vector, to create prey-AD vectors. These were transferred into the bait strain, and cultured on SD/-Leu/AbA plates. The primers used for the Y1H assay are shown in Y1H was also performed using the Matchmaker Gold Y1H System, as described in another previous study . The Y1HIbERF1, IbPGP19, and IbPDC were cloned into pGBKT7, to construct bait-BD vectors. The PGBKT7-bait and PGADT7-empty vectors were co-transferred into Y2HGold cells. The positive control was pGBKT7-53 co-transformed with pGADT7-53, while the negative control was pGBKT7 co-transformed with pGADT7-53. The primers used in the Y2H assay are shown in Y2H assays were performed using the Matchmaker Gold Y2H kit , as recommended by the manufacturer, but with minor modifications. In brief, the PEG/lithium acetate method was used to simultaneously transform the pGADT7 (AD) and pGBKT7 (BD) vectors into Y2HGold cells. The transcriptional activity for IbERF1, IbPGP19, and IbPDC was investigated using the Y2H assay. The full-length coding sequences for IbMYB1-4 promoter. In brief, the full-length cDNAs for IbERF1, IbPGP19, and IbPDC were inserted into the vector pGreen II 0029 62-SK , while the IbMYB1-4 promoter was inserted into the vector pGreen II 0800-LUC . Both these constructs were then transformed into arabidopsis protoplasts, as described in a previous publication [® Reporter Assay (Promega). Three independent experiments were performed for each of the interactions between the TFs and promoters, and three replicates were used for each experiment. The positive control was a Renilla luciferase gene driven by the 35S promoter in a luciferase vector. Mixtures containing each TF with the empty vector 62-SK were also used on promoters as a control. The primers for the dual-luciferase assay are shown in Dual-luciferase assays were performed, to quantify the transactivation abilities of IbERF1, IbPGP19, and IbPDC with the lication . The LUCBamH Ⅰ and Hind Ⅱ. This vector contains the promoter for UBQ, as well as the GFP gene. Both constructs were transformed into Arabidopsis protoplasts, in accordance with a previous publication, while protoplasts were prepared, as described previously, but with slight modifications [For the investigation of subcellular localization, upstream TF CDSs lacking a stop codon were amplified, and cloned into pCambia1300 , with ications . The proIbCHI, IbCHS, IbF3H, IbF3′H, IbDFR, IbANS, and IbUF3GT, in the roots  of the PFSP cv. A5, and the WFSP cv. Yubeibai. The Prime Script™ RT Master Mix (Takara) was employed, to synthesize the first-strand cDNA from the total RNA, and SYBR® Premix Ex Taq™ II (Takara) was used to perform RT-qPCR. The 20 μL reaction volume contained 10 μL of SYBR® Premix Ex Taq™ II, each primer at 0.5 μM, and 100 ng of template cDNA. The amplification program consisted of one cycle for 10 s at 95 °C; then 40 cycles for 5 s at 95 °C, and 30 s at 60 °C, using a Bio-Rad CFX96 Real-Time PCR system , as recommended by the manufacturer. The internal control was IbG14, and the calculations were performed using the Ct analysis method. The primers for the RT-qPCR experiments are shown in RT-qPCR was used to evaluate the expression of the upstream TF IbERF1, the TFs IbMYB1, IbbHLH2, and IbWD40, and the structural genes p < 0.05). Sigmaplot 12.3 was used to draw the figures.Three replicates of each biological sample were evaluated, using one-way analysis of variance (ANOVA). The SPSS 21.0 statistics software package  was used to determine the significant differences, with Tukey’s honest test (IbMYB1 promoter (length 2183 bp) was cut into four segments . A previous study investigated the IbMYB1-1 promoter for upstream TFs [PIbMYB1-2 and PIbMYB1-3, with 400 ng/mL being the minimum inhibitory AbA concentration for the PAbAi-PIbMYB1-4 strains. A total of 519 positive colonies were screened for PIbMYB1-4. The Y1H assay identified 130 binding proteins for the IbMYB1-4 promoter. PIbMYB1-4. The IbERF1, IbPGP19, and IbPDC proteins were found to interact with the promoter of IbMYB1-4. This gene is involved in the biosynthesis of anthocyanin.The  length 23 bp was IbERF1/IbPGP19/IbPDC+pGADT7-empty transformed strains were found to grow on SD/-Trp, SD/-His-AbA plus, and SD/-His-AbA X-a-Gal plus plates, with the color of the emerging yeast colony being blue. The pGADT7-53+PGBKT7 transformed strains were unable to grow on SD/-Trp, SD/-His-AbA plus, and SD/-His-AbA X-a-Gal plus plates  was fused to the C-terminus of IbERF1, IbPGP19, and IbPDC, and transiently expressed in eus only , indicatReal-time PCR was employed to evaluate the expression of the upstream TFs, TFs, and structural genes involved in the biosynthesis of anthocyanin at the different root stages of the PFSP and WFSP . The expPIbMYB1 involved in anthocyanin biosynthesis in the PFSP’s storage roots were screened using a Y1H assay. IbERF1 was found to be an upstream TF for PIbMYB1-4 in the synthesis of anthocyanin. The interactions between IbERF1 and PIbMYB1-4 were confirmed using dual-luciferase and Y1H assays. Moreover, IbERF1 was found in the nucleus, using a subcellular localization method. This indicates that IbERF1 is a nuclear protein, and functions as a TF in regulating the expression of downstream genes.Anthocyanin biosynthesis in plants is regulated via the MBW complex. Other TFs, including ERFs, COP1, WRKYs, SPL9, NACs, and DELLA proteins, are also thought to affect the activity of the MBW complex, and hence the biosynthesis of anthocyanin ,21,22,23DcPAL3 promoter activity, while DcERF2 functions in different ways in anthocyanin biosynthesis [Arabidopsis, a double mutant (aterf4 and aterf8) reduced the light-induced anthocyanin accumulation, indicating the positive regulation of anthocyanin biosynthesis by AtERF4 and AtERF8 [The ethylene response factors AP2/ERFs are a major TF family, involved in multiple physiological processes, including plant growth, and the response to biological and non-biological stress ,6. It haynthesis . In Salvynthesis . TF ERFsynthesis . In Arabd AtERF8 . In peard AtERF8 .MdMYB1, MdMYB9, and MdMYB11 promoters to regulate anthocyanin biosynthesis [MdMYB1 promoter to regulate the drought-stress-induced biosynthesis of anthocyanin at the posttranslational level [IbMYB1-4 promotor, and thus regulate the biosynthesis of anthocyanin in the PFSP. These results help to clarify the underlying mechanism for anthocyanin biosynthesis, and improve our understanding of the regulatory networks in the PFSP.ERF interacts with MYB proteins, and binds to MYB promoters, to regulate the biosynthesis of anthocyanin in plants . In applynthesis . MdERF38al level . In pearal level , while Pal level . IbERF1 IbMYB1-4 in the regulation of anthocyanin biosynthesis in the PFSP.In this research, IbERF1 was identified as a TF for