Schizosaccharomyces pombe, systematic analyses of single transcription factor deletion or overexpression strains have made substantial advances in determining the biological roles and target genes of transcription factors, yet these characteristics are still relatively unknown for over a quarter of them. Moreover, the comprehensive list of proteins that regulate transcription factors remains incomplete. To further characterize Schizosaccharomyces pombe transcription factors, we performed synthetic sick/lethality and synthetic dosage lethality screens by synthetic genetic array. Examination of 2,672 transcription factor double deletion strains revealed a sick/lethality interaction frequency of 1.72%. Phenotypic analysis of these sick/lethality strains revealed potential cell cycle roles for several poorly characterized transcription factors, including SPBC56F2.05, SPCC320.03, and SPAC3C7.04. In addition, we examined synthetic dosage lethality interactions between 14 transcription factors and a miniarray of 279 deletion strains, observing a synthetic dosage lethality frequency of 4.99%, which consisted of known and novel transcription factor regulators. The miniarray contained deletions of genes that encode primarily posttranslational-modifying enzymes to identify putative upstream regulators of the transcription factor query strains. We discovered that ubiquitin ligase Ubr1 and its E2/E3-interacting protein, Mub1, degrade the glucose-responsive transcriptional repressor Scr1. Loss of +ubr1 or +mub1 increased Scr1 protein expression, which resulted in enhanced repression of flocculation through Scr1. The synthetic dosage lethality screen also captured interactions between Scr1 and 2 of its known repressors, Sds23 and Amk2, each affecting flocculation through Scr1 by influencing its nuclear localization. Our study demonstrates that sick/lethality and synthetic dosage lethality screens can be effective in uncovering novel functions and regulators of Schizosaccharomyces pombe transcription factors.In  The physical interactions between transcription factors, their regulators, as well as their target genes form a complex regulatory network that establishes the transcriptome of the cell.Transcription factors are an integral component of the response to external stimuli during growth and development  interaction is when the phenotype of a double deletion mutant has a greater growth defect than expected based on the growth of each single mutant . SDL intS. cerevisiae to further characterize gene function, facilitated by the availability of full-genome deletion and overexpression collections  transcription factors have no assigned gene name and well-defined biological role, although the full complement of single deletion and overexpression strains has been phenotypically characterized under standard lab conditions  composed of 1% (v/w) yeast extract, 3% (v/v) glycerol, and 4% (v/v) ethanol. All media used in the SGA screens were supplemented with 2% galactose to limit cell–cell adhesion and allow more consistent pinning of the yeast arrays. Standard genetic and molecular methods were performed as previously described , followed by lithium acetate transformation into a leu1-32 strain.The NatMX4) with a transcription factor deletion array of 91 strains (KanMX6) and were based on the SpSGA methodology developed by Δleu1::KanMX4 Bioneer strain was subjected to a marker switch with the NatMX4 cassette and crossed to the transcription factor deletion array to obtain an estimate of the single mutant fitness of the array strains. SL screens were performed in 768 colony array format in which each transcription factor deletion array strain was represented 6 times with a border of Δhis5 strains to control for spatial bias at the edges of the plate, as well as at least 1 blank spot. All media used in the SL SGA screens were supplemented with A, L, H, and U to ensure that growth was not affected in the control query strain. The query and array strains were mated by mixing on SPAS plates with a BM3 BioMatrix Robot . The plates were incubated at 25°C for 3 days to allow mating and sporulation and then transferred to 42°C for 3 days to select for spores and remove unmated vegetative cells. The spores were germinated by pinning on YES medium and the vegetative cells were grown for 3 days at 30°C. The cells were subsequently pinned on YES medium containing G418 and clonNAT to select for double transcription factor deletion strains. In this final step, the entire array was pinned in duplicate to a final density of 1,536 colonies per plate in which each double mutant strain was represented a total of 12 times.The SL screens involved crossing 38 transcription factor deletion query strains . Normalization of colony size to correct for spatial biases, resulting from variation in the media or local environment on the plate, was performed with SGAtools  was performed to validate negative genetic interactions from the SGA screens. Parental strains were mated on SPAS medium, incubated for 3 days at 25°C, and then suspended in 0.5% glusalase solution for 6 h at 30°C to remove vegetative cells. The spores were washed twice in sterile water to remove the glusalase, diluted 1,000-fold, and plated at a ratio of 1:2:2:4 on YES, YES + G418, YES + clonNAT, and YES + G418 + clonNAT media . The corrected total cellular fluorescence was calculated as described by Δubr1 and Δmub1 cells was determined by a 2-tailed t-test.All fluorescence cell images in this study were captured with a Zeiss Imager Z1 microscope and AxioCam MRM digital camera . To examine potential additive cell cycle phenotypes from our SL screens, single and double transcription factor deletion strains were exponentially grown in YES medium at 30°C. The cells were then methanol-fixed and treated with DAPI (1 μg/mL) to stain the nuclei and calcofluor white (50 μg/mL) to stain the cell wall material. To identify potential regulators of transcription factors from our SDL screens, the intracellular localization and intensity of Scr1-GFP under the native promoter were compared in wild-type, 7 cells/mL into 20 mL FIM and liquid EMM, respectively, and grown for at least 72 h at 30°C in a shaking incubator. The induction of flocculation by overexpression of +sds23 was determined by incubating a nmt1 promoter-driven +sds23 strain on an EMM + thiamin plate overnight at 30°C, and cells (∼107 cells/mL) were then transferred into 20 mL of liquid EMM and grown for 24 h at 30°C in a shaking incubator. To test if overexpression of +scr1 could repress flocculation, a nmt1 promoter-driven +scr1 strain was streaked on an EMM plate without thiamin and incubated for 24 h at 30°C to allow induction of gene expression. Cells (∼107 cells/mL) were then transferred into 20 mL of liquid FIM and grown for at least 72 h at 30°C in a shaking incubator. After incubation, 5 mL of cell cultures were transferred to 60 mm × 15 mm Petri dishes and placed on an orbital shaker for 10 min to promote floc formation. Images of flocs were acquired using a spImager-M system . Three replicates of the flocculation assay were performed for each strain.Normal and constitutive flocculation of deletion and control strains were assayed by inoculating ∼10S. pombe. Ninety-one nonessential transcription factor deletion mutants were used as the array strains, 38 of which were also selected as query strains  . The scr+h haploids, respectively. The different stresses that cells are exposed to could have various effects on fitness for certain mutant strains, such as thermosensitive strains in our screens. Unlike the S. cerevisiae revealed low correspondence of fitness measurements and genetic-interaction scores between the 2 types of screens  and S phase (+res2 and +tos4) where their loss is known to cause defects in the progression of the cell cycle  had greater cell lengths than the single mutants, indicating a disruption of the cell cycle . These a+prz1 also resulted in double mutant cells that were more elongated than either single mutant  (We developed a systematic SDL screening method using SGA for factors) . Fourteefactors) . Five adnmt1 promoter in the presence of thiamin and a strong genetic interaction with the deletion mutant, which does not tolerate any degree of transcription factor overexpression.We detected 195 putative SDL interactions from 3,906 double-mutant combinations from our SDL SGA screens (4.99%) . Fifty-o+scr1 overexpression with Δsds23, as well as +yox1 overexpression with Δcds1, Δcdt2, and Δgad8  is regulated by Scr1  (ubr1 cells were still significantly higher than wild type (P = 0.0072) (The Scr1 transcription factor functions in catabolite repression by inhibiting transcription of target genes such as  deleted . Two of  0.0001) . While d 0.0072) . Altogetscr1 strain formed small flocs in liquid EMM, indicating that Scr1 functions in the repression of flocculation under noninducing conditions . Therefore, 4 of the 6 confirmed SDL interactions of +scr1 appear to function as putative negative regulators of Scr1, either through degradation by ubiquitination or nuclear exclusion.We hypothesized that hits recovered from our SDL screens of transcription factor genes are likely to represent negative regulators. If this was the case, then the SDL hits of n in FIM . Like sdckground . The chaS. pombe and S. cerevisiae, there is evidence that phosphorylation is a necessary precursor to ubiquitination by the Ubr1/Ubr2 complex. In S. cerevisiae, Sml1, an inhibitor of ribonucleotide reductase, is degraded by the Rad6-Ubr2-Mub1 ubiquitin ligase complex when it is phosphorylated upon exposure to DNA damage . Tos4 is involved in the DNA damage response and its expression is tightly coupled with S phase : Brl1, Rhp6, Brl2, and Shf1. The HULC is responsible for the mono-ubiquitination of H2B lysine 119 and is associated with heterochromatin, enhanced silencing of several genes, and increased pol II occupancy at the centromeric repeats . Interestingly, the Δpef1 and Δcdr1 strains also share a synthetic negative genetic interaction with the Δyox1 strain  to be omitted from the final analysis. Defects in growth, cell adhesion, or mating efficiency could alter the effectiveness of the robotic pinning or mating steps of the SGA procedure. In the future these complications could be alleviated by using a weaker promoter such as nmt41 or nmt81 to overexpress the transcription factor gene or by integrating the pREP plasmid into the genome  and Canada Foundation for Innovation (16855).None declared.jkac194_Figure_S1Click here for additional data file.jkac194_Figure_S2Click here for additional data file.jkac194_Table_S1Click here for additional data file.jkac194_Table_S2Click here for additional data file.jkac194_Table_S3Click here for additional data file.jkac194_Table_S4Click here for additional data file.jkac194_Table_S5Click here for additional data file.jkac194_Table_S6Click here for additional data file.


Transcription factors (TFs) modulate gene expression by regulating the accessibility of promoter DNA to RNA polymerases (RNAPs) in bacteria. The MerR family TFs are a large class of bacterial proteins unique in their physiological functions and molecular action: they function as transcription repressors under normal circumstances, but rapidly transform to transcription activators under various cellular triggers, including oxidative stress, imbalance of cellular metal ions, and antibiotic challenge. The promoters regulated by MerR TFs typically contain an abnormal long spacer between the –35 and –10 elements, where MerR TFs bind and regulate transcription activity through unique mechanisms. In this review, we summarize the function, ligand reception, DNA recognition, and molecular mechanism of transcription regulation of MerR-family TFs. The canonical Class I and Class II transcription activation models suggest that transcription activators interact with both RNAP and the upstream of core promoter region to increase local enrichment of RNAP at their regulated gene promoters and/or to facilitate subsequent promoter unwinding process ,6. DistIn this review, we summarize the physiological function, ligand reception, DNA recognition, and mechanism of transcription regulation of MerR-family TFs. Recent comprehensive reviews are recommended for readers who are interested in mechanism of bacterial transcription and general bacterial transcription regulation ,9,15.2, σ3.1, and σ4 of σ70-type factors; region I and III for σ54-type factors) on the surface of RNAP core enzyme and thread the linker domain  into the active-site cleft . Li+, Ag+, and Au+, or +2 charge, for example Cd2+, Zn2+, Pb2+, Ni2+, and Co2+ (2+ to volatile Hg (MerA), transcription factors (MerD and MerR), a Hg2+ scavenging protein Hg2+ (MerP), and Hg2+ transporters  cluster, of which each of the Fe atoms is stabilized by two coordinate valent bonds made by two cysteine residues  E or at aesidues F. In sumP.aeruginosa BlrR andE.coli EcmrR [The multidrug-resistance MerR TFs have a Gyrl-like ligand-binding domain (LBD) at their C-terminal domain that is much larger than the metal-binding loop of the metal- and redox-responsive members of the family G[119]. i EcmrR ,110.+-CueR/DNA (activated complex) determined by O’ Halloran’s lab provided excellent opportunity to understand the conformational change induced by ligand bindingMost MerR-family TFs interact with DNA both in the absence and in the presence of ligands, while the state of ligand occupancy defines the shape of dsDNA and determines the outcome of transcription ,122. Re+ binding. The folded metal binding loop establishes new interaction with nearby structure units and transmits the ligand-biding signal to the distance change of two DBDs. The refolded metal binding loop wedges into the interface of the nearby dimerization helix and DBD. Such event on one hand causes a slight inward rotation of the nearby DBD, and on the other hand causes a small ‘scissors’ movement of dimerization helix resulting in further inward rotation of DBD at the other end. Because both the half palindromic repeats of DNA are tightly anchored by DBD, the conformational change of DBD forces kinking and under twisting of their associated DNA. Other metal-responsive MerR-family TFs likely use the same strategy to trigger the allosteric movement upon metal binding. However, it is still to be determined how the redox-responsive MerR-family TFs, such as SoxR that coordinates the [2Fe-2S] metal cluster and adopts ordered conformation in both the oxidative and reduced conditions, interchanging their conformations . Thctively ,19. The–20 bp be2 domain and restrains the dsDNA in the inactive shapeBinding of apo MerR-family TFs further inhibits the weak basal transcription activity of their regulated promoter DNA ,65. TheE.coli CueR-TAC,B.subtilis BmrR-TAC, andE.coli MrR-TAC  holoenzyme recognizes the -35 and -10 elements from the opposite face. The architecture explains the paradox that MerR-TFs activate transcription while occupying the core promoter region, an interaction typically represses transcription due to steric hinderance between TFs and RNAP. Intriguingly, although RNAP and MerR-TF occupy the same core promoter regions, BmrR makes interaction with neither RNAP core enzyme nor σA factor in the cryo-EM structure ofB.subtilis BmrR-TACE.coli CueR and EcmrR only contact a small surface patch of the non-conserved region of σ70 factor (σNCR) in the cryo-EM structures ofE.coli CueR-TAC andE.coli EcmrR-TAC  inE.coli are able to activate transcription of their regulated promoters using evolutionarily distant RNAPs  that have completely different σNCR domainsNCR interactions observed inE.coli CueR-TAC and EcmrR-TAC, however, contribute to the transcription activation by providing auxiliary bridge interaction between RNAP and DNA . . 70  during eukaryotic polymerase II transcription initiation. A recent study in Seok’s lab identified FruR, a GalR-LacI family transcription factor in


The relationship between long-range Polycomb-associated chromatin contacts and the linear propagation of histone H3 lysine 27 trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) is not well-characterized. Here, we nominate a role for developmental loci as genomic architectural elements that enable long-range spreading of H3K27me3. Polycomb-associated loops are disrupted upon loss of PRC2 binding and deletion of loop anchors results in alterations of H3K27me3 deposition and ectopic gene expression. These results suggest that Polycomb-mediated genome architecture is important for gene repression during embryonic development. in cis, both locally and megabases away on the same chromosome. In mouse embryos, loop anchor deletion leads to ectopic activation of the partner gene, suggesting that Polycomb-associated loops control gene silencing during development. Further, we find that alterations in PRC2 occupancy resulting from an RNA binding–deficient EZH2 mutant are accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 uses RNA binding to enhance long-range chromosome folding and H3K27me3 spreading. Developmental gene loci have unique roles in Polycomb spreading, emerging as important architectural elements of the epigenome.Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction. This process is essential for embryonic stem cell (ESC) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spreading of H3K27me3 on the linear genome. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remain unclear. Here, we apply H3K27me3 HiChIP, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes. Genetic deletion of H3K27me3 loop anchors results in disruption of spatial contact between distant loci and altered H3K27me3  Regulation of gene expression is crucial for a myriad of biological processes, including embryonic development, tissue homeostasis, and dosage compensation –5. Gene Drosophila melanogaster. In mammalian genomes, hypomethylated CG bases known as CpG islands may function similar to Drosophila PREs (Polycomb-group proteins (PcGs) and their target genes are evolutionarily conserved , but howila PREs , 10 and ila PREs , 11, 12.ila PREs –18. Howeila PREs  and coheila PREs . Polycomila PREs , 22. RNAila PREs –25, likeila PREs . As PcGsila PREs  and the ila PREs  and optiila PREs  combinedSI Appendix, Fig. S1A). We compared high-confidence loops called by HICCUPS  and shared loops represented a higher-confidence subset of loops called by FitHiChIP . Therefore, we focused our analysis on 4,101 high-confidence H3K27me3-associated loops that were robustly detected over background and present on all chromosomes . Comparison of chromatin loops revealed that H3K27me3-associated loops bridge genomic distances spanning dozens of megabases, crossing significantly greater distances than H3K27ac-associated loops which are enriched at enhancer–promoter contact regions , suggesting that cohesin-CTCF may be more important for H3K27ac-associated loops and that other factors may mediate H3K27me3-associated loops.As enhancer–promoter contacts tend to occur specifically within topologically associating domains (TADs) , 34, 35,SI Appendix, Fig. S2B), in contrast to H3K27ac loop anchors and all H3K27me3 peaks including nonlooping peaks . Further, we find that PRC2 nucleation sites are more likely to overlap H3K27me3 loop anchors relative to all H3K27me3 peaks excluding PRC2 nucleation sites . Interestingly, we find that enrichment of EZH2 occupancy and nucleation points at H3K27me3 loop anchors increases with increasing loop distance and is substantially enriched compared with H3K27ac-associated loops . We find that PRC1 components such as Ring1B are also enriched at H3K27me3-associated loop anchors, suggesting that both PRC1 and PRC2 complexes are enriched at these long-range contacts. Prior studies .Given the role of PRC1 complexes in long-range genomic interactions , 39, 40, studies , 40 haveSI Appendix, Fig. S4B). While many of these loops, such as those at Hoxa1, Hmx1, and Wnt6, are also detected in deeply sequenced Hi-C . Many key developmental genes encoding transcription factors (TFs) for cell-type specification and patterning, such as the Hoxa gene cluster, make long-range H3K27me3-associated loops with EZH2 occupancy at both anchors but PRC2 nucleation points occasionally only at one anchor ; and 3) a previously unobserved 3.8-Mb loop .To determine which genes are involved in H3K27me3-associated loops, we performed Gene Ontology analysis for the nearest genes to loop anchors and found significant enrichment of developmentally associated processes with distinct ontology term enrichment compared with all H3K27me3 peaks excluding those at loop anchors  a c-Mb loop  connectiHoxa cluster, as well as a region adjacent to the deletion. Additional probes were placed at the Vax2 loop anchor 31 Mb away and at a control region located at the midpoint of the two loop anchors in linear genomic distance. In order to fully remove the PRC2 nucleation and EZH2 occupancy sites, we deleted the majority of the Hoxa cluster from Hoxa2 to Hoxa13, leaving Hoxa1, which is outside of the loop anchor detected by HiChIP. Hoxa cluster genes Hoxa2 to Hoxa13 included in the deletion are not expressed in mESCs so the effects of deletion should not be attributed to altered dosage of deleted genes. ORCA confirmed the presence of a 31-Mb loop identified by HiChIP in unedited cells, with 40% of cells positive for contact (<150 nm) between Hoxa and Vax2 by high-resolution imaging , Wnt6, and Hmx1 genes are not expressed in mESCs, the effects we observe are most likely to result from deleting the genomic region and not from eliminating expression of these genes. Our data showed reduced H3K27me3 adjacent to deletion breakpoints, consistent with loss of local Polycomb spreading . These regions also tend to colocalize in the same compartment . Interestingly, we observed no significant changes in H3K27me3 at long-range contacts with deleted loop anchors identified by HiChIP, all of which have EZH2 occupancy. These data suggest an intriguing possibility that PRC2 nucleation sites such as the Hoxa gene cluster may distribute the repressive H3K27me3 mark to distant regions within the same compartment which lack intrinsic Polycomb binding. We predict that these interactions are dynamic and transient, as we did not identify significant looping interactions between the deleted nucleation sites and regions with loss of H3K27me3 in our HiChIP data. We also identified sites with increased H3K27me3 at local as well as distant sites (We next asked if deletion of H3K27me3 loop anchors can affect spreading of the Polycomb-mediated H3K27me3 mark. We performed H3K27me3 Cut&Tag (cleavage under targets and tagmentation)  in mESCsdeletion A. Becauspreading B. For th between A and C. nt sites C, consisnt sites .Wnt6, the loop anchor partner of Pax3 . Importantly, this loop is maintained during differentiation in both the developing limb bud and in differentiated neural progenitor cells (NPCs)  embryos in distal limb buds, demonstrating the in vivo relevance of this loop for gene silencing  A .To determine if long-range Polycomb loops are conserved features across evolution, we performed H3K27me3 HiChIP in human iPSCs. We converted genomic coordinates for mESC H3K27me3-associated loop anchors to the human genome and found that ∼30% of high-confidence loops identified by H3K27me3 HiChIP in human iPSCs were shared with mouse ESCs, and conserved H3K27me3 loops in the human genome were enriched for similar developmental Gene Ontology terms as observed in mESCs (RNA−EZH2 hereafter) results in genome-wide alterations in Polycomb binding and H3K27me3 deposition with more pronounced effects at specific sites such as TBX5 and FOXA1 . Because RNA−EZH2 iPSCs have decreased H3K27me3 at these same loci, resulting changes in HiChIP signal can be attributed to either loss of H3K27me3 modification or loss of 3D contact. Therefore, we performed 4C-seq  to its loop partner (TBX5) but minimal effects on chromosome looping at sites such as the LHX5 locus which maintains EZH2 occupancy . These results suggest that RNA binding by EZH2 is required to drive long-range chromosome looping and spread Polycomb occupancy to distant loci.To examine the broader effects of PRC2 binding on genome architecture, we utilized an EZH2 variant with mutations in two regions  that is deficient in RNA binding but otherwise has normal PRC2 complex formation and intact H3K27me3 methylase activity , 46. Thind FOXA1 A, as desnd FOXA1 . To ask d 4C-seq , a targeWT cells B. In EZHccupancy B. SimilaHere, we apply H3K27me3 HiChIP to identify long-range Polycomb-associated loops among developmental genes, and demonstrate that local or global loss of Polycomb binding can mediate long-range effects on H3K27me3 deposition and genome architecture. Polycomb loops link H3K27me3-modified loci from the same chromatin compartment that are separated by tens to hundreds of megabases on the linear chromosome, and they appear orthogonal to the ∼1-Mb-size TADs based on CTCF and cohesin, indicating an intermediary structure in the hierarchical organization of 3D genome folding. This finding is consistent with studies that conclude that PRC1-associated interactions are independent of CTCF and TADs . While cin cis , picking the guides showing the best off-target score. WT G4 mESCs (129/Sv × C57BL/6 F1 hybrid background)  protocol . Brieflykground)  were cotentation . For eacPax3 was generated by PCR amplification using mouse limb complementary DNA. All animal procedures were in accordance with institutional, state, and government regulations .In situ hybridization was performed according to standard protocols. The probe from WT and mutant (MT) iPSC clones were obtained from the T.R.C. lab; MT iPSC clones were generated by Y.L. from the original WTC-11 iPSC from the Coriell Institute . For regular culture and passaging, cells were maintained in Essential 8 Flex medium  using vitronectin  as the coating material and the medium was changed every other day until ready for passaging. To passage the cells, 5- to 10-min incubation with 0.5 mM ethylenediaminetetraacetate acid (EDTA) in phosphate-buffered saline (PBS) after a PBS wash step facilitated detachment and cells were split with a 1:6 (up to 10) ratio into new culture dishes coated with vitronectin. Cells were cryopreserved in Essential 8 Flex medium with 10% dimethyl sulfoxide for long-term storage.6) were fixed in 2% formaldehyde for 10 min at room temperature (RT). HiChIP was performed as previously described  library preparation were performed according to the manufacturers’ instructions using the Qiagen RNeasy Mini Kit and the KAPA mRNA Hyper Prep Kit, respectively. Each condition for WT or MT samples was sequenced in biological triplicates using Illumina HiSeq technology according to standard protocols.HindIII (6-bp cutter) was used as a primary restriction enzyme. NlaIII was used as a secondary restriction enzyme. For each viewpoint, a total of 1.6 mg of each library was amplified by PCR . Samples were sequenced 2 × 75 bp with Illumina HiSeq 4000 technology according to standard protocols.The 4C-seq libraries were generated from fixed cells as described previously . HindIII2O.Cut&Tag experiments were performed according to Kaya-Okur et al. . In shorSI Appendix, Table S1) were designed as previously described  spanning 200 kb of the chromosomes tiled by the experimental probes for image registration purposes. Probes were amplified from the oligopool (CustomArray), and amplified according to the protocol described (d-lysine for at least 1 h and then rinsed with 1× PBS to remove residue. A population of control and deletion mESCs was then plated directly onto the coverslip in two spatially distinct populations and allowed to dry for 7 to 10 min. Once cells were dried and adhered to the slide, hybridization and imaging were performed as previously described (The primary probes tiling the regions of interest (escribed  with theescribed , 56. In escribed . For priescribed . Image aescribed .Supplementary File


The expression of Kr-h1 is fluctuant during insect development with high abundance in juvenile instars and lower levels in the final instar and pupal stage, and reappearance in adults, which is governed by the coordination of JH, 20E, and miRNAs. The dynamic expression pattern of Kr-h1 is closely linked to its function in the entire life of insects. Over the past several years, accumulating studies have advanced our understanding of the role of Kr-h1 during insect development. It acts as a universal antimetamorphic factor in both hemimetabolous and holometabolous species by directly inhibiting the transcription of 20E signaling genes Broad-Complex (Br-C) and Ecdysone induced protein 93F (E93), and steroidogenic enzyme genes involved in ecdysone biosynthesis. Meanwhile, it promotes vitellogenesis and ovarian development in the majority of studied insects. In addition, Kr-h1 regulates insect behavioral plasticity and caste identity, neuronal morphogenesis, maturation of sexual behavior, as well as embryogenesis and metabolic homeostasis. Hence, Kr-h1 acts as a cornerstone regulator in insect life.Insect life cycle is coordinated by hormones and their downstream effectors. Krüppel homolog1 (Kr-h1) is one of the crucial effectors which mediates the actions of the two critical hormones of insects, the juvenile hormone (JH) and 20-hydroxyecdysone (20E). It is a transcription factor with a DNA-binding motif of eight C Krüppel homolog1 (Kr-h1) which in turn regulates other stage-specifying genes. However, Kr-h1 is also regulated by 20E and involved in the 20E-mediated events. Hence, it is a major factor that is critical to life of the insects and that has been repurposed during evolution to regulate many processes. The aim of this review is to summarize the significant advances that have been made in the molecular action of Kr-h1 in insect life during the past few years to get a general understandingof the functions of Kr-h1.Insects represent the largest and most diverse group of animals, with over 70% of all species. They are the first to master flight and establish social societies . The comKr-h1 was initially discovered in Drosophila melanogaster by characterizing a P-element induced mutant that dies during the prepupal period and fails to complete head eversion. Molecular studies revealed that the Kr-h1 locus gives rise to two major transcripts (Kr-h1α and Kr-h1β) which derive from two distinct promoters. The Kr-h1α encodes a 791 amino acid protein while Kr-h1β has an additional 54 residues at the N-terminus. Both protein isoforms contain a central domain with eight C2H2 zinc fingers belonging to the Krüppel-like family of transcription factors , which belong to paleopterans. Alignments of the full protein sequences of the orthologs reveal that the eight zinc-finger DNA binding domains are remarkably well conserved among insect orders, as are two additional regions (LPL/PRKR and RXXSVIXXA) at the extreme C-terminus which are proved to be involved in the transcriptional inhibitory activity of Kr-h1 , but the overall expression patterns of these two isoforms are very similar, although the levels of BmKr-h1β are far lower than BmKr-h1α. The expression of Kr-h1 is fluctuant during B. mori development. Both BmKr-h1α and BmKr-h1β are continuously expressed in B. mori epidermis during the larval stages but disappear completely upon ecdysis to the final larval instar, reappear and peak during the prepupal stage, then disappear again in the pupae. From the late pupal stage, they increase gradually and maintain high levels during the adult stage  in the BmKr-h1 promoter had been identified, which also exist in the promoter region of Kr-h1 genes in other insects, like D. melanogaster, T. castaneum, A. mellifera, A. aegypti, and A. pisum , which then positively regulates their binding with JHRE, thus activating the target gene expression , a protein with acetyltransferase activity (HAT), reduces the JH-driven increase of Kr-h1  inhibitor, causes an increase in expression of Kr-h1  motif, which was first reported to be involved in the regulation of programmed cell death in al stage . The stult stage . Importatularius , N. luge. lugens , and Gryaculatus .Kr-h1 expression. It was initially reported in B. germanica that miR-2 bound the 3′UTR of BgKr-h1mRNA and scavenged Kr-h1 transcripts during the transition from nymph to adult. Depletion of miR-2 prevents the normal elimination of Kr-h1. This study suggests that the abrupt decrease of Kr-h1 mRNA levels in the final nymphal instar results not only from the decline of JH titer but also from the action of miR-2  has emerged as a critical post-transcriptional regulator in of miR-2   in the early juvenile stages, the continuous presence of JH and the low levels of miRNA maintain high expression levels of Kr-h1. 2) in the final larval stage of holometabolans, the decrease of JH and the increased expression of miRNA lead to the downregulation of Kr-h1. 3) at the prepupal stage or the mid-late stage of the penultimate nymphal instar, a surge of 20E and JH gives rise to the transient peak of Kr-h1; 4) upon pupation or ecdysis to the final nymphal instar, gradually decreasing JH and increasing levels of E93 and miRNA lead to the disappearance of Kr-h1   or etaboly) . In mosts subtle , adminiss subtle . Converss subtle . Regardl insects , even in aegypti . Moreove program . Additioastaneum , antepenermanica  or penul apterus  induces the pupa . Depletial stage . On the d cocoon . In D. mreatment . In summBroad-Complex (Br-C) and the adult specifier gene Ecdysone induced protein 93F (E93)  . The rolariation , and in nd adult . JH prevpupation . Likewisnous JHM . This inE93 functions as a universal adult specifier in both hemimetabolous and holometabolous insects is afforded by the research of Urena et al. The authors found that E93 is highly upregulated during the final nymphal instar or the entire pupal period. RNAi depletion of E93 in the penultimate nymphs of B. germanicaor mid final instar larvaeof T. castaneum prevents the nymphal-adult or pupal-adult metamorphic transition, inducing multiple reiterations of nymphal molts or producing a supernumerary second pupa, even in the absence of JH  in their promoters , epigenetic modification of the promoter, or post-translational modification of Kr-h1. A study in mosquitoes showed that Kr-h1 acts either as a transcriptional activator or a repressor in female reproduction through directly binding to the regulatory regions of its target genes in a promoter-specific manner  by the fat body, followed by transportation of Vg through the intercellular spaces in the follicular epithelium (known as patency) and subsequent uptake by maturing oocytes via Vg receptor-mediated endocytosis  from the medial neurosecretory cells of the brain. The OEH activates follicular cells of the ovary to produce ecdysone, which is converted into the active 20E in the fat body. 20E then stimulates Vg expression via the ecdysone pathway. The 5′ upstream regulatory region of the mosquito Vg contains several binding sites for EcR-USP and the early and early-late 20E-inducible genes, such as E74B, E75A, Br-C-Z2, βFTZ-F1, and HR3, which indicates a combinatorial control of Vg expression by 20E  were strongly decreased in the JH signaling-deficient flies. The ECM components are indispensable for ovarian muscle contraction that eventually generates a mechanical force. Therefore, JH signaling promotes ovulation and maintains egg shape by inducing the expression of ECM genes demonstrated that the dual functions of Kr-h1 depend on the property of the recruited cofactors. The high expression levels of the corepressor CtBP and coactivator CBP in the juvenile stage and adult stage, respectively, might explain the distinct Kr-h1 complexes forming in these two stages. However, whether other factors are involved in stimulating the specific complexes remains to be investigated.It is interesting to note that Kr-h1 functions as a transcriptional repressor in insect metamorphosis and switches as an activator in adult reproduction. As mentioned previously, the studies by Wu Kr-h1 is also necessary for many other aspects of insect development, such as neuron fate specification and morphogenesis  neurons both at the time of initial morphological differentiation and during metamorphic neurite remodeling. Overexpression of Kr-h1 in developing MB neurons inhibits axon remodeling and blocks MB neuron morphogenesis  are sequentially produced, with γ, α'β' neurons arising during early larval stages or late larval stages respectively, and pioneer αβ and αβ are generated during early metamorphosis  and EcR is required to enable neuronal maturation to proceed normally . In the noctuid moth, Agrotis ipsilon, newly emerged males are sexually immature and unable to behaviorally respond to the female-produced sex pheromone, and theirMAGs are undifferentiated and have a low protein content. Three to five days after emergence, males become sexually mature and highly attracted to the female sex pheromone; their MAGs exhibit elevated protein synthesis. This increase in behavioral responsiveness is well known to be JH-dependent  Kr-h17.1 and Kr-h17id), obtained by the mobilization of the P element in one of the Kr-h1 mutations (Kr-h17), that removed coding sequences common to both DmKr-h1α and DmKr-h1β rarely eclosed as larvae. However, the mutants showed normal embryonic development and elaborated larval structures by the end of embryogenesis, with occasional ectopic neurons  What is the precise mechanism of Kr-h1 acting as both transcriptional repressor and activator? Is it dependent on the recruited cofactors? And what are these cofactors? So far, interactions of Kr-h1 with the coactivator CBP and the corepressor CtBP important for activating vitellogenesis and preventing metamorphosis have thus far been studied in L. migratoria. As Kr-h1 plays many roles in insect life, we speculate that Kr-h1 interacts with multiple distinct partners, each time regulating a different aspect of insect biology. But how does Kr-h1 discriminate against these partners at different developmental stages? And how is their interaction stimulated? Moreover, besides phosphorylation, whether other post-translational modifications affect Kr-h1 binding with these cofactors? Does the opposite stage-specific regulatory effect of Kr-h1 on Br-C depend on the property of partners or the post-translational modification of Kr-h1? In addition, Krüppel-like transcription factors can epigenetically induce DNA methylation or demethylation of the promoters of their target genes to inhibit or promote their transcription respectively. Does Kr-h1 exert similar functions? If so, what are the DNA methyltransferases and demethylase? 2) The functions of the eight zinc finger domains need to be dissected. Do all of them engage DNA? or which of them are involved in DNA binding and protein interaction? Do different zinc fingers recognize specific DNA sequences? 3) Apart from the already known functions, is Kr-h1 involved in other JH or 20E-regulated physiologies, such as spermatogenesis, testicular development, female mating, and sex pheromone production?In mammals, Krüppel-like transcription factors are critical in many physiological and pathological processes including cell proliferation, differentiation, inflammation, and apoptosis. Does Kr-h1 perform similar functions in insects? With the advent of new genomic technologies and the capacity of using different insect species, we will be in an excellent situation to deepen our understanding of the functions of Kr-h1 and properly exploit it as the target of pest control.Kr-h1 is a conserved transcription factor among insect orders. It acts as a cornestone regulator in insect life, from preventing metamorphosis to promoting female reproduction. In addition, it modulates social insects’ caste identity and behavior plasticity and the associated neuronal morphogenesis. Hence,


Dysregulation of alternative splicing (AS) induced by serine/arginine‐rich proteins has recently been linked to cancer metastasis. Nonetheless, as a member of the serine/arginine‐rich protein family, the involvement of SRSF11 in colorectal cancer (CRC) is unknown.The TCGA dataset and clinical samples were used to assess SRSF11 expression levels in CRC. For SRSF11, functional experiments were conducted both in vitro and in vivo. RNA‐seq technology was used to analyze and screen SRSF11‐triggered AS events, which were then confirmed by in vivo UV crosslinking and immunoprecipitation (CLIP) and mini‐gene reporter assays. Jalview software was used to determine the preferential binding motif with relation to exon skipping (ES) events. Furthermore, coimmunoprecipitation (Co‐IP) and Phospho‐tag SDS‐PAGE experiments were used to investigate PAK5‐mediated phosphorylation regulation on SRSF11, and in vitro kinase experiments validated the interaction.In CRC, SRSF11 was discovered to be overexpressed and associated with a poor prognosis. And SRSF11 played a pro‐metastatic role in vitro and in vivo. By screening SRSF11‐regulated AS events, we identified the binding motif of SRSF11‐triggered splicing‐switching of HSPA12A AS, which specifically regulated HSPA12A AS by directly binding to a motif in exon 2. Mechanistically, the HSPA12A transcript with exon 2 retention increased N‐cadherin expression by promoting RNA stability. Furthermore, the oncogenic kinase PAK5 phosphorylated SRSF11 at serine 287, protecting it from ubiquitination degradation.SRSF11 exerts pro‐metastatic effects in CRC by inhibiting the AS of HSPA12A pre‐RNA. Our findings point to SRSF11‐regulated HSPA12A splicing as a novel relationship between SRSF11‐regulated splicing and CRC metastasis and suggest a PAK5/SRSF11/HSPA12A axis as a potential therapeutic target and prognostic biomarker in CRC. PAK5 facilitates the nuclear translocation and alternative splicing function of SRSF11 on HSPA12A through its phosphorylation regulation.SRSF11 inhibits HSPA12A exon 2 skipping, which benefits CRC metastasis.Exon 2‐inclusive HSPA12A transcripts increase the stability of N‐cadherin mRNA. It is made up of five small nuclear ribonucleic acids (snRNAs) and several hundred protein components. Because of the interaction between SFs and the spliceosome, the AS processes completed the steps of recognition, assembly, catalysis and dissociation.Notably, due to the abundance of arginine and serine sequences in the RS domain, posttranslational modifications such as phosphorylation prefer it.Changes in the expression levels of general or specific SFs are one of the primary causes of abnormal AS processes in cancer, which leads to the synthesis of previously unknown RNAs or changes in the ratios between existing isoforms, with consequent implications on oncogenesis and tumour growth.We sought to explore the expression levels of SRSF11, as well as its clinical correlation, biological function, and underlying AS‐associated function of CRC. SRSF11 was discovered as an elevated gene and an indicator of poor prognosis in analyses of the TCGA database and clinical samples. RNA‐seq technology was used to explore SRSF11‐triggered AS events, which were then validated by in vivo CLIP and mini‐gene reporter assays, revealing a preferential binding motif for ES events. Then, we proved that SRSF11 played a pro‐metastatic role in CRC cells by modulating HSPA12A splicing, which led to N‐cadherin mRNA stability protection. Furthermore, we uncovered PAK5‐regulated phosphorylation, which resulted in the protein stability of oncogenic SRSF11. Our findings shed light on the role of SRSF11 in the progression of CRC metastasis and elucidate the underlying mechanisms in detail.22.12, at 37°C. Actinomycin D (ActD) , MG132  and cycloheximide (CHX)  were used to inhibit transcription, proteasome and protein synthesis, respectively. Alkaline phosphatase (AP)  was used as a mediator reversing phosphorylated protein into unphosphorylated status.LoVo, HCT116, SW480, SW620, DLD‐1, HT‐29 and FHC cell lines were obtained from the General Surgery Department of Xuzhou Medical University. Cells were cultured in DMEM  or RPMI 1640  containing 10% FBS . The incubation conditions of cells are 5% CO#1 Sense: GGAUACCUCUAGUAAAGAAAU, Anti‐Sense: UUCUUUACUAGAGGUAUCCGA; SRSF11#2 Sense: AGAUCAAGAUCACGUUCUAGG, Anti‐Sense: UAGAACGUGAUCUUGAUCUUG; SRSF11#3 Sense: GCAAGAAGAAGAAGAGUAAAG, Anti‐Sense: UUACUCUUCUUCUUCUUGCUU. The sequence of HSPA12A‐exon 2‐specific siRNAs are as follows: Sense: UCAUCAUGGAGGAAGAAGGCG, Anti‐Sense: CCUUCUUCCUCCAUGAUGAAA. The Flag‐mCherry‐SRSF11 and MYC‐PAK5 were constructed with the pcDNA3.1(+) vector from Shanghai Hewu Biotechnology Co. Ltd. The lentiviruses packaged with SRSF11 (NM_004768.5) plasmids and empty vectors were produced by GenePharma. To generate stable strains, cells that had been transfected with lentiviruses for 72 h with the help of polybrene (10 µg/ml) were administrated with puromycin (5 µg/ml).A suite of SRSF11 siRNAs was synthesized by GenePharma, and si‐HSPA12A was synthesized by IBSBIO followed by transfection using jetPRIME  based on the manufacturer's instructions. The sequence of SRSF11 siRNAs are as follows: SRSF112.2Forty‐six pairs of CRC and normal adjacent tissues (Cohort 1) were acquired from CRC patients experiencing surgery at the Affiliated Hospital of Xuzhou Medical University with written informed consent. The resected tissues were rapidly frozen at −80°C for subsequent experiments. Seventy‐seven pairs of CRC tissue microarray chips were customized from TuFeiBio Co. Ltd.2.3After deparaffinization, rehydration and antigenic retrieval, sections were sent to implement antigen repair via the thermal repair method. Then the sections were sent for further H&E staining or antibody incubation according to standard procedure. Halo Version 3.0.311.314 software was applied for analysis following scanning with a PANNORAMIC panoramic slice scanner. The following formula was used to determine the score: Σ (positive rate × intensity).2.4The plasmids were constructed from IBSBIO. The wild‐type or mutant Flag‐SRSF11 plasmids were recombinant plasmids constructed by inserting the human SRSF11 gene (NM_004768.5) or the respective mutants into the pcDNA3.1(+) empty vector plasmid. Therein, S249/277/287/295/348A means the serine (S) of 249/277/287/295/348 site was mutated into alanine (A) by site‐directed mutagenesis.2.5Total RNA was isolated by Trizol Reagent , reverse transcribed into cDNA by First Strand cDNA Synthesis Kit  and utilized as the template for amplification with SYBR Green qPCR Master Mix . The primers of qRT‐PCR were as follows: HSPA12A‐Intron 1, Forward: ATGTGCCTTGGGGAACCAG, Reverse: CAGGAAGCGTTGTGTGCAC; HSPA12A‐Intron 1‐Exon 2, Forward: GTGCACACAACGCTTCCTG, Reverse: CCTTGGGCAAGGTGCTTAG; HSPA12A‐Exon 2‐Intron 2, Forward: ATGAAAACATCTGAGACTCAGGG, Reverse: CGTACTCGTGCTGGTTGAC; SRSF11, Forward: GACTAATGTCTCCCCGAGCG, Reverse: AGACTGGCAAAGGCGAATCA; N‐cadherin, Forward: GTGCATGAAGGACAGCCTCT, Reverse: CCGTGGCTGTGTTTGAAAGG. Amplification of DNA was performed by Fast sTaq PCR Master Mix  following the guidelines provided by the manufacturers. The primers of RT‐PCR were as follows: HSPA12A, Forward: TAGAAGGTGGAGTTGAGCAG, Reverse: TGAAGCGGCTATTTCCCAC. Relative quantitation of RNA was normalized to GAPDH levels; GAPDH, Forward: GTCAGTGGTGGACCTGACCT, Reverse: TGCTGTAGCCAAATTCGTTG.RIPA lysis buffer  was used to extract all the protein, and Enhanced BCA Protein Assay Kit  was used for quantification. Nuclear extraction kit  was applied for the isolation and extraction of cytoplasmic and nuclear proteins. Equivalent protein was separated by 10% SDS‐PAGE electrophoresis, followed by transmembrane using the nitrocellulose filter membranes . Then the membranes were blocked by 5% non‐fat dry milk or BSA for 2 h at room temperature and followed by the corresponding antibodies incubation. GAPDH, β‐actin or PCNA were used as internal controls. TanonTM High‐sig ECL Western Blot Substrate (Tanon) was used to reveal protein bands, followed by analytical processing of Tanon's Image Analysis Software. Then, the gray value of all bands was calculated by the software ImageJ.2.62, 30 µg of protein and 3.3 units of AP, followed by incubation at 37°C for 12 h and then mixed with 100 µl of 3× SDS‐PAGE loading buffer. For Mn2+‐Phos‐tag SDS‐PAGE, acrylamide‐pendant Phos‐tag ligand (50 µM) and two equivalents of MnCl2 were added to the separating gel before polymerization. An acrylamide stock solution was prepared as a mixture of a 29:1 ratio of acrylamide to N,N′‐methylenebisacrylamide. The electrophoresis running buffer (pH 8.4) was 25 mM Tris and 192 mM glycine containing 0.1% (w/v) SDS as in normal SDS‐PAGE. Before a typical SDS‐PAGE transmembrane, the gel undergoing electrophoresis was washed modestly in 1 mmol/L EDTA for 10 min, followed by gentle washing in a general transfer buffer without EDTA for 10 min. Then, the gel was transmembrane using the PVDF membranes followed by a block for 2 h at room temperature and incubated with specific primary antibodies and the following secondary antibodies as in normal Western blotting (WB) analysis.Dephosphorylation of phosphorylated proteins was acquired from a 50 mM Tris‐HCl buffer (pH 9.0) containing 1.0 mM MgCl2.72 . The STK substrate 2‐biotin and the synthetic peptides were treated in a 50 µM solution with distilled water. Streptavidin‐XL665 was diluted to 500 nM. Recombinant human PAK5 protein  was stored at −80°C. The detection buffer was resuspended in distilled water and employed to formulate the working solution of Streptavidin‐XL665 and STK‐antibody‐cryptate. Adenosine triphosphate (Sigma) was diluted to 5 mM with 1× kinase buffer.Phosphorylation assay was performed with a Homogeneous Time‐Resolved Fluorescence (HTRF) KinEASE‐STK (serine/threonine kinase) S2 kit  as per the guidelines provided by the manufacturer. The KinEASE‐STK S2 kit is suitable for measuring the activities of serine/threonine kinases, including PAK5. Briefly, the reagents needed for the assay were prepared following the instructions. The 1× kinase buffer was prepared by distilled water dilution of 5× buffer supplemented with DTT  and MgCl4.The assay was performed with an HTRF 96‐well low‐volume plate . Next, 4 µl of 1× kinase buffer, 2 µl of STK substrate 2‐biotin or synthetic peptide solution, 2 µl of recombinant PAK5 protein (0.1 mg/ml) and 2 µl of ATP were added in turn to 96‐well plates. After sealing the plate and incubating for 30 min at 37°C, 5 µl of STK antibody‐cryptate and Streptavidin‐XL665 were added, respectively. After 1 h of incubation, the acceptor and donor emission signals of each individual well were detected by a VICTOR Nivo multimode microplate reader . The relative phosphorylation level was converted according to the ratio of  ×102.84 cell suspension. Paraformaldehyde  was applied to fix cells for 20 min, followed by a 24‐h culturation, and then the cells were stained using 0.1% crystal violet  for 15 min. Cells that did not reach the chamber's upper layer were wiped off after being thoroughly cleaned with cotton swabs and washed with PBS. Then, we used a Nikon digital camera to take pictures with a magnification of ×200 followed by the software, ImageJ  to compute the number of cells penetrating through the pores.For the migration and invasion assays, Transwell inserts (Corning Incorporated) without or with Matrigel (BD Biosciences) coating were used. A 24‐well plate containing inserts of 8 µm size was put in place, and each well was prefilled with 480 µl of culture medium together with 120 µl FBS. Then, each insert was filled uniformly with 100 µl of 5 × 10OE) (5 × 106 cells suspended in 100 µl PBS). In accordance with the Institutional Animal Care and Use Committee's rules, all animals were raised in conditions free of pathogens. Six weeks later, mice were euthanased, and the isolated lung tissues were resected and used for subsequent experiments.Five‐week‐old, male nude mice (BALB/c) that were acquired from Nanjing GemPharmatech Co., Ltd were used to construct the xenograft tumour formation assay. The mice were intravenously inoculated via the tail veins with the stable strains . Goat serum (ZSGB‐BIO) was used for blocking for 30 min. The cells were incubated with the anti‐Flag primary antibody  or anti‐N‐cadherin primary antibody  overnight at 4°C, and subsequently with the respective secondary antibody conjugated with CoraLite488  after being washed thrice in PBST (PBS with 1‰ Triton X‐100). Phalloidin  staining was used to observe the actin cytoskeleton. The DNA dye DAPI  was applied for nucleus staining. Fluorescent scanning analysis was employed through the STELLARIS 5 confocal fluorescence microscope  under established methods.2.10g and 4°C. The supernatants were incubated with the indicated antibody  overnight at 4°C, followed by 2 h of incubation with 50 µl Protein A+G Agarose beads at 4°C. The suspension was collected by centrifugation followed by extensive washing with IP lysis buffer. After then, the sediment was dissolved in 30 µl of 1× SDS loading buffer . The antibodies used are listed as follows: SRSF11 , PAK5 , MYC  and Flag .The coimmunoprecipitation (Co‐IP) method was carried out via the Protein A+G Agarose . Collected cells were resuspended in IP lysis buffer  for a 30‐min lysis, followed by a 15‐min centrifugation at 12 000 × 2.11The in vivo crosslinking and immunoprecipitation (CLIP) assay was performed according to the previous description with fine‐tuned adjustments.2.12Following the manufacturer's guidelines, 150 bp paired‐end RNA‐seq using an Illumina NovaSeq platform was performed on total RNA isolated from LoVo cells transfected with negative control (si‐NC) and si‐SRSF11, and rMATS (3.2.5) software was used to analyze the differential AS events. The rMATS software can classify the AS events into five categories , and can conduct differential AS analysis on samples with biological duplication. Each AS event corresponds to two isoforms, which are exon inclusion (EI) isoform and ES isoform. The expression level of the two isoforms is counted and divided by their effective length to receive the accommodated expression level. The ratio of the total expression amount of EI isoform in the two isoforms is calculated, namely IncLevel. Finally, the significance of the differences was analyzed. Under the accession number of GSE199094, the raw sequence data have been uploaded to the Gene Expression Omnibus.2.13All sequences of skipping exon events regulated by SRSF11 according to the RNA‐seq data described above were sorted out by FASTA format. The sequence alignment was then performed using Jalview Version 2.11.2.0 software. The aligned base was visualized according to the sequence consensus, in which the topmost represents a larger proportion and the bottommost represents a smaller proportion.2.14RNA‐seq data (RPKM) from 434 COAD and READ and normal samples were obtained from the TCGA database. The correlation scatter plot of SRSF11 expression levels between tumour and normal tissues was generated by GraphPad Prism 9. The correlation scatter plot between SRSF11 and N‐cadherin was downloaded from the TCGA database.2.15Mini‐genes were established according to the previous description.2.16t test, Student t′ test, or Student–Neuman–Keuls (SNK) tests. Non‐parametric Mann–Whitney U tests or Kruskal–Wallis tests were followed with non‐parametric Wilcoxon two‐group tests. The associations between SRSF11 and CRC patients’ clinical‐pathological characteristics were performed by two‐sided Fisher's exact tests. In addition, survival curves were evaluated by Kaplan–Meier methods, with comparisons operated using the log‐rank test. For correlation, Pearson correlation test was used. p < .05 was defined as statistically significant.SPSS 26.0 was applied for the statistical analysis, and GraphPad Prism 9 was utilized to visualize all results. Data were presented as mean value ± SD after three times repeating each experiment. Parametric comparisons were conducted with Student 33.1p < .0001)    and found that the gene expression of SRSF11 was significantly higher in CRC compared with normal tissues, with a statistically significant difference  Figure . Based o) Figure . Additio) Figure , and thr) Figure .p < .0001), pN status (p = .011), pM status (p < .0001) and recurrence (p = .008). Additionally, Kaplan–Meier analysis revealed that patients with higher levels of SRSF11 possessed inferior OS and progression‐free survival (PFS) than those with lower levels   Figure . Togethe3.2To fully examine the biological role of SRSF11 in CRC in vitro, we knocked down SRSF11 in the LoVo cell line, and qRT‐PCR analysis determined the knockdown efficiency Figure . Then, wOE) and the corresponding control (NC) cells through tail vein injection and assessed lung metastatic potential 6 weeks later. H&E staining revealed that the SRSF11OE group had more lung metastases than the NC group ClueGO analysis of SRSF11‐regulated genes indicated probable biological roles for SRSF11, and their molecular function was enriched for adherens junction‐associated pathways Figure . Further3.4Given that SRSF11 is implicated in CRC metastasis and promotes HSPA12A exon 2 inclusion, we hypothesized that HSPA12A exon 2‐inclusive transcript (HSPA12A‐Ex2+) could contribute to CRC metastasis. To verify this hypothesis, we designed a set of siRNAs selectively targeting the exon 2 of HSPA12A (si‐HSPA12A) and RT‐PCR validated the efficiency of interfering HSPA12A‐Ex2+ transcript in both LoVo and SW480 cell lines Figure . Then, whttp://www.cuilab.cn/lnctar) was applied to predict the possibility of HSPA12A‐Ex2+ interacting with E‐cadherin or N‐cadherin mRNA,Because of the importance of EMT progression in tumour metastasis, we aimed to assess the impact of SRSF11 on E‐ and N‐cadherin expression levels, two important epithelial and mesenchymal gene markers of EMT progression. The results from WB analysis revealed that SRSF11 overexpression considerably decreased E‐cadherin protein levels while increasing N‐cadherin, whereas SRSF11 knockdown significantly reversed these changes Figure . We alsohttp://service.tartaglialab.com/page/catrapid_omics2_group)To discuss the mechanism of HSPA12A‐exon 2 stabilizing N‐cadherin mRNA, we focused on the AGO2 protein, which catalyzes the RNA‐induced silencing complex (RISC) engine and guides gene silencing processes. RIP assays were conducted as shown in Figure 3.5p < .001)  Figure . Followi1 Figure . We corr1 Figure .333.62+‐Phos‐tag SDS‐PAGE was carried out, and the shift in blotting indicated that wild‐type PAK5 overexpression (PAK5‐WT) could raise the expression level of phosphorylated SRSF11 (p‐SRSF11); however, the inactivated mutant PAK5 plasmid (PAK5‐K478M) was unable to increase p‐SRSF11  because the RS domain is abundant in serine and arginine within serine/arginine‐rich family proteins. To clarify, we first performed Co‐IP analysis and confirmed that PAK5 interacted with SRSF11 at both endogenous Figure  and exog1 Figure . To avoi2+‐Phos‐tag SDS‐PAGE. The blotting showed a distinct drop in the Mut‐3 group in both LoVo and SW480 cells, implying that the serine 287 (S287) was the trigger for PAK5 phosphorylation  Figure , indicat3.7It has been observed that the phosphorylation status of serine/arginine‐rich proteins could affect their subcellular distribution and splicing activity. We wanted to find out how PAK5 affects SRSF11 subcellular localization based on our phosphorylation‐related interaction between PAK5 and SRSF11. As shown in Figure To determine the effect of PAK5‐regulated phosphorylation on SRSF11‐triggered AS, we co‐transfected si‐SRSF11 and PAK5 plasmids into CRC cells. As shown in Figure 4In this study, we investigated in‐depth SRSF11 and SRSF11‐triggered AS events, as well as upstream phosphorylation regulation in CRC. We discovered that SRSF11 induces critical splice‐switching of HSPA12A, which was linked to CRC metastasis in part through maintaining N‐cadherin RNA stability. SRSF11 was also demonstrated to be a substrate of PAK5 kinase and to be protected from Ub‐dependent degradation. These data suggest that SRSF11 promotes metastasis by activating tumour‐related AS events, emphasizing the importance of AS as a key regulator of metastasis.Serine/arginine‐rich proteins, as members of the SF family, play a fundamental role in controlling AS events. So far, 12 members have been identified, with the 11th member, SRSF11, being discovered to be elevated with the most significant difference in CRC compared with normal adjacent tissues by our qRT‐PCR experiments (data not shown). SRSF11 has been reported to be implicated in the regulation of many AS events, but no comprehensive studies of its regulation in malignancies have been conducted.Furthermore, we illustrated the SRSF11‐regulated AS landscape in CRC cell lines using RNA‐seq. SRSF11 expression affected 70 439 AS events, with ES being the most common splicing type (76%). One SF has been shown to perform antagonistic roles in activating or inhibiting AS processes in cancer cell lines.Canonical AS‐associated studies focused on the SF‐protein isoform axis. SETMAR pre‐mRNA, for example, was reported to be alternatively spliced to two protein isoforms in bladder cancer by NONO, leading to lymphatic metastasis.In addition, we applied a consensus analysis of the ES events regulated by SRSF11 using RNA‐seq data and found two peaks with overlapping sequences that matched a 6‐mer motif (GACC/TCA) in exon 2 of HSPA12A. Further mini‐gene reporter experiments with mutant plasmids confirmed the necessity of the 6‐mer motif for SRSF11 AS on HSPA12A. Certain serine/arginine‐rich proteins, including SRSF1, SRSF2, SRSF5 and SRSF6, have a preferred motif via which they exert the splicing processes.https://www.alphafold.ebi.ac.uk/entry/Q05519),The serine‐rich proteins piqued our interest in examining the phosphorylation regulation between SRSF11 and PAK5 kinase, the latter of which is a Ser/Thr protein kinase that has oncogenic functions in diverse types of cancer.In conclusion, we discovered that upregulated SRSF11 and elevated exon 2‐inclusive HSPA12A were both related to enhanced metastatic ability and worse prognosis in CRC patients. Our findings demonstrate that SRSF11 can be phosphorylated by PAK5 and hence retains protein stability. Advanced research in different cancer types was required to determine whether modulating AS by targeting serine/arginine‐rich family members be widely employed in pan‐cancer. Overall, our findings implicate SRSF11 in mediating CRC metastasis by suppressing the ES of HSPA12A pre‐RNA , suggesting that it might serve as both a potential therapeutic target and a prognostic indicator in CRC patients.National Natural Science Foundation of China, Grant/Award Number: 81872080The authors declare no potential conflicts of interest.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes. Here the authors build different regulatory landscapes in an inactive chromatin environment and observe that enhancers can both carry out their classic role of stimulating transcription initiation and elongation at target gene promoters and recruit cohesin to create contact domains, engage CTCF sites and activate distant genes. Genome-wide, a general correlation between enhancer-promoter (E-P) contact frequencies and transcriptional activity has been uncovered by these methods7. Also, non-coding disease-associated genetic variants, identified by genome-wide association studies (GWASs), could be successfully linked to target genes when considering their 3C-based chromatin contacts8. As an enhancer has been found to activate expression when it was forced to loop to a distant gene, E-P looping seems a requirement for, more than a consequence of, long-range gene activation9.Tissue-specific and developmentally restricted expression of genes is often controlled by enhancers, regulatory DNA elements that can act over distance to activate gene expression. In the mammalian genome, enhancers may be located near their target gene, but they can be separated over hundreds of kilobases. Chromatin looping is thought to enable distal enhancers to approach and activate target genes. Indeed, individually studied developmental genes have been found to form preferred contacts with distal enhancers for their activation11, exactly as it has been predicted to create chromatin loops in vivo13. DNA-bound CTCF is the most dominant factor that binds and halts the DNA-extruding cohesin complex on chromatin and to anchor DNA loops in mammalian cells18. CTCF-anchored loops are the most readily detectable loops in genome-wide Hi-C contact maps16, highlighting their frequent recurrence across cells. They span sub-megabase domains, also known as topologically associating domains (TADs), containing sequences that preferentially self-interact, and they form boundaries that hamper contacts between neighboring domains.Cohesin is responsible for chromatin loop formation in interphase chromosomes. In vitro, the ring-shaped cohesin complex can extrude DNA to form loops20. This raised doubts about whether chromatin looping was really necessary for enhancers to control gene expression over distance, doubts that were further fueled by live-cell microscopy studies that have sometimes21, but not always23, found enhancers in closer proximity to the genes that they activated. Yet other studies did observe that cohesin depletion caused loss of E-P contacts25 and reduced enhancer-dependent expression27 of many, but not all, genes, suggesting that both cohesin-dependent and cohesin-independent mechanisms exist for long-range gene regulation28. Acute depletion of WAPL, the factor that releases cohesin from chromatin, caused repositioning of cohesin from tissue-specific enhancers to CTCF boundaries, disrupted E-P looping and downregulated expression of tissue-specific genes controlled by such enhancers24. This suggests that these enhancers can serve as cohesin entry sites and that active cohesin loading is required for their productive interaction with target genes24. Support for this model came from findings that NIPBL, the cohesin loading factor, is preferentially associated with enhancers30 and appears to be required for long-range gene regulation20. Looping between CTCF sites, which depends on cohesin, has been found to correlate with the presence of active promoters and enhancers in the intervening chromatin32, further suggesting that active regulatory DNA elements can act as entry sites for the loop-extruding cohesin machinery.Without cohesin, both CTCF loops and contact domains dissolve in Hi-C chromatin contact maps, but overall steady-state gene expression levels seem remarkably stable36. Within a contact domain, a proximal gene in principle has an advantage over a distal gene for activation by a shared enhancer38. Promoter mutations that interfere with enhancer contacts can re-direct the enhancer to contact and activate more distal genes40.CTCF boundaries insulate chromatin domains not only physically but also functionally, as they can obstruct enhancers to activate genes in neighboring domains. Consequently, cognate enhancers of a given gene are typically found in the same contact domainCollectively, this suggests an intricate interplay between enhancers, CTCF sites and cohesin to form contact domains and control the expression of distant genes. To experimentally investigate this further, we took a bottom-up approach and built a large series of different regulatory landscapes in an inactive chromatin environment. In this chromatin setting, we find that a developmental enhancer can recruit the cohesin loop-extrusion machinery to promote longer-range chromatin contacts, build contact domains and enable long-range gene activation. Since the same E-P pair relies on cohesin for gene activation strongly when separated over large distances (>100 kb), mildly when separated over 47 kb and not at all in proximal (<11 kb) configurations, our data show that linear distance is important in determining whether an enhancer requires cohesin for ‘long-range’ gene regulation.41 (μLCR), a prototype of a strong tissue-specific enhancer, as a regulatory DNA sequence, and a β-globin gene (HBG1) promoter-driven green fluorescetn protein (GFP) reporter as its target gene , the enhancer conferred long-term stable expression: even after 5 weeks of culturing, nearly all cells (>95%) showed very high GFP expression that was enhancer-dependent. With the enhancer integerated at increasing distances from the promoter , however, the enhancer correspondingly lost the capacity to protect the reporter gene from silencing (~20% (E11), 30% (E47) and 50% (E100) silenced cells after 5 weeks) Fig. . At all Given the high local levels of H3K27me3 in this locus Fig. , we aske36, suggested a relationship between enhancer-mediated gene activation and E-P contact frequencies. We used 4C-seq44 to more directly analyze E-P contact frequencies. 4C-seq involves quantifying the competitive ligation events between a genomic site of interest and its spatially most proximal DNA fragments inside each cell nucleus, which then provides a semi-quantitative measure for contact frequencies in a cell population. We first selected for each E-P distance pair a clonal cell line , which we genetically confirmed and validated to display representative transcriptional activity and stability  cells. Contact profiles were almost identical Fig. , which scis, it became obvious that the distantly located enhancer stimulated the gene to engage bi-directionally in longer-range contacts. Gene contacts were stimulated not only with the enhancer itself, but also with intervening and surrounding sequences, across a defined genomic interval  selected from the human genome. We successfully integrated the μLCR together with the 3×-hCTCF cassette at 0, 11, 47 and 100 kb from the reporter gene, with the CBSs oriented convergently, facing the enhancer and  reporter gene (we failed to find integrations at 407 kb). We generated bulk GFP-positive cell populations, as described before, and found that, at all four integration sites, the presence of flanking CBSs resulted in higher levels of transgene expression, even after hemin induction Fig. . Furtherdid Fig. . The intdid Fig. . We thendid Fig. . Howeverdid Fig. . Here, tdid Fig. . Thus, n19. Tissue-specific enhancers have been proposed to recruit cohesin32. To directly test whether our enhancer recruited cohesin to induce intra-domain chromatin interactions, we used CRISPR interference (CRISPRi) to deplete the cohesin components SMC1A and RAD21 in the E100, EC100 and E407 clones. In all clones, following depletion, we observed a loss specifically of the enhancer-induced chromatin contacts , exclusive analysis of its characteristics on the targeted allele was not possible. Despite this technical limitation, 4C-seq directed to this position confirmed that this normally unengaged CTCF site now formed a novel chromatin loop with the integrated CTCF sites in both cell lines  was placed in identical orientation at position 0 (C0). One line then carried the μLCR downstream of the one CTCF cassette (EC0–C100), while the other line had the enhancer downstream of the other CTCF cassette (C0–EC100). When we applied 4C-seq to the endogenous left boundary site, we found that the E0 stimulated left boundary contacts mostly with C0, creating a small domain, whereas the E100 enabled the left boundary to select the distal C100 as its preferred contacting partner , as before, and another CTCF cassette  of mediator50, a protein complex important for enhancer function. Different from cohesin, GATA1 and mediator were required for transcriptional activation irrespective of enhancer distance to the promoter , knockdown of all three cohesin subunits, RAD21, SMC3 and SMC1A, led to strong down-regulation of reporter-gene expression Fig. . At E47,ter Fig. . Thus, t52 in which the concerted action of tissue-specific transcription factors at enhancers serves two purposes: (1) they enable recruitment of co-factors, together with which they can stimulate initiation and elongation of the transcriptional machinery at gene promoters, and (2) they enable recruitment of cohesin, which, presumably through DNA extrusion, locally stimulates looping and contacts with and between more distal sequences, to form contact domains. We here show that cohesin recruitment is necessary for enhancers to activate distant, but not proximal, genes. Since cohesin forms chromatin loops, this supports the debated idea that long-range gene activation, certainly in an inactive chromatin context, requires chromatin looping. Importantly, developmental genes often rely on distant tissue-specific enhancers located in inactive chromosomal regions. Interestingly, when separated over short linear distances  our E-P pair functioned perfectly well (or even better) under conditions of reduced cohesin. This and similar observations made in a parallel study on the Shh locus53 argue that we may need to better define the meaning of ‘long range’ when discussing mechanisms of gene activation. We postulate that the type (strength) of the enhancer and the genomic context determines whether the enhancer relies on cohesin-dependent or cohesin-independent mechanisms28 to fully activate a target gene over intermediate distances (approximately 0–100 kb). Molecular condensates formed through non-specific interactions between intrinsically disordered domains of enhancer- and promoter-associated transcription factors and mediator may enable cohesin-independent E-P communication over such distances57. Over farther distances, we expect that enhancers increasingly need cohesin for gene activation.By using a unique, bottom-up approach of building many different regulatory landscapes in an inactive chromatin environment, we provide experimental evidence for an emerging model46. Preformed contacts established prior to gene activation have been observed at, for example, the Hoxd locus, and such ‘permissive’ structures58 may be reminiscent of the topologies that we detect here. It was striking to see that gene silencing or hyper-activation (by stimulating cells with hemin) only mildly decreased or increased (respectively) E-P contact frequencies, as measured by 4C-seq. Importantly, our data consistently showed that the enhancer stimulated the distal gene not only in E-P contacts, but also in contacts with intervening and immediately surrounding sequencing. This may suggest that recruited cohesin does not stay anchored at the enhancer, but extrudes and simultaneously migrates away from its entry site. Interestingly, modeling showed that local chromatin loops can stimulate E-P communication, even if they are not directly anchored at the regulatory elements59.We further find that the genomic location of the enhancer determines which flanking pair of convergent CTCF sites is selected as boundaries of the contact domain. If boundaries immediately flank the gene or enhancer and place them in a small domain, they can support the distal enhancer in conferring strong and stable expression to the target gene. We propose this is a consequence of concentrated loop extrusion activity. We, like others, find that transcriptional activity is controlled by more than just E-P contact frequencies60.Finally, a particularly clear E-P distance effect was seen when we followed expression over time in our cell cultures: the further apart on the chromosome, the more difficult it was for the enhancer to protect the target gene from silencing. Silencing most likely was polycomb-mediated and  took place at the reporter gene promoter, not at the enhancer. We concluded the latter from cell lines with two reporter genes controlled by a shared enhancer. Almost always, it was either one  or the other reporter gene that silenced, but not both Fig. , as woulIn summary, our work demonstrates that an enhancer can recruit cohesin to stimulate the formation of a self-interacting domain, engage flanking CTCF sites in loop engagement and activate expression of distal target genes.Technical replicates were done for all experiments. All main conclusions are based on observations reproducibly made across multiple independent clones, that is clones with the enhancer, reporter gene and/or ectopic CTCF sites integrated at different locations. No statistical method was used to predetermine sample size, no data were excluded from the analyses, the experiments were not randomized and the investigators were not blinded to allocation during experiments or outcome assessment.2 in RPMI 1640 (Gibco) with 10% FBS (Sigma) and 1% penicillin–streptomycin (Gibco). Density was kept between 1 × 105 and 5 × 105 cells per ml medium. Cells were routinely tested for mycoplasma. Prior to every experiment, cells were weekly bulk-sorted for 2 consecutive weeks on Becton Dickinson SORP FACSAria FUSION Flow Cytometer. Gating was based on cells without an enhancer (no-E cells) or fluorescence-minus-one controls. Flow cytometry analysis was done 3 days after sorting on a Beckman Coulter Cytoflex S. For differentiation, culture medium was supplemented with porcine hemin  1 day after sorting. Hemin and medium were refreshed 1 day later. We prepared 4 mM hemin stock solutions according to ref. 62 and kept them at −20 °C. For long-term culturing and FI monitoring, at each indicated time point an aliquot of cells was taken and treated with hemin for 2 days, and FI was monitored. To obtain irreversibly silenced populations of cells, GFP-negative cells were weekly sorted for 6 (E100) or 9 (E407) consecutive weeks.Human erythroleukemia K562 cells were used in this study . Cells were grown at 37 °C at 5% CO41, µLCR–3×mCTCF48, µLCR–3×hCTCF (described below), dsRed–pHBG63) of interest flanked by two ~1-Kb-sized site-specific homology arms that were amplified from genomic DNA with primers as indicated in Supplementary Table Targeting constructs contained one or two regulatory elements  were selected from ref. 64. The µLCR enhancer was removed by transient transfection of a CRE-recombinase encoding plasmid. The clone that had the transgene integrated at chromosome 18 (position: 19609009) was selected for further experiments and designated ‘no E’.The founder cell line containing the d2eGFP reporter driven by human pHBG1 and µLCR enhancer was generated by Tol2 transposition. The founder µLCR is flanked by sleeping beauty (SB) terminal inverted repeats (ITRs) and LoxP sites which split a puromycin gene driven by pSV40 such that it is not functional. GFP-expressing cells upon transgene transposition were single-cell sorted, and integration sites were mapped using TLARegulatory elements were integrated in target cell lines using CRISPR–Cas9 and homology-directed repair. Cas9 plasmid (pSpCas9(BB)-2A-BFP (a modified version of PX458 (Addgene plasmid no. 48138), in which eGFP is replaced for tagBFP)) containing a position-specific single guide was co-transfected with a targeting plasmid, and GFP-expressing cells were single-cell sorted using flow cytometry, expanded, genotyped and Sanger-sequenced for correct integration of the transgene. Guide sequences specific for position of interest were cloned between BbsI sites and are listed in Supplementary Table The µLCR enhancer was targeted to no-E cell line to positions 11 kb and 47 kb upstream of the eGFP reporter to obtain the respective clonal cell lines. µLCR inserted at position 407 kb carried an additional Anch4 imaging platform (kind gift from K. Bystricky). µLCR–3×hCTCF was targeted to positions 0 Kb (pos0) and 100 Kb (pos100) to obtain EC0 and EC100, with loxP sites flanking the 3×-hCTCF cassette and FRT sites flanking the μLCR. Additionally, µLCR–3×mCTCF was targeted to no E to obtain EmC0. E0 and E100 were generated by transient transfection of a CRE-recombinase (Cre)-encoding plasmid. C0, mC0 and C100 were made by expressing the recombinase Flippase (Flp) in the respective EC-lines. The C0-EC100 clonal cell line was generated by targeting µLCR–3×hCTCF to pos100 in cell line mC0. For EC0-C100, µLCR×3×mCTCF was targeted to C100, pos0. C-0 E100 was generated by deleting the 3′ part of eGFP and subsequently repairing it with a targeting construct containing 3×hCTCF–eGFP flanked by homology arms. Homology arm primers and genotyping primers are indicated in Supplementary Table To place the E407 enhancer immediately upstream of the GFP reporter gene, the genomic region between the integrated eGFP and the integrated μLCR at position 407 kb was removed by transient transfection of two Cas9 plasmids (pSpCas9(BB)-2A-BFP), each containing a specific single guide targeting the upstream or downstream sites of this region. Guides are listed in Supplementary Table +dsRed+ double-positive cells were selected to obtain single clones. The intervening region between dsRed reporter and right boundary was removed as described above.Double reporter cell lines were generated by targeting dsRed–pHBG1 to pos100 in a separately generated E50 cell line that also had a flanking Anch4 sequence. GFPµLCR or µLCR–3×hCTCF was targeted as for the clonal cell lines on the same day to noE cell line at positions 0 Kb, 11 Kb, 47 Kb and 100 kb upstream of the eGFP reporter to obtain the respective E- and EC-lines. After 5 days, GFP-expressing cells were bulk-sorted weekly for 2 consecutive weeks and analyzed in the presence or absence of hemin 3 days after the second sort. Cells that were not treated with hemin were kept in culture and induced with hemin prior to analysis at days 10, 17, 24 and 31 after the second sort.65. dCas9–KRAB-containing target cells were first sorted for GFP for 2 consecutive weeks and transduced with sgRNA-coding virus 2 days after the second sort. Cells recovered for 1 day before puromycin selection  was started. Crosslinking for 4C and analysis by flow cytometry was performed 5 days following puromycin selection. sgRNA sequences are listed in Supplementary Table For gene knock-down experiments, dead-Cas9 (dCas9) fused to Krüppel-associated box (KRAB) was randomly integrated in the genome of the target cell line via lentiviral transduction of a modified version of pHR-SFFV-dCas9-BFP-KRAB (Addgene plasmid no. 46911) carrying a P2A blasticidin selection and UCOE element (kind gift of M. Tanenbaum). The top 50% BFP-expressing cells were bulk-sorted and kept under 0.7 μg/ml blasticidin selection (Sigma). sgRNA sequences against genes of interest were cloned into lentiviral targeting plasmid pU6-sgRNA EF1Alpha-puro-T2A-BFP (Addgene plasmid no. 60955). Optimal target sequences were selected from ref. Flow cytometry standard (FCS) files were acquired with CytExpert 2.3.0.84 and analyzed with FlowJo 10.5.2 software. Fluorescence compensation was applied on the basis of single-fluorophore samples. Cells were transferred to a 96-well plate for flow cytometry analysis directly from the culture dish. Cells were gated for live single cells on the basis of FSC-A, SSC-A and FSC-W. For every experiment, at least 10,000 cells within the live single-cell gate were recorded. For silencing experiments, cells were considered GFP-positive on the basis of gating on the no-E cell line treated with hemin. For KD experiments, cells were considered sgRNA-positive on the basis of control cells containing dCas9–KRAB but no sgRNA as indicated in Supplementary Information Figure For fluorescence-assisted cell sorting, ten million cells were collected, centrifuged and resuspended in 1 ml of culture medium. Cells were considered GFP positive on the basis of gating on the noE cell line. Gates were drawn such that more than 99.9% of noE cells fell into the GFP-negative gate. To enrich for KRAB–BFP-containing cells prior to knock-down experiments, cells were gated to be BFP-positive. From this population, the top 50% BFP-expressing cells were sorted. For analysis of knock-down experiments, cells were gated to be BFP sgRNA positive as compared with cells that contained only KRAB–BFP.44. Eight to ten million cells were cross-linked with formaldehyde. DNA was digested in situ with Csp6I (first cutter) and NlaIII (second cutter). Indexed Illumina sequencing adapters were introduced to ligation fragments of interest with a two-step PCR strategy. All viewpoint-specific primers are listed in Supplementary Table 4C-seq was performed as described44 (https://github.com/deLaatLab/pipe4C) Reads were mapped against versions of hg19 that were modified to contain the aforementioned insert sequences (modified genomes) at the experimentally validated coordinates.FastQ files were demultiplexed and processed as in ref. https://www.R-project.org/). Blind fragments were omitted for analysis. Read counts were then normalized to a million mapped intra chromosomal reads  excluding the two highest covered fragments and 21 fragment end rolling mean scores were calculated for every fragment end. For plotting of overlay profiles of distinct modified genomes, positions of fragment ends were shifted on the basis of the coordinates of the inserted sequences, such that common fragment ends were aligned.Plotting and contacts counting was done in R  as per the manufacturer’s instructions. Chromatin was sheared to 200- to 500-bp fragments on a Bioruptor Plus . Immuno-precipitation was carried out by adding 5 μg of the appropriate antiserum  to approximately 30 μg of chromatin, which was incubating on a rotator overnight at 4 °C in the presence of protease inhibitors. Following addition of protein G agarose beads (SMC1) and magnetic G beads  and washing, DNA was eluted using DNA purification elution buffer (Active motif). The eluted DNA was used in qPCR (Bio-Rad CFX Manager 2.1) using primers targeting putative CTCF/cohesin bound positions and primers targeting the GFP reporter gene for cohesin and H3K27me3, respectively. Putative CTCF/SMC1a and H3K27me3 sites were normalized over a tested bound site . All primers are listed in Supplementary Table 3O4 and protease inhibitor cocktail . Next, samples were centrifuged at full speed (12g) for 10 minutes, and supernatants were collected and processed by standard SDS–PAGE Immunoblot. The following immunoblot antibodies were used: SMC1 , γ-tubulin, goat anti-rabbit HRP  and goat anti-mouse HRP . Membranes were developed using SuperSignal West Dura (Thermo Fisher). Source Data Fig. Protein lysates were obtained after incubation for 30 minutes with RIPA buffer , 1 mM NaF and NaVFurther information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-022-00787-7.Supplementary InformationSupplementary Tables 1 and 2 and Supplementary Figure 1.Reporting Summary.Peer Review File.


Transcription factors are regulators of the cell’s genomic landscape. By switching single genes or entire molecular pathways on or off, transcription factors modulate the precise timing of their activation. The Forkhead (Fkh) transcription factors are evolutionarily conserved to regulate organismal physiology and cell division. In addition to molecular biology and biochemical efforts, genome-wide studies have been conducted to characterize the genomic landscape potentially regulated by Forkheads in eukaryotes. Here, we discuss and interpret findings reported in six genome-wide Chromatin ImmunoPrecipitation (ChIP) studies, with a particular focus on ChIP-chip and ChIP-exo. We highlight their power and challenges to address Forkhead-mediated regulation of the cellular landscape in budding yeast. Expression changes of the targets identified in the binding assays are investigated by taking expression data for Forkhead deletion and overexpression into account. Forkheads are revealed as regulators of the metabolic network through which cell cycle dynamics may be temporally coordinated further, in addition to their well-known role as regulators of the gene cluster responsible for cell division. Transcription factors are proteins that bind, relatively close to the TSS, to specific DNA sequence elements (‘motifs’) that typically span 5–12 base pairs. They then interact with histones and other transcription factors on the DNA, thus impacting the assembly of a TSS protein complex. In addition, enhancer elements may influence transcription distantly from a TSS.Saccharomyces species Mapping the binding sites of transcription factors across the genome in living cells has been made possible through Chromatin Immunoprecipitation (ChIP), the first genome-wide methodology with microarray detection of bound DNA (ChIP-chip) ChIP data provide a list of targets (i.e. genomic locations where binding occurs) of transcription factors that may be subsequently tested, thus ‘predicting’ their possible novel cellular functions. In the analyses of ChIP data, false-positives binding events have to be identified, in order to retrieve a comprehensive, but reliable picture of a transcription factor’s functions. Transcription factors may bridge multiple spatial, temporal and functional scales across cellular layers of regulation, such as cell cycle, metabolism, signalling, etc. Thus, they may be hubs, i.e. nodes in a network characterized by a high connectivity, at the interface between cellular layers in multi-scale models that aim to understand how biological functions emerge from networks of interactions Forkhead (Fkh) transcription factors are highly conserved across eukaryotes. In humans, they play a role in a number of cellular pathways that, when dysregulated, may lead to development of pathologies such as cancer Here, we focus on the complexity of the ChIP-based interaction landscape that has recently emerged for the yeast Forkheads. Specifically, we point out both the validity and uncertainty of target genes retrieved for the transcription factors Fkh1 and Fkh2.2Microarray-based RNA profiling maxPeak – which is not sensitive to a relatively low number of strong peaks obtained by the ChIP-exo as compared to other existing “peak detection methods” such as GEM maxPeak, GEM, and MACE were selected. Our analyses provided the high-confidence genes whose expression may be modulated by Fkh1 and Fkh2 Recently, we have shown that the yeast Fkhs targets promoters of novel target genes, among which cell cycle genes as well as genes involved in metabolism and signal transduction CLB2 gene whose transcription levels peak in the early mitotic phase (G2/M transition) of the cell cycle to control cell division through the activity of Clb2/Cdk1 kinase complexes SWI5 gene CLB1 that promotes cell division CLB2; the cyclin gene CLN1 that promotes budding events CLN2; and the transcription factor ACE2 that promotes SIC1 transcription SWI5.Well-known Fkh targets involved in cell cycle control were retrieved: (i) the Intriguingly, transcription factors and metabolic enzymes that play a role in central carbon metabolism and are crucial for cell growth and division were also identified as Fkh targets in vivoAfter our study, two new binding studies have been recently published that report on the spectrum of targets of transcription factors in budding yeast among which Fkh1 and Fkh2 https://www.yeastgenome.org/) was queried – following engagement with SGD curators who have conducted a dedicated update of the YeastMine database To further explore the potential relevance of Forkheads in the regulation of yeast cellular networks, among which cell cycle and metabolic processes, we first thoroughly analysed the six binding studies, i.e. three ChIP-chip using DNA microarrays As we highlighted in our previous study a priori clear that all functional targets should respond to both deletion and overexpression of each transcription factor. In fact, alternative transcriptional regulators may exist that can be involved in the regulation of a specific gene  was upregulated. Similarly, 47 out of 53 fully validated Fkh1 targets were downregulated at the end of the overexpression time course, four of which were upregulated. Conversely, and in agreement with the overexpression experiments, the deletion experiments revealed that most of these target genes were upregulated (40 out of 53 for Fkh1 and 20 out of 24 for Fkh2). An exception is CLB2, the major Fkh target gene. CLB2 is – as expected – downregulated upon Fkh2 deletion; however, notably, it first rises before dropping below wild type levels in the overexpression time course. This result suggests that the genes transcriptionally regulated after CLB2 activation  validated 4x+ target genes, 104 out of 188 Fkh1 targets and 51 out of 63 Fkh2 targets are CCR genes AMN1, ASE1, ATG42, BUD4, CAR2, CIN8, CLB2, CTS1, CWP2, DIF1, ENV9, FRK1, GIC1, GLN1, HOS3, HSL7, IDI1, KIP2, RNR1, RPL37B, SED1, SGO1, SIM1, SLM4, SNA2, SRL1, TEM1, VAC17, WSC2, WSC4, WTM1, YBR138C, YCG1, YHP1) – more than half of the Fkh2 targets – that are mostly confined in the replicative phases of the cell cycle, specifically in S, G2, G2/M and M, with the majority in S and M phases  validated 4x+ target genes in common ; RPE1 (pentose phosphate pathway); and CIT1, CIT2 (citrate/TCA cycle). Of note, these targets were all retrieved in the analyses we carried out earlier In the fully validated 4x+ target genes , the majATG42, HOS3 and SIM1 are fully validated CCR targets for both Fkh1 and Fkh2. Strikingly, ATG42 and HOS3 are found as targets in all the six binding studies and are fully validated with respect to the deletion and overexpression experiments.Among the targets, the metabolic enzymes CLB5 and CLB6 and the G2/M cyclins CLB1–CLB4) and their M/G1 stoichiometric inhibitor SIC1  CLB3 being upregulated upon Fkh2 deletion CLB3 promoter is only shown by one of the ChIP-chip studies CLB3 does not score above the stringent threshold imposed in any of the three peak detection methods  employed to analyze ChIP-exo data, thus resulting in its exclusion from being retrieved as Fkh2 target. This case indicates that genes exhibiting a low DNA binding signal in ChIP studies should not be disregarded as potential targets but require further investigation through independent experimental testing.Conversely, ChIP studies highlight some incongruences with respect to the molecular mechanisms that have been validated through independent molecular biology and biochemical investigations. Though a Systems Biology, integrative strategy combining predictive mathematical modeling and dedicated experimental testing, we have recently discovered a role of Fkh2 as temporal coordinator of mitotic Clb waves, identifying Fkh2 as a controller molecule of the sequential activation of SIC1 for Fkh1 CLB5 for Fkh2 CLB5 expression levels CLB4 promoter in a genome-wide location analysis CLB4 promoter, Fkh1 deletion resulted in an increased CLB4 transcription and in a change of RNA pol II occupancy CLB4 promoter region and CLB4 ORF CLB4 promoter, leads to suspect a lower affinity that this multiple, overlapping binding sites at CLB4 promoter may have for Fkh1.On the one hand, the high stringency that we have used to analyze ChIP data calls for an experimental validation of the high-scoring target genes. On the other hand, it does not confirm previously identified Fkh targets through ChIP-chip, such as This observation finds a parallel with a ChIP-based genome-wide study of human Fox transcription factors, which have revealed that an extensive overlap in chromatin binding can dictate their recruitment to chromatin 4Altogether, the evidence presented here for the Fkhs in budding yeast suggests that complementary studies, from genome-wide, ChIP-based studies to independent molecular biology- and biochemistry-based experiments are needed to exclude false positives and point to the effective targets of transcription factors. Specifically, to interpret ChIP data, identification of stable method(s) for data analysis accuracy in the identification of functional targets shall be taken into account.In humans, the FoxM1 and FoxP transcription factors are homologs of the yeast Fkh1 and Fkh2 ChIP-based methodologies have the power to generate hypotheses, which predictions may be directly tested experimentally through molecular biology and biochemical investigations. Moreover, this data can be integrated into computational approaches 10.13039/501100003513University of Surrey to M.B.This work was supported by the Systems Biology Grant of the Matteo Barberis: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Thierry D.G.A. Mondeel: Methodology, Formal analysis, Resources, Data curation, Writing – review & editing, Visualization.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.