Of these 45 CGs, 47% were hypomethylated and 53% were hypermethylated. Interestingly, 5 out of the 7 genes had correlated gene upregulation with CG hypermethylation at CTCF and gene downregulation with CG hypomethylation at CTCF. We also investigated active H3K27ac surrounding the same CTCF binding sites within these seven genes. We observed a significant increase in H3K27ac in four genes (FDR < 0.05). Three genes  showed increased CTCF binding in response to rotenone. Our data indicate that rotenone alters regulatory regions of PD-associated genes through changes in epigenetic patterns, and these changes impact high-order chromatin organization to increase the influence of non-coding variants on genome integrity and cellular survival.CCCTC-binding factor (CTCF) is a regulatory protein that binds DNA to control spatial organization and transcription. The sequence-specific binding of CTCF is variable and is impacted by nearby epigenetic patterns. It has been demonstrated that non-coding genetic variants cluster with CTCF sites in topological associating domains and thus can affect CTCF activity on gene expression. Therefore, environmental factors that alter epigenetic patterns at CTCF binding sites may dictate the interaction of non-coding genetic variants with regulatory proteins. To test this mechanism, we treated human cell line HEK293 with rotenone for 24 h and characterized its effect on global epigenetic patterns specifically at regulatory regions of Parkinson’s disease (PD) risk loci. We used RNA sequencing to examine changes in global transcription and identified over 2000 differentially expressed genes . Among these DEGs, 13 were identified as PD-associated genes according to Genome-wide association studies meta-data. We focused on eight genes that have non-coding risk variants and a prominent CTCF binding site. We analyzed methylation of a total of 165 CGs surrounding CTCF binding sites and detected differential methylation (|>1%|,  Parkinson’s disease (PD) is the second most common neurodegenerative disorder in the United States . More thCCCTC-binding factor can play a long-range cis-regulatory role that insulates genes from their surrounding signaling environment by directing chromatin looping . FunctioApproximately 85% of PD cases cannot be explained by genetic predisposition alone . TherefoIn our pesticide-induced cellular model, we used rotenone, a naturally occurring insecticide and potent inhibitor of complex I in the mitochondrial electron transport chain. The primary use of rotenone today is as a piscicide to terminate invasive or noxious species of fish. Permissible application concentrations up to 250 ppb can be applied to public and recreational waters . RotenonDNA methylation and histone acetylation are epigenetic modifications implicated in rotenone-induced neurotoxicity . DNA hypIn this study, we examined rotenone-induced changes in DNA methylation and histone acetylation patterns at CTCF binding sites adjacent to PD-associated genes. Eight selected genes had identified disease-risk SNPs in a non-coding region and were indexed by a meta-data analysis of over seven million human polymorphisms . We hypoAll media reagents and chemicals in cell culture were purchased from Sigma . Human cell line HEK293 was grown in Dulbecco’s Modified Eagle Medium with high glucose, L-glutamine, and sodium pyruvate. Media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin. HEK293 cells were confirmed by ATCC. Cells were treated at approximately 70% confluency with 200 nM rotenone or DMSO vehicle control (<0.001%) for 24 h. Cell viability was measured with trypan blue (0.4%) staining, and cells were counted manually with a hemocytometer. Cells used for experiments were at least 85% viable relative to the vehicle control.Genomic DNA was extracted from two replicates of DMSO or rotenone-treated HEK293 using 1:1:1 phenol: chloroform: isoamyl alcohol . Global DNA methylation was first measured by dot blot analysis. Bisulfite-treated DNA (30–60 ng/μL) was denatured at 95°C for 5 min and then cooled at 4°C for 5 min in a conventional thermocycler . DNA was spotted onto 0.45-micron nitrocellulose paper as 1- or 2-μL drops and dried for 30 min at room temperature. The membrane was UV cross-linked at 3000 Hz and incubated in anti-5methylcytosine (5mC) primary antibody overnight at 4°C . The membrane was washed with TBST and incubated with a secondary antibody conjugated to HRP for 1 h at room temperature . The membrane was washed again with TBST after secondary incubation and visualized with chemiluminescence . We used a serial dilution of 100% 5mC standard  to create a standard ladder .t-test (p < 0.05).HEK293 cells were collected after a 24-h treatment, and histones were extracted using the Abcam Histone Extraction kit according to kit instructions . Histone protein concentration was measured by Qubit Protein Assay from Thermo Fisher Scientific . Protein (5 μg) was loaded onto 4–15% Bio-Rad Page Gels and transferred to 0.45 μm nitrocellulose . The blots were incubated with H3K27ac primary antibody  overnight at 4°C and anti-rabbit IgG conjugated secondary antibody  for 1 h at room temperature. The histone protein was normalized to total histone 3  and quantified with ImageJ software. We tested significance by comparing the ratio of H3K27ac/H3 with a two-tailed Student’s paired Total RNA was extracted from two replicates of DMSO or rotenone-treated HEK293 using the TRIzol method . A total of 2 μg per sample was used for library construction using the TruSeq Sample Preparation kit from Illumina . Poly-A-containing mRNA molecules were isolated from total RNA using oligo-dT attached magnetic beads. Isolated mRNA was then fragmented and synthesized into double-stranded cDNA according to kit instructions. Ligation of unique Illumina adapter indices was completed for each sample before bead purification. Libraries were loaded onto a 2% agarose gel, and library products between 200–800 bp were purified using the mini-Elute gel extraction kit from Qiagen . Approximately 150 ng was sent for sequencing on a HiSeq 2000 platform with 100-bp paired-end reads.Adapter sequences were removed from the raw sequencing data, and individual libraries were converted to the fastq format. Sequencing reads were aligned to the human genome (hg19) with TopHat2 (v2.0.9) . For mRN–ΔΔCT).Total RNA was extracted from an additional replicate of HEK293 treated with DMSO or rotenone using the same procedure as stated above. A total of 500 ng RNA was converted to cDNA with the PrimeScript RT reagent kit with gDNA eraser from Takara . We selected 10 genes for quantitative PCR (qPCR) analysis using primers listed in UBOX5, was among the most significant polymorphisms identified by the meta-analysis . A total of 200 ng DNA was bisulfite-converted using the Sigma DNA Imprint Modification kit two-step protocol. Bisulfite-converted DNA (BS-DNA) was amplified with primers for selected regions designed with MethPrimer  Supplem. Amplifi1. Duplicates and singletons identified in alignment were ignored from the methylation call. Minimum and maximum per-base depths were 1000× and 100,000×, respectively. The output was selected for methylKit format. Coverage statistics and differentially methylated regions were calculated for CG sites with methylKit installed in R (v3.5)  . Differeon model . The cha6 cells/mL in a conical tube. Cells were fixed with 1% formaldehyde for 10 min at room temperature. Reaction was stopped with 0.2 M glycine and incubation at room temperature for 5 min. Fixed cells were centrifuged for 5 min at 300 × g and 4°C and washed with 1 mL cold PBS. Fixed cell pellet was stored at -80°C until chromatin immunoprecipitation (ChIP).All chemicals were purchased from Sigma unless otherwise noted . HEK293 cells were harvested after a 24-h treatment and resuspended in fresh media at 10 × 106 cells/0.1 mL with PBS + 0.5% Triton-X + 1% protease inhibitor cocktail and incubated on ice for 10 min prior to centrifugation for 5 min at 400 × g 4°C. The pellet was resuspended in TE buffer pH 8.0 with protease inhibitor and PMSF. Cells were sonicated at high intensity for 30 s on/60 s off until DNA fragments were within 200–800 bp as checked by 2% agarose gel. After sonication, samples were centrifuged for 15 min at 14,000 × g 4°C to pellet insoluble material. Sheared chromatin was transferred to RIPA buffer, and 10% of total chromatin was saved for input DNA extraction.Cell pellets were resuspended at approximately 1 × 10Chromatin immunoprecipitation was done with Dynabeads Protein A  and 4 μg of primary ChIP-grade antibody . Beads were washed with lithium chloride (LiCl 0.25 M) buffer, and immunoprecipitated DNA was extracted from beads using the phenol: chloroform method. DNA was quantified using Qubit dsDNA high-sensitivity assay .We selected eight genes for quantitative real-time PCR (qPCR) analysis using primers listed in p-value threshold of p < 10–3. Among enriched biological processes, we observed a significant induction of the oxidative stress response, transcription factor activity, and chromatin organization (<10–3) (–7) and DNA binding (<10–5) with the nuclear cell component being most represented (<10–5)  . We obse (<10–5) , 6. We a (<10–5) . Three oR2 = 0.96)  and three additional genes from the PD gene meta-analysis  for further examination of epigenetic patterns  . We selepatterns .2) were also analyzed with Regulomedb, a database that annotates SNPs with known or predicted interactions with regulatory elements in intergenic regions   .FER cg143 at chr5:102025097 and CHCHD2 cg217 at chr7:56174103, were significantly hypomethylated (FER cg143Δ = −4.4) and hypermethylated (CHCHD2 cg217Δ = 1.7) at the predicted CTCF binding sequence  and hypermethylated (UBOX5Δ = 0.33) across the entire CTCF binding region with p < 0.05 but did not remain significant after multiple hypothesis testing (FDR > 0.05)  . CollectGPRIN3, UBOX5, FER, and BMP4) had significantly increased H3K27ac at CTCF binding motifs with FDR < 0.05. One gene, CNKSR3, had reduced H3K27ac at its CTCF binding motif but was not statistically significant  . FER was downregulated despite increased H3K27ac in its first intron but may be related to changes in DNA methylation within that region, altered posttranscriptional regulation, or limitations of targeted ChIP analysis. Interestingly, the H3K27 region amplified in qPCR overlapped at least one differentially methylated cytosine for all four significantly enhanced genes  . H3K27aced genes . Only onPARK2, GPRIN3, and BMP4)  . BMP4 haed genes .We selected rotenone based on its ability to model gene–environment interactions in rodents and non-mammalian models of PD . It is eTo determine if the gene expression changes discussed above were due to changes in global levels of epigenetic patterns, we performed dot blots and Western blots to examine global levels of DNA methylation (5mC) and H3K27ac . DNA metITGA8 0.6 fc, GPRIN3 1.5 fc, FER 0.6 fc, CNKSR3 1.6 fc, BMP4 1.5 fc, UBOX5 1.6 fc, PARK2 0.6 fc, and CHCHD2 1.9 fc) that remained significantly associated in at least two studies with their most significant variant lying in a non-coding region . We discovered 13 genes with significant association to PD using GWAS meta-data. We focused on eight genes   and (UBOX5-hypermethylated)  . We saw hylated) . OverallOne of the primary functions of CTCF is to act as an insulator by blocking enhancer–promoter interactions . CTCF isHistone acetylation patterns are vulnerable to environmental factors and like DNA methylation are heritable . It is lPARK2 is a well-known genetic factor in PD as described earlier. Increased CTCF binding at its upstream enhancer decreased its expression, thereby affecting its role in the ubiquitin proteasome system. BMP4 is a gene that encodes bone morphogenetic protein 4, and it regulates neurite outgrowth and axonal transport through the activation of the TGFβ/Smad pathway which is disrupted according to RNA sequencing reactome enrichment data .RNA sequencing data has been submitted to the Gene Expression Omnibus (GEO) repository at NCBI Databank with record number GSE147617. Bisulfite-amplicon sequencing data is publicly available on the sequence read archive (SRA) at NCBI Databank with Bioproject ID DF designed and performed experiments with supervision from ZW. Both authors analyzed and interpreted data and prepared the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human cytomegalovirus (HCMV) is a ubiquitous herpesviral pathogen that results in life-long infection. HCMV maintains a latent or quiescent infection in hematopoietic cells, which is broadly defined by transcriptional silencing and the absence of de novo virion production. However, upon cell differentiation coupled with immune dysfunction, the virus can reactivate, which leads to lytic replication in a variety of cell and tissue types. One of the mechanisms controlling the balance between latency and reactivation/lytic replication is the regulation of the major immediate-early (MIE) locus. This enhancer/promoter region is complex, and it is regulated by chromatinization and associated factors, as well as a variety of transcription factors. Herein, we discuss these factors and how they influence the MIE locus, which ultimately impacts the phase of HCMV infection. Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects 40–60% of the population in developed countries and up to 100% in underdeveloped nations . For immCurrently, there are five antiviral therapies approved for the treatment of HCMV-associated disease by the FDA, including ganciclovir, valganciclovir, foscarnet, cidofovir, maribavir, and letermovir. In the transplant setting, there are two therapeutic strategies to combat HCMV-associated disease: (1) prophylaxis, where the drug is given to the patient from the time of transplant for a specific, pre-determined treatment course following the transplant; or (2) preemptive therapy, where patients are first monitored for HCMV infection and treated once they reach a detectable level of viremia . GuideliL) and unique short (Us), which are flanked by terminal repeats, terminal repeat long (TRL) and terminal repeat short (TRS), and internal repeats, internal repeat long (IRL) and internal repeat short (IRs). Upon infection, this linear genome circularizes and exists as a circular genetic element called an episome within the nucleus of the host cell . T. T84]. Tnscripts ,86,87. Ived IE19  and UL12ved IE55  and IE18ved IE55 . IE72 isved IE55 , suggestved IE55 ; if thisr assays . IE18, ar assays , displayr assays . IE40 anr assays ,93,94,95r assays ,97. Later assays . As mentr assays ,100. Howr assays , thus prr assays ,102,103.r assays ,106,107,r assays ,110,111.Gene expression is a complex process that is partially regulated by chromosome organization, which at the smallest unit, is made up of DNA and histones. The negatively charged DNA binds the positively charged amino acids of the histones. The amino-terminal histone tails protrude from the nucleosome core and are then subject to a variety of post-translational modifications (PTMs), predominantly on H3 and H4 histone tails . These hMethylation of histones is associated with transcriptionally inactive regions of the human genome . SpecifiAcetylation of histone H3 (K9 and K14) and H4  lysine tails is associated with active gene transcription ,152,153.Transcription factors play a major role in the expression and regulation of genes, and transcriptional control of the MIE locus is no exception. Because expression of the MIE genes is paramount to infection of the host cell, regulation of this region is under tight control. There is a unique balance between activation and repression of this enhancer/promoter region that is partially controlled by cellular transcription factors , allowinLatency is characterized by transcriptional silencing, which is due in part to the association of repressive transcription factors that work alongside chromatin remodeling factors discussed above. This silencing allows the virus to evade the host immune response while quiescent, thereby securing life-long infection. As with chromatin remodeling, regulation of the MIE locus via transcription factors is a dynamic process that allows the MIE enhancer/promoter to toggle between an active (lytic), repressed (latent), and de-repressed (reactivated) state. Gfi-1 mRNA expression is enhanced in the bone marrow and lymphoid tissue and is essential for hematopoiesis [Most of the transcription factors that bind in the MIE locus do so within the enhancer region . The proopoiesis . As HCMVThe distal enhancer, consisting of 300 bp upstream of the proximal enhancer region, has binding sites for both YY1 and Ets-2 Repressor Factor (ERF) transcription factors. YY1 is a zinc finger DNA-binding transcription factor with the ability to activate, repress, or initiate transcription depending on the promoter. Its function is partially dependent upon different binding partners and the recruitment of varying cofactors of YY1 . YY1 binUL127. Similarly, SATB1  binds the unique region between −593 and −549 [Four transcriptional repressors bind the unique region of the MIE locus, including CUX1/CDP , HMGB/SBand −549 . How SATand −549 . Thus, pSeveral other host factors have binding sites within the MIE locus that likely contribute to its regulation. Pancreatic duodenal homeobox factor 1 (PDX1) regulates HCMV gene expression by repressing MIE activity via a 45-bp element at position −593 to −549 upstream of the transcription start site . A decreReactivation from latency is characterized by the de-repression of the MIE locus to initiate lytic gene transcription. While our understanding of the repertoire of factors controlling this is likely incomplete, differentiation of myeloid cells to macrophages or DCs triggers viral reactivation, which in the face of a weakened immune system, leads to lytic replication and viral dissemination . De-reprcis-acting 19 bp repeats. Mutation of the CRE element at position −137 significantly decreases viral gene expression measured by a CAT reporter assay in primary fibroblast, primary aortic endothelial, telomerase-immortalized retinal pigmented epithelial, and primary hepatocyte cells [UL123 and UL122 expression in reactivating CD14+ monocyte-derived DCs [In addition to NFκB binding sites, ATF/CREB also binds the MIE proximal and distal enhancer, with an additional binding site in the core promoter. There are a total of five CRE sites in this locus , charactte cells , suggestte cells , bindingived DCs , which iived DCs . Collectcis-acting DNA binding sites called the RA-responsive elements (RAREs) [The MIE enhancer region also contains binding sites for ligand-inducible transcription factors, retinoic acid receptors (RAR), and the retinoid-X receptor (RXR-A), both of whose ligand is retinoic acid (RA) . RAR and (RAREs) ,203,204. (RAREs) . Within  (RAREs) ,203,204. (RAREs) . In muri (RAREs) . It is i (RAREs) , making  (RAREs) . Given tThere are seven predicted Specificity Protein 1 (Sp1) binding sites in the MIE enhancer . Two of O-tetradecanoylphorbol-13-acetate (TPA) response elements (TRE) with the consensus sequence 5′-TGAC/GTCA-3′ [UL127 and does not have an effect on MIE-driven genes [+ HPCs [The AP-1 transcription factor binds two sites in the MIE proximal enhancer ,160. Thi/GTCA-3′ , while t/GTCA-3′ ,160,215./GTCA-3′ ,160, AP-/GTCA-3′ . Additio/GTCA-3′ , suggest/GTCA-3′ . These sen genes . Indeed, [+ HPCs . FOX tra [+ HPCs , charact [+ HPCs . This isUL123 and UL122 gene expression in lytically infected, fibroblasts [Multiple transcriptional activators bind sites only within the MIE distal enhancer. Two of these transcriptional activators include Serum Response Factor (SRF) and Ets transcription factor/ETS-like kinase-1 (ELK-1) . ELK-1 iroblasts , suggestroblasts , which croblasts . This suroblasts . An IFNγroblasts . The delroblasts . Howeverroblasts . Upon IFroblasts . Thus, iLess well-studied transcription factors that activate the MIE locus include nuclear factor-1/CAAT box-binding transcription factor (NF-1/CTF), peroxisome proliferator-activated receptor-gamma (PPARγ), and methylated DNA-binding protein family (MDBP). NF-1/CTF has two sites in the unique and modulator regions of the MIE locus ,171 and microRNAs (miRNAs) are non-coding RNAs that regulate protein expression through interaction primarily with the 3′ or 5′ untranslated region (UTR), where the net effect is the inhibition of the target gene’s translation . Many viUL122, and when overexpressed in lytically infected fibroblasts, results in IE86 downregulation. Furthermore, when the hsa-miR-200 cluster cis site within the IE86 3′UTR was mutated, this virus failed to maintain latency in primary CD34+ cells, suggesting HCMV uses this host miRNA cluster to effectively “mop up” any aberrant transcription from the MIE locus during latent infection [In addition to the virally-encoded miRNAs, HCMV co-opts cellular miRNAs to its advantage during latency. The hsa-miR-200 cluster specifically targets the 3′ UTR of nfection . In suppnfection , thus itnfection . Finallynfection . CollectUL123 expression when induced with TNFα to stimulate reactivation [+ HPCs, which is further bolstered by TNFα treatment [It is clear from our discussion above that a complex balance of transcription factors and chromatinization tightly regulates the MIE locus. These players and their regulators, therefore, help to control the stage of HCMV infection. The transcription factors and chromatin remodeling enzymes are themselves regulated by both viral and cellular proteins to orchestrate HCMV latent and lytic infection . Upon HCtivation , suggesttivation ,258,259,tivation . Furtherreatment . AlthougIn addition to the host encoded regulators of the MIE locus, HCMV encodes proteins to modulate this region during latency, including UL138 ,261,262 + monocytes [UL123 and UL122 gene transcription. Further, lncRNA 4.9 plays a role in viral DNA replication through activity at the HCMV origin of replication (oriLyt), as knockdown inhibits viral DNA replication and viral growth in HCMV infected fibroblasts [Another virally encoded regulator is the long non-coding (lnc) RNA 4.9, which binds the MIE locus and recruits the repressor complex, PRC2 , a methyonocytes . This reroblasts . The abiStrict regulation of the MIE locus through chromatin remodeling and transcription factor binding is necessary to balance latent versus lytic infection. As the “molecular switch” dictating the “on/off” state of the virus, it is not surprising there are a multitude of host and viral proteins regulating this region. Continued research aimed at understanding a complete repertoire of these factors, as well as their upstream regulators, will further elucidate the mechanisms underlying HCMV latency and reactivation.


Eucalyptus HD-Zip family has not been reported until today. Here, we systematically identified 40 HD-Zip genes in Eucalyptus . Besides, we comprehensively analyzed the HD-Zips of Eucalyptus by studying the homology, conserved protein regions, gene structure, 3D structure of the protein, location of the genes on the chromosomes and the expression level of the genes in different tissues.The HD-Zip transcription factors are unique to plants and play an essential role in plant growth, development and stress responses. The HD-Zip transcription factor family consists of a highly conserved homeodomain (HD) and a leucine zipper domain (LZ) domain. Although the HD-Zip gene family has been extensively studied in many plant species, a systematic study of the Eucalyptus has four subfamilies, which is consistent with other plants such as Arabidopsis and rice. Moreover, genes that are in the same group tend to have similar exon-intron structures, motifs, and protein structures. Under salt stress and temperature stress, the Eucalyptus HD-Zip transcription factors show a differential expression pattern.The HD-Zip family in Eucalyptus HD-Zip transcription factors.Our findings reveal the response of HD-Zip transcription factors under salt and temperature stresses, laying a foundation for future analysis of  Transcription factors are also an essential participant in the processes of eukaryotic growth and differentiation [Transcription factors (TFs) are essential proteins that bind to a specific ntiation , 2. By fntiation , 4. Tillntiation , 6.Medicago truncatula, grape (Vitis vinifera), rice (Oryza sativa), maize (Zea mays), potato (Solanum tuberosum), wheat (Triticum aestivum) and banana [cis-elements and biological functions [Eucalyptus also has four subfamilies, HD-Zip I to HD-Zip IV. Among them, the HD-Zip I contains both of the two basic motifs; however, the HD-Zip II, III and IV families possess other motifs in addition to HD and LZ motifs. 1) CPSCE  exist in the HD-Zip II subfamily [The homologous domain leucine zipper (HD-Zip) gene family is reported only in the plants and regulates plant-specific growth and development processes , 8. HD-Zd banana –17. The unctions , 19. Simubfamily , 21. 2) ubfamily , 23. HD-ubfamily , 21, whiubfamily , 24.Arabidopsis, when overexpressed, an HD-Zip I member ATHB12, results in big leaves and enlarged cells, displaying its role in leaf growth. While another HD-Zip I member, ATHB1 contributes to cell wall composition and elongation [Medicago truncatula HD-Zip I TF HB1 is required for lateral root development [Arabidopsis [Ralstonia solanacearum [HD-Zip I proteins are reported for their function in plant light signal transduction , leaf anongation , 26. Beselopment , 18. Morbidopsis . In sunfbidopsis . Besidesbidopsis –31. HD-Znacearum .HD-Zip II subfamily possesses a conserved domain CPSCE, which plays an essential role in mediating plant response to changes in light quality and shading , 34, andThe structure of the HD-Zip III proteins is most complex among HD-Zips. The HD-Zip III family possesses the START domain, homeodomain-START associated domain (HD-SAD) and Met-Glu-Lys-Hi-Leu-Ala (MEKHLA) domain. The START domain is ABA-responsive, and the function of the HD-SAD domain is currently unknown. It is worth noting that the HD domain of HD-Zip III has two altered amino acid residues compared to the other subfamilies, and this change may be related to the unique MEKHLA domain . The MEKArabidopsis developing roots, cross-talk of at least six different phytohormones dynamically regulates the spatio-temporal expression pattern of miR165/166 and HD-Zip III. Besides, HD-Zip III mediated root development is modulated transcriptionally through phytohormones and KANs, and post-transcriptionally by miR165/166 [Arabidopsis [Arabidopsis jabba-1D (jba-1D) mutant plant affects the transcripts of class III homeodomain-leucine zipper family genes [microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the gene expression . miRNAs R165/166 . Class Ibidopsis , 49. Alsly genes .The HD-Zip IV subfamily contains four conserved domains identical to the HD-Zip III subfamily, the HD, Zip, START and HD-SAD domains. However, HD-Zip IV lacks the MEKHLA domain , 52. TheEucalyptus, a native plant of the southeastern coast of Australia, is a member of the genus Myrtaceae. In China, Eucalyptus is mainly planted in Guangxi province. It enjoys moist and fertile river loam soil and red soil weathered by basalt. Eucalyptus has high economic value due to its short growth cycle and other advantages. The leaves of Eucalyptus can be used to extract aromatic oils [Eucalyptus wood has corrosion resistance characteristics, and it is widely used in such construction, papermaking and fuel industries. Also, it is commonly used in artificial afforestation around the world, and as a unique commercial tree species, Eucalyptus is planted on a large scale. However, Eucalyptus is often affected by abiotic stresses such as drought, low temperature, and salt stress during its growth, resulting in a decline in the yield [Eucalyptus to improve the excellent traits and breeding.tic oils . Besidestic oils . Eucalyphe yield –61. TherEucalyptus, analyzed the relationship between the HD-Zip family and HD-Zips responses to temperature and salinity stress. These results provide the necessary basis for the functional characterization of the Eucalyptus HD-Zip family and a theoretical basis for the subsequent improvement of Eucalyptus varieties against stress.Here, we identified and classified the HD-Zip proteins of Eucalyptus and named as EgHD-Zip 1–40. To further analyze the selected sequences, a phylogenetic tree consisting of the 40 sequences was constructed. Also, to better highlight the difference between Eucalyptus and other plants, Arabidopsis and rice were included in the evolutionary analysis. The results showed that Eucalyptus, like Arabidopsis and rice, also had 4 subfamilies of HD-Zip genes. Among them, the HD-Zip I subfamily has the maximum members (15 Nos), and the HD-Zip III subfamily has the least members (4 Nos)  Fig. .Fig. 1PIn the II subfamily, EgHD-Zip16 and AT1G70920 (ATHB18), EgHD-Zip40 and AT4G16780 (ATHB2) showed a high homology. Besides, in the subfamily I, EgHD-Zip36 had high homology with AT1G26960 (ATHB23). In the subfamily III, EgHD-Zip19 and AT1G52150 (ATHB15) were highly homologous, so it can be speculated that EgHD-Zip19 may also have a similar role in inducing the development of vascular bundles. Similarly, EgHD-Zip20 and AT4G32880 (ATHB8) were on the same branch, so it can be speculated that EgHD-Zip20 could also induce the development of vascular bundles. In subfamily IV, EgHD-Zip4 had high homology with AT1G79840 (ATHB10), EgHD-Zip5 and AT4G00730 (ANL2), suggesting that EgHD-Zip4 may be involved in the epidermal cell differentiation process, and EgHD-Zip5 can affect flower development. The accumulation of glucoside in the leaf epidermis, and when the mutation occurs, it will inhibit the accumulation of anthocyanin. At the same time, it may also be involved in determining the identity of cells in the root [Eucalyptus HD-Zip genes are all localized in the nucleus.As shown in Table Multiple Em for Motif Elicitation (MEME) and Gene Structure Display Server (GSDS) were used to understand the relationship between EgHD-Zip proteins and their structure. It is worth noting that all of the EgHD-Zip proteins possessed the conserved motif encoding the HD and LZ domains. Highly homologous members were composed of the same motif, indicating that HD-Zip proteins of the same subfamily could have similar functions. The results also suggest that all EgHD-Zip proteins except EgHD-Zip30 have motifs 1, 2 and 5. Motifs 1 and 2 represent the conserved motifs encoding the HD domain, and motif 5 represents the encoding LZ conserved motifs of the domain Fig. .Fig. 2PEucalyptus  was used to analyze the structural diversity of the HD-Zip genes in tus Fig. . The resTo obtain intron gain/loss information for all sister pairs, we also compared the intron/exon structure of genes clustered at the end branches of the phylogenetic tree. Among them, 5 pairs showed intron/exon structural changes, including HD-Zip6/− 10, HD-Zip3/− 5, HD-Zip7/− 11, HD-Zip35/− 37, and HD-Zip39/− 30 Fig. , these cEucalyptus HD-Zip structure of the Swissmodel database, the structures of the members from four subfamilies of Eucalyptus HD-Zip were predicted. The N- and C-termini of the 3D structure of EgHD-Zip protein are shown in Fig. Using the protein homology modeling method based on the Eucalyptus. We found that the distribution of HD-Zip genes in Eucalyptus was uneven. Chromosome 10 had only one HD-Zip gene, while the remaining chromosomes had two to five genes  and PlantTFDB v4.0 database (Plant Transcription Factor Database). Also, the Eucalyptus HD-Zip HMM search [http://smart.embl-heidelberg.de/) and EMBL pfam (https://pfam.xfam.org/) database was used to detect the conserved domains. Finally, the wrong sequence was removed by manual screening. The 40 candidate sequences after the screenings were considered to be the Eucalyptus HD-Zip gene (EgHD-Zip). The chromosomal locations of the EgHD-Zip genes were obtained from the genomic annotation information of phytozome v12.1.6 , and using the Mapchart software it was visualized.HD-Zip protein sequences from M search  was carrEucalyptus, rice and Arabidopsis HD-Zip protein sequences were performed using MUSCLE v3.8.31 [n = 1000. After that, the results were imported to iTOL (https://itol.embl.de/) online software for processing.Multiple sequence alignments of  v3.8.31 . The IQ-Eucalyptus HD-Zip gene. We downloaded the annotation file in the format of GFF from Phytozome and retrieved the annotation information of the HD-Zip gene. The annotation information was uploaded to GSDS according to the default parameters of the exon-intron structure of the HD-Zip gene. Multiple Em for Motif Elicitation (MEME) was used to further determine the composition of motifs that might not be recorded in the public database [Eucalyptus.The Gene Structure Display Server (GSDS) , 69 was database , 71. TheEucalyptus RNA-Seq data included the following tissues: immature xylem, mature leaf, phloem, shoot tips, xylem. The heat maps of EgHD-Zip expression profiles from different tissues were obtained using TBtools software [RNA-Seq data were downloaded from public websites and Phytozome . Among tsoftware .Eucalyptus grandis clone Eg5, was collected from the College of Forestry, Fujian Agriculture and Forestry University. Eucalyptus grandis plants were grown on local soil for ten months under outdoor conditions. The annual average temperature in the growing area ranged from 16 to 20 °C, the yearly average rainfall from 900 to 2100 mm, and the annual relative humidity was about 77%. The red soil, which has the organic matter content from 2.57 to 6.07%, and the pH value 5, was used for cultivation.The experimental material, Eucalyptus seedlings were treated with 100 mmol/L sodium chloride and 200 mmol/L sodium chloride for 0, 6, 12, and 24 h for salt stress, and the control seedlings were treated with distilled water. For the temperature stress, Eucalyptus seedlings were placed at 4 °C and 40 °C, respectively, while and the control group was kept at room temperature (at 25 °C) for 0, 6, 12, and 24 h. After the stress, the samples were stored at − 80 °C for the subsequent analysis. For each treatment, five different seedlings were used, and the experiment was repeated at least three times.Annual EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix following the manufacturer’s protocol .and qRT-qPCR was conducted using TransStart® Top Green qPCR SuperMix . As a reference gene actin was used. Relative transcript abundance was calculated using the comparative 2–ΔΔCT method [Total RNA was extracted using RNA extraction Kit  from control and stress treated samples. cDNA was synthesized with the T method . All expA two-tailed Student’s t-test was applied to find statistical significance. Results are depicted as the mean values ± SE of three biological replicates.Additional file 1: Figure S1. Phylogenetic classification of HD-Zip gene in Eucalyptus grandis, Arabidopsis thaliana and Oryza sativa.Additional file 2: Figure S2. Phylogenetic classification of HD-Zip gene in Eucalyptus grandisAdditional file 3: Figure S3. The motif composition of HD-Zip proteins.Additional file 4: Figure S4. Sequence information of each motif identified by MEME.Additional file 5: Figure S5. Relative expression levels of EgHD-ZipI genes in various tissues.Additional file 6: Figure S6. Relative expression levels of EgHD-Zip II genes in various tissues.Additional file 7: Figure S7. Relative expression levels of EgHD-Zip III genes in various tissues.Additional file 8: Figure S8. Relative expression levels of EgHD-Zip IV genes in various tissues.Additional file 9: Table S1. Details of primers used in this study.Additional file 10: Table S2. The expression profile of pineapple bHLH genes in different tissue and developmental stages.Additional file 11: Table S3. Eucalyptus HD-Zip sequences used in current study.


Eukaryotic cells are complex biological systems that depend on highly connected molecular interaction networks with intrinsically disordered proteins as essential components. Through specific examples, we relate the conformational ensemble nature of intrinsic disorder (ID) in transcription factors to functions in plants. Transcription factors contain large regulatory ID-regions with numerous orphan sequence motifs, representing potential important interaction sites. ID-regions may affect DNA-binding through electrostatic interactions or allosterically as for the bZIP transcription factors, in which the DNA-binding domains also populate ensembles of dynamic transient structures. The flexibility of ID is well-suited for interaction networks requiring efficient molecular adjustments. For example, Radical Induced Cell Death1 depends on ID in transcription factors for its numerous, structurally heterogeneous interactions, and the JAZ:MYC:MED15 regulatory unit depends on protein dynamics, including binding-associated unfolding, for regulation of jasmonate-signaling. Flexibility makes ID-regions excellent targets of posttranslational modifications. For example, the extent of phosphorylation of the NAC transcription factor SOG1 regulates target gene expression and the DNA-damage response, and phosphorylation of the AP2/ERF transcription factor DREB2A acts as a switch enabling heat-regulated degradation. ID-related phase separation is emerging as being important to transcriptional regulation with condensates functioning in storage and inactivation of transcription factors. The applicative potential of ID-regions is apparent, as removal of an ID-region of the AP2/ERF transcription factor WRI1 affects its stability and consequently oil biosynthesis. The highlighted examples show that ID plays essential functional roles in plant biology and has a promising potential in engineering. Cells are dynamic and complex systems in which extensive molecular networks, functioning as part of signaling pathways, regulate biological processes such as development and responses to environmental cues. Gene-specific transcription factors (TFs) are terminal components of these signaling pathways and bind to target gene promoters to induce or repress gene transcription . TFs alsKd range [Thirty to 50% of eukaryotic proteins contain large intrinsically disordered regions (IDRs) ,4. DespiKd range ,17. SLiMKd range ,19,20. MKd range , which uKd range ,22. TogeKd range ,23.Plants are constantly challenged by stressors, and to cope with and adjust to these, they rely on complex transcriptional networks regulated by a large number of TFs from different TF families . IDRs anArabidopsis thaliana contain extended (≥30 residues) IDRs, which is comparable to 92% for human TFs [Early global scale analysis of ID in TFs from model organisms revealed that 91% of the TFs in xtended ≥ residuesuman TFs . In a reuman TFs . This reuman TFs ,33. In auman TFs ,35. To cIn this review, we focus on selected plant TF families, which have been studied with respect to ID. The families include the Nam/Ataf 1/Cuc 2 (NAC), Apetala 2/ethylene-responsive element binding factor (AP2/ERF), basic leucine zipper (bZIP), myeloblastosis (MYB), basic helix-loop-helix (bHLH), Teosinte Branched1, Cycloidea, Proliferating Cell Nuclear Factor (TCP), and auxin response transcription factor (ARF) TF families. They all follow the general domain structure of eukaryotic TFs with at least a family-designating DNA-binding domain (DBD) and a TRD .Arabidopsis [Arabidopsis showed that most NAC genes are induced by at least one type of abiotic stress signal [Arabidopsis and crop NAC TFs have been shown to be mostly intrinsically disordered using biochemical and biophysical methods such as size exclusion chromatography and various types of spectroscopy [Arabidopsis NAC TRDs showed that the conformational ensembles of the ANAC046, NAP and ANAC019 TRDs correspond to pre-molten globules, whereas the ANAC013 TRD is more compact. This leaves the potential for NAC TRDs to undergo function-related changes in secondary structure and compaction. Supporting this, naturally occurring protective osmolytes, which help organisms counteract the protein denaturing effects of environmental stress, induce structure in the long ANAC046 TRD [The NAC family is one of the largest plant-specific TF families with 110 genes in bidopsis  and 150 bidopsis . NAC TFsbidopsis ,39,40,41s signal . Most NAs signal  and longs signal , as shows signal  and PONDs signal  for disos signal . Since ttroscopy ,47,48. Dtroscopy ,49. HoweA few NAC TFs deviate from the characteristic NAC structure. Thus, Suppressor of Gamma Response 1 (SOG1)  belongs Arabidopsis and rice, respectively [Arabidopsis, rice, and moss, suggested that for the Arabidopsis ERF1DBD, the β-strands are ordered, while the α-helical region is structurally flexible, less conserved, and of low complexity [The AP2/ERF plant-specific TF family has 147 and 180 members in ectively ,55. AP2/ectively ,56,57. Tectively . The AP2ectively . A recenmplexity . The AP2mplexity , and themplexity , which implexity .Arabidopsis and rice, respectively [bHLH TFs are present in all eukaryotes and are encoded by 162 and 167 genes in ectively . They arectively ,64. The ectively ,66. Famiectively . The bHLectively . MYC2 alectively   plant-specific TF family contains only 24 members in elopment . The TCPelopment . TCP8 coelopment , the laselopment . A coileelopment , and ID elopment .Arabidopsis and rice, respectively [The auxin response transcription factors (ARFs) comprise another small TF family in plants with 23 and 25 genes in ectively . ARF TFsectively ,95. As iectively . For ARFectively , as descID is dominating in TFs from multiple TF families, as shown by both computational and experimental studies, with ID found mostly in the non-DBD regulatory regions . HoweverIDRs evolve fast with mutational patterns differing from those of globular proteins ,101,102.HvNAC005, which is a strong positive regulator of senescence in barley [The large and divergent intrinsically disordered NAC TRDs A containn barley . The NACn barley .The ID patterns of the NAC TFs are not conserved at the family level. However, specific sub-groups have conserved ID-patterns ,36, in aArabidopsis AP2/ERF TF family revealed multiple group-specific sequence motifs in the non-DBD regions [CBF1 from tomato confers increased freezing and salt tolerance and late flowering phenotype to transgenic Arabidopsis. The ability of the LWSY SLiM to mediate transcription depends on an aromatic SLiM-residue—tryptophan—as typical of SLiMs implicated in transcriptional activation [Several years ago, phylogenetic analysis of the  regions , and rec regions . As for  regions ,125, and regions . The LWS regions . Thus, etivation . The MCGtivation , plays mtivation ,129. Thetivation . All thetivation , emphasiConserved motifs in the non-DBD regions of MYB TFs are also useful for dividing the TFs into sub-groups with similar biological functions. In many cases, the sub-group motifs overlap with predicted MoRFs ,73. SeveSo far, family-scale ID predictions for the bZIP TFs are lacking. However, the non-DBD regions also contain sequence motifs present as conserved islands in IDRs, and several of the motifs have the same characteristics as the dominating NAC and AP2/ERF motifs with conserved aromatic and/or hydrophobic residues surrounded by acidic residues ,86. LikePlant TFs contain family- and group-specific SLiMs in non-DBD regions, many of which overlap with MoRFs. Although many of the SLiMs have known biological functions, numerous remain orphans without known functions ,27,36. MThe structural flexible associated with ID enables proteins to participate in interactomes, which require efficient adjustments whenever needed ,12,13. TArabidopsis RCD1-interactome represents a well-characterized example of an interactome with a central folded hub protein—RCD1—interacting with numerous, mostly disordered, TFs [rcd1 mutant displays pleiotropic phenotypes in development and stress responses and has a changed expression pattern of more than 400 genes [The red, TFs ,140. Somred, TFs   domain for its many interactions with TFs ,108,140.The RST hub domain adopts a unique helical conformation consisting of four α-helices flanked by disordered segments B and shaRCD1 interacts with TFs from at least seven different TF families including the NAC, AP2/ERF, zinc finger, bHLH, bZIP, MYB, and WRKY families ,140, allKds) binding to RCD1. This suggests that the RCD1-complexes are structurally heterogeneous with the DREB2A-RBS bound as an α-helix and the ANAC013- and ANAC046-RBSs bound as disordered regions or extended structures [Coupled folding and binding is paradigmatic for ID-based interactions and can take place either through conformational selection or induced fit. In conformational selection, a specific conformation from the TF ID ensemble is bound by the partner protein. In induced fit, the TF recognizes its partner in a disordered state that then folds over the surface of the partner ,22. Bindructures   is a key transcriptional co-activator in eukaryotic cells, which links information from TFs with the central transcriptional machinery, thereby fine-tuning transcriptional reprogramming ,153. In Arabidopsis MED15A was mapped and shown to contain 11 TFs including MYB63  play important roles in the regulation of cells, and IDRs are predominant sites of PTMs due to their conformational flexibility increasing the accessibility to modifying enzymes ,184. PTMPhosphorylation and ubiquitination are two prominent PTMs controlling TF activity . IncreasSOG1 plays essential roles in DDR, resulting in cell cycle arrest, DNA repair, and apoptosis ,123,196.The functions and regulatory mechanisms of SOG1 are comparable to those of the human oncogene TF p53 ,198. ManORE1, undergo early senescence. A C-terminal IDR of ORE1, containing eight serine and threonine residues, was suggested to be a phosphorylation hotspot [Calcium-regulated protein kinases are key components of intracellular signaling in plants and mediate rapid stress-induced responses to environmental changes. Recently, an in vivo phosphoproteomics screen with inducible CPK1 identified the NAC TF ORE1 (ANAC092), a key regulator of senescence and programmed cell death ,200, amo hotspot ,202  results in enormous agricultural losses making unraveling of the HS-related networks of uttermost importance. As part of large TF networks, DREB2A is a key TF regulator of HS and drought tolerance ,214,215.DREB2A is also regulated by SUMOylation  Figure B of Lys1MYC2 is also subjected to UPS-dependent degradation, and during JA-responses the levels of MYC2 correlate positively with the expression of early wound-response genes and negatively with the expression of late pathogen-responsive genes . A 12 reArabidopsis DREB2A provides an excellent example of this. Thus, a splice variant of DREB2A, which lacks the RBS  ,241. Howeactions . In addieactions . Finallyrylation .TFs are usually composed by one or more DBDs and one or more ADs that are typically located within an IDR ,246,247.Vernalization 1 (VRN1) is a plant TF composed by two B3-DBDs connected by a low complexity ID-region. The protein has a key role in plant vernalization  and undeLiquid-like properties have been considered key characteristic of membraneless organelles, whereas solid-state properties have often been related to pathological phenotype. The auxin response factor 7 and 19 (ARF7 and ARF19) G form asArabidopsis transcriptional regulator ELF3 reversibly forms liquid droplets in response to temperature increases, acting as a thermo sensor that regulates protein transcription at high temperatures. The sequence of ELF3 includes a poly-glutamine repeat that is part of a prion-like domain (PrLD), named from its sequence similarity to yeast prions [Recently, Jung et al.  showed tt prions . PrLD ist prions . The EL3t prions  (Figure t prions .ELF3 LLPS does not represent the first example of membraneless organelle formation triggered by temperature changes . In addiStudies of LLPS only began in the last decade , and manThe interplay between IDRs and structured domains, such as DBDs, also plays important roles in the functional regulation of TFs and can be mediated by allosteric coupling between different regions of the TFs ,261. SucFor ORE1, it was analyzed if remote IDRs affect DNA-binding. Both the ORE1 NAC-DBD and full-length ORE1 A were usNAC TFs bind DNA as homo- or hetero-dimers, and dimerization is required for DNA-binding . A conseANAC083 gene, encoding a NAC TF implicated in xylem cell specification [ANAC083 expression in protoplast cells is inhibited by the His135→Lys substitution in ANAC019, which mimics protonated His135. The mutant protein is defective in dimerization and target-gene binding. This indicates that protonation of His135 in ANAC019 affects the regulation of ANAC019 target genes, also in plants. For most of the more than 100 NAC TFs [The physiological relevance of the proposed pH-dependent DNA-binding mechanism of ANAC019 is supported by studies of the ANAC019-dependent regulation of the fication . Inducti NAC TFs , the net NAC TFs . Therefo NAC TFs .Examination of the specific role of ID across different phyla can improve the functional understanding of IDRs and IDPs . The hisArabidopsis TFs containing a functional RBS [Alignment of orthologues and inparalogs of the 10 onal RBS  revealedonal RBS , which ional RBS . The DBDThe MoRF-oriented analysis of the AP2/ERF TF family suggests that MoRFs can be shuffled between different protein families during evolution . In seveAs for the NAC TFs, ex nihilo SLiM evolution is likely for the MYB family. For sub-group 12 MYB TFs, the MIM  is respoIn this review, we summarize ID and protein dynamics properties for gene-specific TFs in relation to their function in plant biology. Although it is obvious that the flexibility associated with both ID and protein dynamics are advantageous for molecular functions in signaling networks of extreme biological importance, the molecular and mechanistic details remain elusive. For example, how are folded hub domains, as the RST domain of RCD1, able to bind TFs via the same TF SLiM but to different TF structures, and how does this influence putative competition scenario? To what extent are the IDRs of TFs, which are themselves hubs in the center of large interactomes, as in the case of DREB2A, responsible for the many interactions of the TF hub? For DREB2A, several molecular features of its IDRs are already known to be functionally important. Furthermore, what are the functional roles and interaction partners of the many orphan SLiMs and MoRFs of the TF IDRs? So far, many single case studies have addressed the effects of PTMs, e.g., phosphorylation, of IDRs, but consensus with respect to overall mechanistic aspects remains to be determined. For example, what are the molecular mechanisms linking phosphorylation and degradation, and do the effects of the ANAC019-AD on DNA-binding represent a generic mechanism for fine-tuning DNA-binding? Plant systems are useful models for mechanistic studies addressing these questions, since they allow analyses at the whole organismal level, as shown by effects of the SLIM-based MYB29:MYC4 interactions on glucosinolate accumulation in planta. Moreover, studies of phase separation in plants have revealed unique TF regulation pathways. Thus, plants may react to environmental changes through the formation of membraneless organelles, and additional novel mechanisms for modulations of transcription through phase separations may become apparent through additional studies. Studies of TF ID will also generally improve the basic understanding of molecular signaling in plants, and may reveal promising targets of engineering, as in the case of WRI1. Therefore, both basic and applied research in plant TF ID remains important future research goals.


G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment. The guanine quadruplex G4) structures play a role in DNA replication, transcription, recombination, and DNA repair . G4s may structur3+NG3+NG3+NG3+, but all of them are G-rich and may overlap G4-prone sites. To clarify whether G4s can be recognized by CTCF, in vitro binding assays are needed.Similarly to other zinc-finger (ZF) transcription factors, CTCF binds DNA in a sequence-specific manner, but its cognate sites are rather diverse . A motifCTCF sensitivity to DNA methylation is an open question. Hypomethylation was initially shown to promote CTCF binding ; but subMethylation is not the only epigenetic factor at play in CTCF positioning. Moderate nucleosome density and regular nucleosome positioning ,16 are rThis study was designed to partially verify the links between G4s, CGIs, chromatin compactness and CTCF recruitment. First, we used ChIP-seq, in silico modeling and in vitro binding assays to test CTCF affinity for G4s. Next, we analyzed previously reported ChIP-seq and bisulfite sequencing data to determine whether G4s account for CTCF recruitment to CGIs and whether DNMT1 plays a substantial role. Finally, to determine whether G4s affect accessibility of CTCF binding sites in internucleosomal DNA, we explored G4 interactions with linker histone analogs and other architectural chromatin proteins.CTCF is presumed to be incapable of recognizing G4s . Howeverp-value < 1 × 10−300 and OR = 3.2, p-value < 1 × 10−300 for PDS-treated and control cells, respectively). In contrast, BG4 peaks were clearly enriched in CTCF-bound sites irrespectively of PDS (The resulting CTCF occupancy profile was similar to that obtained for non-treated (control) cells A,B. Bothy of PDS D.We next questioned whether prolonged incubation with an excess of G4-stabilizing ligands would enhance CTCF occupancy at G4 sites. For that, we treated K562 cells with 20 μM PDS for 48 h. Phen-DC3, a more potent G4 stabilizer than PDS , was tesWe first investigated direct CTCF-G4 interactions in silico. The well-characterized G4 from MYC NHE, previously referred to as Pu27 G4 , was selTo verify affinity for CTCF in vitro, we picked three sequences predicted to form stable G4s  but not vice versa.The frequency of colocalization with CTCF peaks was similar for BG4 peaks inside  and those outside  CGIs. On the other hand, BG4-harboring and BG4-lacking CGIs showed markedly different distributions. In most cases  the latter were CTCF-free, while 9% (1962/21399) flanked and 29% (6188/21399) overlapped with CTCF peaks. In contrast, most BG4-harboring CGIs  overlapped with CTCF peaks, while 7% (439/6319) flanked CTCF peaks and 21% (1318/6319) were CTCF-free. To summarize, G4s colocalized with CTCF-bound sites irrespectively of CGI presence, while CGIs colocalized with CTCF if they contained G4s B. These We next assessed the contribution of DNMT1. Overlap with CTCF peaks was observed for 62% (858/1385) of DNMT1-lacking BG4-harboring CGIs and 75% (3704/4934) of DNMT1- and BG4-harboring CGIs. Analogously, DNMT1-harboring BG4 peaks within CGIs overlapped CTCF peaks at a slightly higher frequency  than those lacking DNMT1 . To summarize, DNMT1-bound sites colocalized with CTCF-bound sites only slightly more frequently than DNMT1-free sites B. This rFinally, to clarify the role of CpG methylation, we compared methylation levels within CTCF-bound sites, CGIs, and G4 sites in K562 cells using available whole-genome bisulfite sequencing data C. The soThe total average BG4 peak methylation level and those of BG4 peak-overlapping fragments of CTCF and CGI were similar (median: 30%). Although both CGIs and CTCF peaks were hypomethylated, methylation was enhanced at their intersections . To summarize, CTCF-bound sites and G4s have slightly higher methylation levels than CGIs in general. These results argue against the decisive role of hypomethylation maintenance in CTCF recruitment to CGIs and supports the role of local methylation.Apart from sequence and methylation, CTCF recruitment depends on nucleosome arrangement ,16. We sWe ranked the proteins based on their scores in the reported microarray-based assay with model G4s  and pickTo verify the association between the HMG proteins and G4s in the human genome, we examined available ChIP-seq data for HMGN3 (K562 cells), HMGN1 (CD4+ T cells), and HMGB2 (IMR90 cells). Average G4-seq coverage of protein-bound sites with flanks (±200 bp to account for the nearest nucleosome) was approximately 22%, 5%, and 4% for HMGN3, HMGN1, and HMGB2, respectively. These values were substantially higher than the average whole-genome G4-seq coverage (1.8%), indicating that G4s are enriched in HMG protein-bound sites. For HMGN3, frequent colocalization with G4s became even more apparent when we switched from G4-seq to BG4 peaks in K562 cells B: 77% ofHMGB2 showed the least pronounced association with G4s, supposedly because it interacts non-specifically with any DNA kinks , and the2 statistics. The observed G4-protein peak intersection frequencies exceeded those predicted by chance (p < 0.005), with the X2 statistic decreasing in the order HMGN3 >> HMGB2 (IMR90p10) > HMGN1 , demonstrating that HMG proteins tend to bind G4 sites both at the whole-genome level and in open chromatin.Given that G4s are associated with active transcription  and HMG The previously reported protoarray assay  allowed Several motif-matching ODNs  were obtODN interactions with HMG proteins were analyzed by MST; binding curves and Kd values are shown in 2 = 5574.1, p < 0.01]. Switching from G4-seq to G4-ChIP-seq (BG4) peaks increased the frequency of triple intersections (HNGN3-G4-CTCF) in K562 cells. Those triple intersections accounted for 60% of BG4 peaks. We conclude that HMGN3 recruitment to G4 sites may partially contribute to subsequent CTCF positioning.To verify whether HMG protein recruitment to G4 sites contributes to CTCF binding, we compared relative distributions of HMG and CTCF ChIP-seq peaks. Overall, there was low to moderate colocalization: CTCF peaks overlapped with 53% of HMGN3 peaks, 16% of HMGB2 peaks, and 8% of HMGN1 peaks in the respective cell lines. Substantial portions of the colocalized sites  harbored G4-seq peaks, although overall frequency of triple intersections was low B. StatisWe showed that CTCF binds folded G4s and consensus duplexes with comparable affinities . This isOur findings support G4 involvement in chromatin organization and partially explain G4 enrichment at TAD boundaries . These f® Plus Enzymatic Chromatin IP Kit with magnetic beads  following the manufacturer’s protocole. Briefly, crosslinking was performed via formaldehyde treatment and quenched with glycine. Cells were harvested and nuclei were prepared by incubation with the lysis buffer. Chromatin digestion was performed by treatment with micrococcal nuclease and stopped by EDTA treatment. Next, the lysate was sonicated to obtain DNA fragments of 200–500 bp. Per chromatin immunoprecipitation (ChIP) reaction, ∼5–10 μg of digested, cross-linked chromatin was incubated with 2–4 μg CTCF antibody pAb  overnight at 4 °C. Normal Rabbit IgG  was used as a negative ChIP control. On the next day, Protein G Magnetic Beads  were added in each sample and incubated for 6 h at 4 °C. Immobilized complexes were washed two times for 10 min at 4 °C in low salt (1X ChIP buffer) and high salt (1× ChIP buffer supplemented with 350 mM NaCl) solutions. Samples were incubated with RNase A  in TE buffer  for 30 min at room temperature. The DNA was eluted from the beads and decrosslinked by proteinase K digestion for 2 h at 65 °C. Next, the DNA was purified using DNA purification spin columns and analyzed by Illumina Next Generation sequencing or qRCR.K562 cells were cultured in RMPI-1640 medium  supplemented with 0.4% fetal bovine serum . Cell viability was quantified via trypan blue staining. The G4-stablizing ligands (PDS or PhenDC3) were added to the cell suspensions (1–1.25 mln/mL) to final concentrations of 10 or 20 μM (PDS) and 8 μM (Phen DC3). After 24 h (10 μM PDS) or 48 h (20 μM PDS or 8 μM PhenDC3) of incubation, cells were fixed, and chromatin immunoprecipitation (ChIP) was performed using SimpleChIPhttps://github.com/shawnzhangyx/PePr, accessed on 20 December 2020) with a p-value cutoff of 0.05 and a sliding window size of 100 bp [Paired-end libraries were prepared according to the manufacturer’s recommendations using NEBNext Ultra II DNA Library Prep Kit . The libraries were indexed with NEBNext Multiplex Oligos kit for Illumina . Size distribution for the libraries and their quality were assessed by Agilent Bioanalyzer using Agilent DNA High Sensitivity Chips . The libraries were subsequently quantified by Quant-iT DNA Assay Kit, High Sensitivity . DNA sequencing was performed on the HiSeq 2500 platform  according to the manufacturer’s recommendations, using the following reagent kits: HiSeq Rapid PE Cluster Kit v2, HiSeq Rapid SBS Kit v2 (200 cycles), HiSeq Rapid PE FlowCell v2 and a 1% PhiX spike-in control. The experiment was repeated twice. Reads for each biological replicate were mapped to the human genome (version hg19) using Bowtie2 (version 2.2.3) with the ‘—very-sensitive’ preset . Non-unif 100 bp . The big2, enhancers, and stabilizers —were mixed to give a final volume of 25 μL. The thermocycler program was 95 °C for 5 min (1 cycle) followed by a 2-step reaction of 95 °C for 10 s and 60 °C (for primer pairs STAT3 and VEGFA) or 62 °C  for 30 s (40 cycles). Amplification curves were analyzed using Thermofisher software and ΔΔCq values were calculated in Microsoft Excel to express the relative DNA amplification of the CTCF-occupied region in ligand-treated/non-treated samples to inputs:For qPCR analysis of the immunoprecipitated samples and inputs, primers intersecting or flanking the CTCF occupancy sites of interest  were designed using NCBI primer-BLAST and Eurofins Genomics primer design tools . qPCR exΔΔCq = 2^(−ΔCq_sample)/2^(−ΔCq_input);ΔCq = Cq_region of interest—Cq_control regionThe control region was a G4-lacking site USP24. All primers are specified in For G4 docking to CTCF, we used the available model of CTCF zinc fingers 4–10 . A two-sG4 motif and i-motif mining was performed using the updated version of imGQFinder  and G4/iOligonucleotides (ODNs) were obtained from Litekh, Moscow, Russia . Recombinant mouse CTCF with polyhistidine (His) tag was purchased from MyBioSource, San Diego, CA, USA. Recombinant human HMGB2 with His tag was purchased from Abcam, Cambridge, UK. Recombinant human HMGN1 and HMGN3 with His tags were purchased from LifeSpan BioSciences, Seattle, WA, USA. For microscale therophoresis (MST) assays, the proteins were labeled with the His-Tag Labeling Kit RED-tris-NTA  according to the manufacturer’s protocol. G4 ligands pyridostatin (PDS) and bisquinolinium-derivatized phenanthroline-dicarboxamide (Phen DC3) were obtained from Sigma-Aldrich .ODN solutions (40 μM) in 140 mM potassium phosphate buffer (pH 7.2 or 6.7) containing 10 mM NaCl were heated to 95 °C for five min and ice-cooled on ice (for G4s and i-motifs) or cooled slowly to room temperature (for duplexes) to facilitate correct folding. These annealed samples were used as stock solutions for microscale thermophoresis assays. For circular dichroism (CD) measurements and polyacrylamide gel electrophoresis (PAGE), the pre-annealed samples were diluted with the respective buffers to a final concentration of 1–5 μM. CD spectra were recorded at room temperature using a Chirascan spectrophotometer  and a quartz cuvette with an optical path length of 10 mm. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was performed in a standard Tris–borate–EDTA (TBE) buffer (pH 8) at a gel concentration of 20%. Low molecular weight marker 10–100-nt ssDNA  was used as a control. The gels were run for 2 h at 200 V at room temperature with a 1× TBE with 10 mM KCl buffer. Oligonucleotide bands were stained with SYBR Gold  and visualized using a Gel Doc scanner .Labeled proteins were mixed with two-fold serial dilutions of unlabeled ODNs to a final protein concentration of 50 nM and varying ODN concentrations (from 20 µM to 0.61 nM). The mixtures were stored at room temperature for 15 min prior to MST measurements. MST curves were registered using Monolith NT.115, equipped with a RED/GREEN detector, and standard capillaries  at 22 °C with MST monitoring by fluorescence of the labeled protein. The dependence of its normalized fluorescence on the concentration of the unlabeled oligonucleotide was analyzed using MO.Affinity Analysis software . To obtain dissociation constant values, experimental data were fitted to the Kd model.


Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that  Meis1/2 in the limb forming region of mouse embryos results in the complete absence of limb, while embryos developed with one functional Meis allele, so identifying the role of Meis in antero-posterior and proximo-distal patterning.Double conditional knockout of  Limb induction requires Tbx4/5 and Fgf10 activation in the lateral plate and their elimination produces limbless embryos2. Tbx5 in the forelimb and Tbx4 in the hindlimb is directly regulated by several inputs, including nuclear ßcatenin, retinoic acid, and specific combinations of axial Hox expression5. In cooperation with persistent nuclear ßcatenin, Tbx factors, in turn, induce limb-bud mesenchyme to express Fgf10. Fgf10 signaling to the overlying surface ectoderm activates several FGF and Wnt factors, which signal back to the mesenchyme to maintain Fgf10 expression, forming a positive-feedback loop essential for limb-bud establishment, growth, and distalization9.Limbs initiate as swellings from the lateral body wall that form by the localized epithelial-to-mesenchymal transition of the somatic coelomic epithelium11. AP prepatterning of the limb-forming region is required for Shh activation and starts simultaneously with limb induction, much earlier than the onset of Shh expression. The posterior field of the limb bud is established by AP-regulated expression of axial Hox9 paralogs in the forelimb and Islet1 in the hindlimb, which induce posteriorly restricted expression of Hand213. Hand2, in turn, restricts Gli3 mRNA expression to the anterior region of the limb bud, producing its first AP compartmentalization14. Hand2 is induced not only by Hox9 paralogs, but also by Hoxd13, which binds to the Hand2 locus15, suggesting direct regulation. At later stages, Shh expression is induced in the ZPA through cooperation between Hand218 and posterior HoxD proteins23.Progression along the proximo-distal (PD) axis is concomitant with limb patterning along the antero-posterior (AP) axis. The main regulator of limb AP patterning is the secreted factor Shh, which is expressed from the zone of polarizing activity (ZPA), a discrete mesenchymal region at the posterior end of the limb bud25 and indirectly with anterior Hox proteins through Pbx, another TALE homeodomain family member26. Meis factors associate extensively but not exclusively with Hox-binding sites in the genome and also act as cofactors for non-Hox transcription factors30. Meis1 and 2 are homogeneously expressed in the lateral plate mesoderm from pre-limb stages and, following limb initiation, become expressed in a proximal-to-distal gradient in the growing limb bud31. Meis overexpression inhibits limb-bud distalization34, whereas Meis elimination after limb induction results in distalization and strong disruption of proximal segments31. How limbs develop in the complete absence of Meis expression in the lateral plate has not been explored. Meis1 loss-of-function mutants have hematopoietic, vascular, and ocular defects37 but show no alterations in limb development. Limb skeletal patterning is similarly unaltered in Meis2 mutants39. Given that Meis1 and Meis2 encode highly similar proteins, both able to inhibit limb distalization and co-expressed during limb development, we reasoned that the lack of phenotype in the single mutants is likely due to redundancy. Here, we describe double Meis1 and Meis2 elimination in the limb-forming region and report Meis functions in limb induction and AP and PD patterning.Meis1 and 2 belong to the TALE (three amino-acid loop extension) homeodomain transcription factor family and regulate Hox activity by interacting directly with posterior Hox proteinsMeis1 and Meis2 in limb development, we conditionally deleted both genes. For this, we generated a new Meis2-targeted conditional allele  injection at E8.5  and the whole lateral plate posterior to the FL, including the HL forming region  or complete (N = 6/9) elimination of limb-bud growth and chondrogenic condensations is observed  embryos die around E13.5-E14.5, due to hematopoietic defects.To determine the combined role of 8.5 Fig. . HoxB6Crion Fig. . The eli42. In regard to cell proliferation, no significant changes were observed in the mesoderm or ectoderm of either FLs or HLs   Fig. . SkeletaKO) Fig. . Along tKO) Fig. .Fig. 2Skp-value < 0.005  −19,1), Fgf10 , and Lef1  , Alx3 , Shox2  and Tbx15 46, and upregulation of distal markers, such as Tfap2b  and Pknox2 48 . This analysis identified 196 downregulated and 242 upregulated transcripts with an adjusted ,4) Fig. . In addi,4) Fig. . M1KO;M2)48 Fig. . These c49, characterized by highly conserved sequences and a preference for distal enhancers  identified the Hedgehog and Wnt pathways as highly significant gene sets associated with Meis-binding sites  or very weak (N = 1/10) in E10.5 M1KO;M2KO hindlimb buds  to reduced expression (N = 3/6) . In HL buds, where Meis genes are completely eliminated, Fgf10 expression was absent or very reduced throughout the buds (N = 5/6)  and HLs (Tbx4)   Fig. . Activat/6) Fig. . During ly) Fig. .Fig. 4ExFgf10 by two mechanisms: direct activation of Fgf10 enhancers and activation of the canonical Wnt signaling pathway effector Lef/Tcf. Both Tbx4/5 and Lef/Tcf then cooperate in Fgf10 activation55. Lef1 expression was completely absent from the posterior part of mutant FLs buds (N = 4/4) and from the whole-mutant HL buds (N = 4/4), demonstrating that the Wnt pathway is inactivated in M1KO;M2KO limbs   Fig. . The disce) Fig. . The prece) Fig. . Nonethece) Fig. , suggest24 and the evidence of cooperation between Hoxc10 and Tbx factors in limb buds60, we investigated the coincidence between Meis, Tbx, and Hoxc10-binding profiles  is consistent with the established role of Meis as a preferential cofactor for Hox proteins24 and was more extensive than the overlap between Hoxc10 and Tbx5-binding sites. Binding sites common to Tbx5, Hoxc10, and Meis appeared at a higher frequency than predicted by the pairwise interactions  contains Meis, Tbx5 and Hoxc10-binding sites, whereas the more 3′ deleted region (3′−0.4∆) contains only Meis and Tbx5-binding sites. We also generated a 14.5 kb deletion (14.5∆) that includes both 0.4 kb deletions and the region between them   , absent (N = 2/4 HLs) or misplaced (N = 1/4 HLs) . In addition, expression of the Shh-repressed factor Cdon, normally restricted to anterior limb-bud regions62, extended toward the posterior limb region (4/4)  Fig. , which i/4) Fig. .Fig. 7MeShh regulators. These include Gli3 and Hand2, whose antagonism establishes an AP prepatterning required for Shh activation. In M1KO;M2KO mutant FLs, Hand2 expression was lost or very reduced (6/6), while Gli3 expression, normally confined to the anterior part of the limb bud, extended posteriorly (2/2) . This was accompanied by the presence of low, homogeneous Gli3 expression across the HL bud AP axis (2/2)  Fig. . Strikin/2) Fig. . Thus, aShh activation. Hoxd10-13 genes are required for Hand2 activation and cooperate with Hand2 in direct Shh activation23.Different phases of Hox expression have been implicated in the generation of the limb AP prepattern and subsequent Hoxd13 is confined distally, at early stages it is found in the posterior limb bud, in agreement with its role in regulating Hand2 and Shh expression. In M1KO;M2KO FLs, Hoxd13 expression was either absent (4/8) or strongly reduced (4/8)  .Although in late-stage wild-type limb buds /8) Fig. . In cont/4) Fig. . InsteadShh activation through its role in regulating genes involved in AP limb prepatterning, including Gli3, Hand2, and 5′ Hoxd genes. The ChIP-seq results support a direct regulatory role for Meis in some of these interactions. Meis-binding sites were detected in the vicinity of Hand2, Ptch1, and the HoxD complex , whereas homozygosity led to limb-specific complete absence of Hand2 expression (N = 4/4 in both FLs and HLs)  Fig. .Shh locus, nor the ZPA regulatory sequence (ZRS), essential for Shh activation . Mice were handled in accordance with the CNIC Ethics Committee, Spanish laws, and the EU Directive 2010/63/EU for the use of animals in research. All mouse experiments were approved by the CNIC and Universidad Autónoma de Madrid Committees for “Ética y Bienestar Animal” and the area of “Protección Animal” of the Community of Madrid. The targeting vector used to introduce loxP sites flanking Meis2 exon 3 was generated by GeneBridges following these steps: (1) insertion of a loxP-flanked neoR cassette into BAC bQM241K9 by Red/ET recombination; (2) subcloning of a 17 kb BAC fragment into minimal vector pMV and subsequent Cre recombination to obtain a single loxP site; (3) insertion of a loxP-FRT-PGK-gb2-neo-FRT cassette into a second location in the sub-cloned DNA. The targeting vector was SalI-released from the backbone and electroporated into ES cells. ES cell clones were screened by Southern blot. Chimeras were generated by ES cell aggregation. The neomycin resistance cassette was eliminated with a Flpe deleter strain69. Mice were subsequently genotyped by PCR. All primer sequences for genotyping, qPCR, and CRISPR/Cas deletions are shown in Supplementary data Experiments were performed using mice (HoxB6CreER mice were as previously described41. Meis1flox mice (Meis1tm2Ngc)70 were generated by Kenneth Humphries  and kindly provided by Dr. Hesham Sadek .4-hydroxy-tamoxifen (4HT) (Sigma) was dissolved in corn oil (5 mg/ml) and injected intraperitoneally (0.8 mg single dose) at E8.5.71. Samples were fixed in 4%PFA, dehydrated in methanol, rehydrated, then a bleaching step (30 min with 6% H2O2) before proteinase K (PK) treatment. Samples were then hybridized with the probe overnight. After SSC-CHAPs and TBST wash samples were developed with BM purple (Roche).Whole-mount in situ hybridization was performed according to standard protocols72. Embryos were eviscerated and fixed in 10% formaldehyde overnight and then washed in acid alcohol (3% HCl in 70% ethanol) several times. Embryos were stained for 3 h with 0.5% w/v Victoria Blue (Sigma) in acid alcohol and after staining embryos were washed in acid alcohol until the embryos were white, then they were washed in 70% ethanol and 95% ethanol. Finally, embryos were clarified with increasing concentrations of methyl salicylate in ethanol  and stored in 100% methyl salicylate.Victoria Blue staining was performed following standard proceduresMeasurements of long bones were done with ImageJ and individually normalized by each embryo’s crown-rump length. Images were taken by using Nikon microscopy camera acquisition software.p-value < 0.05.E10 hindlimbs were dissected from wild-type embryos (5 samples) and M1KO;M2KO embryos (6 samples). Each sample included both HLs of an individual embryo. RNA was isolated from tissue samples using the RNeasy Mini Kit (QIAGEN). Samples were sequenced using Illumina HiSeq technology. An amplification step (NuGen Ovation) was included in the sample preparation protocol. For the analysis, reads were first preprocessed with Cutadapt 1.3 to eliminate Illumina adaptor remains, and to discard reads that were shorter than 30 bp, using FastQC 0.8.0 to assess read quality after each preprocessing step. The resulting reads were then aligned against a mouse transcriptome reference  with RSEM 1.2.3 to estimate gene-level expression levels. Finally, estimated count matrices were used as input in a pipeline for differential expression analysis that included TMM (contained in R package Limma), ComBat (contained in R package SVA 3.20.0 and Limma 3.28.21) as main components. The analysis was restricted to a selection of 13,720 genes that were expressed at a minimal level of 1 CPM in at least 4 samples. Changes in gene expression were considered significant if associated with Benjamini and Hochberg adjusted They are detailed in Figure legends. In all cases, individual measurements were taken from distinct samples. Prism 9 software was used for the analyses.73 (dilution 1/800) and anti-HoxD9 (H-342 Santa Cruz Biotechnology) (dilution 1/100). Images were taken by using Leica microscopy camera acquisition software.Standard procedures were used for immunofluorescence with antigen retrieval step. Anti-Meisa antibody was produced in the lab, recognizing C-terminal short isoform of Meis1 and Meis2HoxB6CreER transgenic embryos were fixed for 30 min, rinsed in PBS, and incubated in the presence of X-gal as described74. 75 using two single-guide RNAs (sgRNAs) flanking the region of interest. See Supplementary Data CRISPR/Cas9 gene editing was performed in the pronuclei of B6CBAF1xB6 zygotes76 were used according to the manufacturer′s instructions. Quantification was performed manually counting TUNEL or pH3 positive cells and normalizing with DAPI positive nuclei (ITCN plugin in ImageJ) in each section. Five WT FLs, 4WT FL, and 6 M1KO;M2KO limbs were considered and 3 sections of each limb were quantified. Statistical comparisons were performed using a Mann–Whitney test by using Prism 9 software.Rabbit polyclonal anti-phospho-histone 3 antibody (06-570 Sigma-Aldrich) (dilution 1/200) and TdT-mediated biotin-dUTP nick end labeling (TUNEL) detection kitt-test. See Supplementary data Both FLs of E9.5 embryos containing the different deletions and somite-staged corresponding WT FLs were dissected and frozen individually. RNA purification was done by Qiagen miRNeasy Mini Kit and retrotranscribed by Invitrogen SuperScript™ II Reverse Transcriptase. qPCR was then performed using Sybr Green PCR Master Mix and run in an ABI Prism 7000 from Applied Biosystems. Statistical comparisons were performed using a one-sample 73. ChIP DNA was sequenced with an Illumina HiSeq 2500 system by NDeep sequencing at the CNIC Genomics Unit. Single-end reads were mapped with BWA 0.7.15 software against version mm9 of the mouse genome. The alignments were then used for peak calling by MACS 2.1.1, using p = 0.0005 as the cutoff for detection, except for the FL-HL common peaks, for which the cutoff p-value was 0.0005 for the compound occurrence of the same peak in both data sets. Detection of peak-to-gene association and functional association of the identified gene sets were performed with GREAT 3.0.0.77. A basal regulatory domain was assigned to each gene of extending from 5000 bp upstream of the transcriptional start site to 1000 bp downstream. The regulatory domain is extended in both directions to the nearest gene′s basal regulatory domain but no more than 1000 kb from the TSS in each direction and curated regulatory domains were added to the basal regulatory domain. Known and de novo motif identification was performed with HOMER 4.878.Around 500 E10.5 (36so) wild-type FLs and 500 E10.5 wild-type HLs were fixed according to standard procedures for ChIP experiments. For the IP, we used ChIP-IT High Sensitivity Columns (Active Motif). Two anti-Meis antibodies were used simultaneously, one recognizing Meis1a and Meis2a,c and the other recognizing all Meis1 isoformshttps://usegalaxy.org) were used to compare and map data sets for different transcription factors. Analyses were visually represented using IGV software79, which is publicly available from the Broad Institute website (https://software.broadinstitute.org/software/igv/).Bedtools at the Galaxy public server (Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Peer Review FileReporting Summary