RUNX1/RUNX1T1 is the most common fusion gene found in acute myeloid leukemia. Seminal contributions by many different research groups have revealed a complex regulatory network promoting leukemic self-renewal and propagation. Perturbation of RUNX1/RUNX1T1 levels and its DNA binding affects chromatin accessibility and transcription factor occupation at multiple gene loci associated with changes in gene expression levels. Exploration of this transcriptional program by targeted RNAi screens uncovered a crucial role of RUNX1/RUNX1T1 in cell cycle progression by regulating CCND2. This dependency results in a high vulnerability toward inhibitors of CDK4 and CDK6 and suggests new avenues for therapeutic intervention against acute myeloid leukemia. Fusion genes and their underlying chromosomal alterations are a hallmark of acute myeloid leukemia (AML). They are particularly prevalent in pediatric AML, where more than 50% of all cases harbor a fusion gene ,2. As fuBCR/ABL1 or FLT3-ITD and is thought to promote proliferation and survival, fusion genes encoding transcription factors such as MLL/AF9, PML/RARA or RUNX1/RUNX1T1  have been classified as type 2 mutations that drive self-renewal and interfere with differentiation. Recently, a third class has been suggested that separates mutated epigenetic modifiers and readers from class 2. However, more recent investigations of transcriptional networks underlying the leukemic phenotypes have blurred this clear distinction, with type 2 members also promoting proliferation by controlling growth factor and cell cycle genes such as FLT3, CDK6, and CCND2Most of these translocations are initiating events in the process of leukemogenesis and are expressed in all leukemic cells of a patient. The generation of fully transformed leukemic cells usually requires the acquisition of several mutations that act in concert to drive self-renewal and proliferation, sustain viability, and avoid differentiation and immune surveillance HOXA cluster  and those that do not  [Importantly, perturbation studies have revealed that human leukemic cells are dependent on continuing expression of their respective type 2 fusion gene, with its transient loss leading to impaired proliferation and increased senescence gements) . Thus, tBecause of their biological significance and exclusive expression in preleukemic and leukemic cells, fusion genes are highly attractive targets for any precision medicine approach. However, their direct targeting by more conventional drug discovery approaches, such as small molecules and monoclonal antibodies, has proven to be challenging, CBFB/MYH11 being so far the only example of a truly fusion protein-specific targeting . An alteAlternatively, the leukemic programs and transcriptional networks associated with fusion gene-encoded transcriptional regulators offer new options for targeted therapeutic interventions. Pathways under the direct control of the corresponding fusion protein are likely to contain elements amenable to pharmacologic interference. The challenge here is to have sufficient insight into the composition of these fusion protein-driven programs and the relevance of its components for leukemic maintenance. These programs can be unraveled by fusion protein knockdown or inhibition of its function, followed by analysis of changes in chromatin occupation and gene expression. Integration of RNA-seq, ChIP-seq, and DHS-seq data revealed the compositions of several AML networks including those for RUNX1/RUNX1T1, CBFB/MYH9, and CBFA2T3/GLIS2 ,17,18. CThese transcription factor nodes add additional levels of complexity with respect to the transcriptional regulation of the whole network. Fusion proteins may affect transcription directly by physical interaction with promoters, enhancers, or silencers, indirectly via regulation of expression and activity of transcription factors, or even in a mixed mode involving both direct binding of the fusion protein and recruitment of downstream transcription factors, consequently exacerbating the identification of direct target genes. Generally, direct target genes are defined by changes in transcript levels upon perturbation of the transcription factor in combination with binding of the transcription factor to the gene locus or to a more distant regulatory element such as an enhancer or silencer RUNX1/RUNX1T1 fusion gene, is the most prevalent chromosomal rearrangement, with an incidence of 15% in children and young adults The chromosomal translocation t, generating the The transcriptional network established and maintained by RUNX1/RUNX1T1 is one of the best characterized leukemic networks. RUNX1/RUNX1T1-exerted control is modulated by several posttranslational modifications including acetylation and ubiquitylation by EP300 and UBE2L6, respectively, and depends on the recruitment of epigenetic modifiers, in particular class I histone deacetylases via SIN3A and NCOR via its RUNX1T1 moiety B 28, 29,Similar to wild-type RUNX1, RUNX1/RUNX1T1 heterodimerizes with CBFβ, increasing its DNA binding affinity, although the relevance of this interaction for the function of RUNX1/RUNX1T1 has remained a matter of debate ,36. FurtA main feature of RUNX1/RUNX1T1-mediated dysregulation of gene expression is its competition with wild-type RUNX1 binding. RUNX1/RUNX1T1 shares with RUNX1 about 75% of its binding sites. A basal level of RUNX1 is required for RUNX1/RUNX1T1 to maintain cell growth, and complete suppression of the differentiation genes is not tolerated Loss of RUNX1/RUNX1T1 severely compromises malignant self-renewal, but hardly affects viability of t AML cells, while depletion of RUNX1 induces apoptosis in this leukemic context ,47. ThisDepletion of RUNX1/RUNX1T1 by fusion site-specific siRNAs has provided some fundamental insights into the transcriptomic program driven by this fusion protein ,50. RUNXConsequently, RUNX1/RUNX1T1 reprograms a massive transcriptional network to establish and maintain leukemia. In agreement with repressing gene function by recruiting class 1 HDACs, RUNX1/RUNX1T1 impairs expression of a set of about 1,400 genes that is linked to myeloid and, in particular, neutrophil differentiation. However, although RUNX1/RUNX1T1 is frequently described as a transcriptional repressor, the vast majority of these genes are rather modestly downregulated, suggesting that a basal expression of these “repressed” genes may not only be tolerated, but actually may be required for the leukemic phenotype. Furthermore, almost 50% of all genes affected are maintained or activated by RUNX1/ETO. This upregulated gene expression signature is associated with several “Hallmarks of Cancer” processes including cell cycle progression at multiple stages, glycolysis and oxidative phosphorylation, MTOR signalling, RNA processing, and ribosome biogenesis ,50.RPS6KA1 or PARL, thus adding an additional layer of complexity to the RUNX1/RUNX1T1 transcriptional network In particular, the latter two processes emphasize additional mechanisms of gene expression control exerted by leukemic fusion proteins. RUNX1/RUNX1T1 regulates the generation of alternative transcripts both by affecting alternative splicing and by repressing the activity of alternative promoters of genes such as MIR126, which reduces apoptosis and increases proliferation, is overexpressed in CBF AMLs EGFL7 gene, a direct target gene of RUNX1/RUNX1T1. Furthermore, repression of MIR223 by RUNX1/RUNX1T1 impairs granulocytic differentiation and activation of leukemic cells and may also support cell cycle progression by enhanced expression of the MIR223 target E2F1 [Finally, RUNX1/RUNX1T1 also controls mRNA translation and stability by perturbing the expression of multiple miRNAs. Genome-wide analysis of AML patient samples revealed that t patients have a distinct miRNA signature compared with other chromosomal translocations get E2F1 ,58. ThesThis large and complex RUNX1/RUNX1T1 network offers both challenges and opportunities for precision medicine approaches. On the one hand, it is predicted to comprise multiple potential targets amenable to conventional drug discovery and development and also a number of candidates for which already clinically approved drugs or at least experimental substances at a preclinical stage are available. On the other hand, because its high complexity, it is challenging to identify those crucial targets whose modulation will lead to a collapse of the net and a potential therapeutic effect. Promising approaches here are RNA interference and CRISPR screens for the functional identification of network components.To identify crucial components of the leukemic network, we assumed that interference with target genes of RUNX1/RUNX1T1 is less likely to be compensated by altered expression of alternative genes than that of indirect target genes. Direct target genes were selected according to the “guilt by proximity” principle by choosing genes with detectable RUNX1/RUNX1T1 binding within or in the neighborhood of their loci. This approach is likely to underestimate the direct impact of a fusion protein, as distances between gene loci and their regulatory elements may span several hundred kilobases. Furthermore, we focussed on genes whose expression was significantly downregulated by RUNX1/RUNX1T1 knockdown because of their potential involvement in leukemic self-renewal.Integration of RNA-seq, ChIP-seq, and DNase I hypersensitive site (DHS)–seq experiments identified a set of 133 genes CCND2. Validation experiments demonstrated a strict dependency of leukemic clonogenicity and competitiveness on CCND2 expression CDK6 locus, and its loss was associated with reduced CDK6 expression. Finally, RUNX1/RUNX1T1-positive AML cell lines and patient cells are very sensitive toward the CDK4/6 inhibitor Palbociclib, exhibiting half-maximal responses at nanomolar concentrations. Thus, RUNX1/RUNX1T1 drives cell cycle progression through the G1 phase by promoting the expression of both components of the CDK6–CCND2 kinase complex.This study provided several key findings. Firstly, the in vivo arm provided many more candidates than the in vitro assays, a result that was also described in a similar RNAi screen in glioma CCND3 mutations were found in MLL-rearranged AMLs, but not in t AML. Although RUNX1/RUNX1T1 occupies the loci of all three D-cyclin genes, RUNX1/RUNX1T1 knockdown reduced CCND1 and CCND2, but not CCND3 transcript levels CCND1 is at least tenfold lower expressed than CCND2 across AML subtypes and neither scored in RNAi screens nor compensated for loss of CCND2 in t cells Independent confirmation of the significance of CCND2 came from the Cancer Dependency Map project, where genome-wide RNAi screens identified CCND2 as an essential gene in the two 2 RUNX1/RUNX1T-expressing AML cell lines within a total of 501 cancer cell lines CDKN1A (p21) in a p53-independent fashion, thereby interfering with the CDK2-mediated progression from the G1 into the S phase of the cell cycle [CCND2 expression possibly contributing to it. However, CDKN1A has also been reported to support DNA repair and self-renewal of leukemia stem cells upon DNA damage It is also interesting to note that RUNX1/RUNX1T1 upregulates ll cycle ,68. It hCCND2 locus revealed that RUNX1/RUNX1T1 binds mainly to an element 30 kb upstream of CCND2’s transcriptional start site (TSS) [CCND2 expression by modulating a three-dimensional interaction between its promoter region and a silencing element. Interestingly, the histone acetyl transferase EP300 binds to the promoter region of CCND2. As EP300-mediated acetylation of lysines located at the N-terminus of RUNX1/RUNX1T1 can support its self-renewal function, it is tempting to speculate that this modification may be involved in the activation of CCND2 transcription, similar to what has been described for other upregulated genes such as ID1 Furthermore, the activation of direct target genes by RUNX1/RUNX1T1 seems to be surprising, given that the translocation replaces the RUNX1 transactivation domain by several repressor domains of RUNX1T1. However, detailed analysis of the impact of RUNX1/RUNX1T1 depletion on chromatin accessibility, histone modifications, and transcription factor occupation of the te (TSS) ,19. Hi-CCCND2 transcription indirectly via the JUN-FOS heterodimer AP-1. Knockdown of RUNX1/RUNX1T1 reduces expression of both FOS and JUN members and blocks JUND binding to the CCND2 promoter. In line with these findings, expression of transdominant negative FOS, which interferes with all AP-1 isoforms, reduces CCND2 transcript levels and severely impairs proliferation of the RUNX1/RUNX1T1-expressing AML cells CCND2, where loss of RUNX1/RUNX1T1 directly changes chromatin accessibility and the three-dimensional communication between promoter and distal elements, and impairs chromatin occupation by AP-1 members.In addition, RUNX1/RUNX1T1 also maintained CDK6 and CCND3 loci are direct target genes of MLL/AF4, the most frequent fusion protein found in infant leukemia, and several NUP98 fusion proteins drive CDK6 transcription by binding to its promoter [CDK6 expression is a common theme for leukemic fusion proteins  and the U.S. Food and Drug Administration (FDA) have approved palbociclib, ribociclib, and abemaciclib, three different compounds with similar activities and properties, for the treatment of breast cancer. In addition, nine trials testing palbociclib or ribociclib in acute leukemias are listed in the ClinicalTrials.gov database. This number does not consider trials such as the European Proof-of-Concept Therapeutic Stratification Trial (ESMART), in which leukemias are part of the tumour portfolio examined. Recent data suggest that all three inhibitors do not directly inhibit CDK4/6 activity but prevent the association of the CDKs with their CCND partners CDK4, CDK6, or CCND1 predict outcome, high levels ofCCND3 indicate worse outcome. The reason for this is currently unknown but may possibly reflect distinct substrate preferences of the different CDK–CCND complexes.However, high CCND2 expression is associated with a better clinical outcome in AML, suggesting that CCND2 may contribute to the relatively high chemosensitivity of t AML. Interestingly, although neither Because of their cytostatic action, CDK4/6 inhibitors will be ineffective as single agents and, thus, need to be applied as part of combination treatments with targeted compounds such as MTOR inhibitors or as part of a scheduled regimen with classic chemotherapeutics The authors declare no conflicts of interest.


During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5′-GAT-3′ sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation. GATA 3 functions as a pioneer factor during cellular reprogramming. Here the authors delineate nucleosome positioning relative to GATA3 binding motifs and describe the structure of a GATA3–nucleosome complex; providing insight into how a pioneer factor interacts with nucleosomes and catalyze their local remodelling to produce an accessible enhancer. The GATA family of proteins are zinc finger transcription factors that have established pioneering functions in multiple developmental pathways, including mammary epithelial cell differentiation5. GATA3 functions to establish new cell fates in cellular reprogramming systems8 including driving of a mesenchymal to epithelial transition in breast cancer cell lines10. In these processes, GATA3 is proposed to bind target DNA sequence wrapped tightly within a positioned nucleosome and to induce chromatin remodeling, probably by recruiting a nucleosome remodeler11.Formation of new enhancers to activate transcriptional programs downstream of the signaling events of development, cellular reprogramming, and response to the environment require penetration of the chromatin barrier. A special class of transcription factors, termed pioneer transcription factors, has the capacity to bind nucleosomal DNA and nucleate the formation of a new enhancer13. The consensus recognition motif derived for GATA3, WGATAR, is believed to be engaged by zinc finger 214. However, elegant structural studies of the GATA3 zinc fingers complexed with its target DNA revealed that the two zinc fingers bind to two neighboring 5′-GAT-3′ sequences located within the same major groove of the DNA15. Should this DNA-binding specificity be encountered on a nucleosome surface, one of the two zinc fingers would sterically clash with the histone core in the nucleosome. Therefore, the details by which the GATA3 zinc fingers bind their cognate recognition motif within a nucleosomal context remains poorly understood.GATA proteins use zinc finger motifs as DNA-binding domains11, we expressed GATA3 at physiologic levels in a GATA3-negative breast cancer cell line, tracking GATA3 binding across the genome by chromatin immunoprecipitation-sequencing (ChIP-seq) and chromatin accessibility using the assay for transposase-accessible chromatin using sequencing (ATAC-seq). We identified three scenarios for GATA3 interaction with the chromatin fiber  libraries , in ten different positions around SHLs 2 and 5  was purified as a recombinant protein . Specific bands corresponding to the GATA3znf–nucleosome complexes were observed with all nucleosomes containing the GATA consensus sequence in different nucleosomal locations  resembling the sequence used elsewhere in this work positioned near the dyad axis around SHL2 in vivo  complex by the GraFix method in the presence of paraformaldehyde , also as expected. In comparison across binding classes, both G1  and G3  have higher frequency of occurrence of 5′-GATNxGAT-3′ sites than G2  precisely as observed in the biochemical experiments, while there is less obvious spacing preference in the G1 category  or G3  . This spacing preference is less obvious, but still significant in G2  and G3 sites   of chromatin architectural proteins, such as linker histones, could potentially influence outcome of transcription factor/chromatin transactions23. The details of specific DNA sequence within recognition motifs and how these are displayed on the surface of a nucleosome are expected to influence the physical properties of transcription factor binding  and hence outcome27. Finally, chromatin and nucleosomes are not static structures in living cells30 and the dynamics of chromatin organization at individual loci likely influence the nature of transcription factor/DNA transactions. These possible influences on outcome at individual loci within the genome are certainly not mutually exclusive and some combination(s) of the above (or other) factors may be critical to the biology.The concept that productivity in nucleation of an enhancer by a pioneer transcription factor differs based on genomic location leads to questions about what distinguishes productive from non-productive loci. In theory, loci within the genome could be distinguished at multiple levels. Higher-order chromatin structure differs as the genome is partitioned by accessibility34, suggesting a potential link between binding site location and transcriptional outcome that is intertwined with the biochemical properties of chromatin remodelers. We also observed preferential positioning of GATA3 near the periphery of the nucleosome after its expression at non-productive loci. A wealth of literature supports the notion that DNA near the periphery of the nucleosome is more accessible for transcription factor binding than more central positions37.Here we have investigated specifics of GATA3-binding motif sequence and position relative to local nucleosome structure as possible determinants of outcome of the GATA3/chromatin transaction. We mapped nucleosome boundaries at high resolution within a set of GATA3 bound loci before and after introduction of the transcription factor. These analyses, using the consensus GATA3 motif WGATAR, revealed substantial differences in the relationship between orientation on the nucleosome surface and outcome. Surprisingly, we observe strong enrichment for GATA3 motifs at position SHL2 in productively remodeled sites. SHL2 is known to be a binding site for multiple chromatin remodelersznf–nucleosome complex. The biochemical experiments confirmed that the GATA3znf can recognize GATA motifs at positions SHL2 and SHL5, as we observed in the genomics data. The cryo-EM structure of the transcription factor/nucleosome complex with GATA3 positioned near the nucleosome periphery indicated that, in the nucleosome, the GATA3 zinc fingers bind the target 5′-GAT-3′ sequences located in consecutive major grooves separated by a minor groove at SHL5.5 and 6.5. This specific geometry avoids steric hindrance between GATA3znf and nucleosomal histones , but facing the histone surface, did not function as the GATA3znf binding site  were mixed in denaturing buffer , and the mixture was rotated at 4 °C for 1.5 h, followed by dialysis against refolding buffer . The resulting histone octamer was further purified by Superdex200  gel filtration chromatography.All human histones  were expressed and purified from bacteriaEscherichia coli cells and was purified for the cryo-EM analysis. The DNA sequences used in this study are derived from the Widom 601 sequence17 and native human genome sequence. The native human genomic region  was selected based on the Capture MNase-seq data . The SHL5e DNA fragment was amplified in 43, with the histone octamer and a DNA fragment. The reconstituted nucleosomes were further purified by native polyacrylamide gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad).The nucleosomes were reconstituted by the salt dialysis methodznf encoded in the pET15b vector was expressed and purified44. The GATA3znf encoded in the pGEX-6P-1 vector was expressed in the E. coli BL21 (DE3) codon plus RIL strain (Stratagene), with induction by Iisopropyl β-d-1-thiogalactopyranoside. After the cells were disrupted, the glutathione S-transferase (GST)-tagged GATA3znf was purified using Glutathione Sepharose 4B beads . The GST-tag was removed by a treatment with PreScission protease. After the GST-tag was removed, GATA3znf was purified by MonoS  column chromatography. GATA3znf was further purified by Superdex200  column chromatography with 20 mM Tris-HCl (pH 7.5) buffer, containing 300 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 1 μM ZnSO4. The purified GATA3znf was stored at −80 °C.The DNA fragment encoding human GATA3 261-371  was inserted into the pET15b and pGEX-6P-1 vectors. The GATA3znf in 6 μl of reaction buffer, containing 13.3 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6.7% glycerol, 1.3 mM dithiothreitol (DTT), 0.67 mM 2-mercaptoethanol, and 0.3 μM ZnSO4, at 25 °C for 30 min. After the incubation, the samples were analyzed by native-PAGE. The gel was stained with ethidium bromide and imaged with an LAS4000 image analyzer  or an Amersham Imager 680 QC .The purified nucleosome was incubated with GATA3znf (6.5 μM), in 240 μl of reaction buffer containing 13.3 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3.3% glycerol, 1.3 mM DTT, 0.67 mM 2-mercaptoethanol, and 0.3 μM ZnSO4. The reaction mixture was incubated for 2 h at 25 °C. After the incubation, the sample was purified and stabilized by the GraFix method45. A gradient solution was formed with buffer A  and buffer B , using a Gradient Master (BioComp). The GATA3znf-nucleosome sample was applied onto the top of the gradient solution, and was centrifuged at 89,800 × g  at 4 °C for 16 h, using an SW41 Ti rotor (Beckman Coulter). After the ultracentrifugation, fractions (0.6 ml each) were collected from the top of the gradient, and were analyzed by native-PAGE. The collected samples were desalted on a PD-10 column , equilibrated with 10 mM HEPES-NaOH (pH 7.5) buffer containing 1 mM DTT and 1 μM ZnSO4, and were concentrated with an Amicon Ultra centrifugal filter unit (Millipore).The SHL5e nucleosome (2.6 μM) was mixed with GATA3znf–nucleosome complex, 2 µl portions of the sample (0.8 µg DNA/µl) were applied to glow-discharged Quantifoil R1.2/1.3 200-mesh grids. The grids were blotted for 8 s under 100% humidity at 4 °C in a Vitrobot Mark IV chamber , and were immediately plunged into liquid ethane.For the cryo-EM specimen of the GATA3znf–nucleosome complex in the vitrified ice were collected using the EPU  auto acquisition software on a Krios G3i cryo-electron microscope , operated at 300 kV at a pixel size of 1.05 Å. Images were recorded with 64 s exposure times on a Falcon 3EC  direct electron detector in the electron counting mode, retaining a total of 51 frames with a total dose of ~50 electron/Å2. The two data collections produced 3,139 images and 2,471 images of the GATA3znf–nucleosome complex.Images of the GATA3znf–nucleosome complex were aligned using MOTIONCOR246 with dose weighting. The contrast transfer function (CTF) was estimated by CTFFIND447 from digital micrographs with dose weighting. All subsequent image processing of the GATA3znf–nucleosome complex was performed with RELION3.048. The particles of the GATA3znf–nucleosome complex were picked semi-automatically with a box-size of 180 × 180 pixels, and were processed by two-dimensional (2D) classification followed by 3D classification. The crystal structure of the nucleosome (PDB ID: 3LZ0) was low-pass-filtered to 60 Å, and was used as the initial model for the first round of classification for the first dataset. The 3D map, which contained the GATA3znf density on the nucleosome, was low-pass-filtered to 60 Å, and used as the initial model for the 3D classification for the second dataset. The 3D classes of the GATA3znf-nucleosome complex from the two datasets were combined, and 2D classification using 1,026,599 particles was performed to remove junk particles. A second round of 3D classification using the combined dataset containing 760,411 particles was performed, followed by particle polishing and two rounds of CTF refinement. The resolution of the refined 3D map of the GATA3znf-nucleosome was 3.15 Å, which was estimated by the “gold standard” Fourier Shell Correlation (FSC) at an FSC = 0.14349. The map of the GATA3znf-nucleosome was normalized with MAPMAN50 and visualized with UCSF Chimera51 and UCSF ChimeraX52. Detailed processing statistics values for the GATA3znf–nucleosome complex are listed in Supplementary Table All movie frames of the GATA311 by washing MDA-MB-231 cells with PBS(−) and treating with a hypotonic buffer  for 15 min on ice. After centrifugation, 4 U of MNase was added to nuclei and incubated for 4 min at 37 °C. Digested DNA fragments were purified using a spin column (Zymo). Mono-nucleosomal fragments were extracted by a QIAGEN gel extraction kit. As a control, the same amount of chromatin was sonicated by COVARIS and the similar length of DNA fragments were purified by the gel extraction kit. Purified (naked) MDA-MB-231 genomic DNAs were also digested by MNase in a same manner and used as another control. Sequencing libraries were prepared by NEXTflex Rapid DNA-Seq kit. Seven hundred and fifty nanograms from each library was used to enrich targeted GATA3 peaks following the instruction in the SureSelect kit (Agilent). Biotinylated RNA probes were hybridized with MNase-seq libraries and enriched by Streptavidin-coupled dynabeads (Thermo). Captured DNAs were further amplified by a 12-cycle PCR.MNase-seq libraries were prepared16. Each fragment midpoint was considered as dyad and used to calculate dyad frequency around the GATA3 motif . The dyad frequency was normalized by the midpoint frequency obtained from capture MNase-seq data in sonicated fragments and plotted on the graph (metaplot). For the heatmap view of motif enrichment analysis, only the fragment midpoints that were located within ± 75 bp from the closest GATA3 motifs were used. The colors were plotted on a log2 scale.The obtained sequence reads were mapped to hg19 genome by bowtie. All paired-end reads were converted to a single fragment, and fragment midpoint and endpoint were extracted for the metaplot analysis. Smoothed nucleosome signals were obtained by iNPS softwareRNA probes were obtained from Agilent . 309 GATA3 peak regions  were selected based on the mappability of the loci. Designed probes cover 1.5 kbp/peak centered on each GATA3 peak center.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Three-dimensional chromatin loop structures connect regulatory elements to their target genes in regions known as anchors. In complex plant genomes, such as maize, it has been proposed that loops span heterochromatic regions marked by higher repeat content, but little is known on their spatial organization and genome-wide occurrence in relation to transcriptional activity.Here, ultra-deep Hi-C sequencing of maize B73 leaf tissue was combined with gene expression and open chromatin sequencing for chromatin loop discovery and correlation with hierarchical topologically-associating domains (TADs) and transcriptional activity. A majority of all anchors are shared between multiple loops from previous public maize high-resolution interactome datasets, suggesting a highly dynamic environment, with a conserved set of anchors involved in multiple interaction networks. Chromatin loop interiors are marked by higher repeat contents than the anchors flanking them. A small fraction of high-resolution interaction anchors, fully embedded in larger chromatin loops, co-locate with active genes and putative protein-binding sites. Combinatorial analyses indicate that all anchors studied here co-locate with at least 81.5% of expressed genes and 74% of open chromatin regions. Approximately 38% of all Hi-C chromatin loops are fully embedded within hierarchical TAD-like domains, while the remaining ones share anchors with domain boundaries or with distinct domains. Those various loop types exhibit specific patterns of overlap for open chromatin regions and expressed genes, but no apparent pattern of gene expression. In addition, up to 63% of all unique variants derived from a prior public maize eQTL dataset overlap with Hi-C loop anchors. Anchor annotation suggests that < 7% of all loops detected here are potentially devoid of any genes or regulatory elements. The overall organization of chromatin loop anchors in the maize genome suggest a loop modeling system hypothesized to resemble phase separation of repeat-rich regions.Sets of conserved chromatin loop anchors mapping to hierarchical domains contains core structural components of the gene expression machinery in maize. The data presented here will be a useful reference to further investigate their function in regard to the formation of transcriptional complexes and the regulation of transcriptional activity in the maize genome.The online version contains supplementary material available at 10.1186/s12864-020-07324-0. Genomic DNA, the largest molecule in a cell, is packed with histone to form chromatin. Recent improvements in the molecular characterization of chromatin have shown that its spatial structure can be dissected into separate functional domains, ranging in sizes from a few Kbps to Mbps –3. ThoseIn plants, the understanding of chromatin organization is mainly derived from a relatively small number of studies in species that include Arabidopsis , rice 88 and maiWhile long-range loop formation between chromatin domains has been shown to link together gene-rich and distal regulatory regions in complex plant genomes, no study yet has been performed to determine the extent of such mechanism and its genome-wide correlation to TAD-like domains and gene transcription in maize. In addition, while Hi-C is known as a “low-resolution” method capturing mainly long-range chromatin loops , its relWhole maize B73 leaf tissue was collected at development stage v04. The same batch of plants, at the same stage (v04) and divided into four biological replicates (four plants per replicate) was used for gene expression profiling , three-dimensional chromatin profiling  and accessible chromatin .The ultra-deep sequencing of two Hi-C biological replicates led to a total number of 3,435,596,872 and 3,424,795,714 raw paired reads, for replicates 1 and 2, respectively. Filtering of the raw data and mapping to the B73 AGPv4 reference genome sequence led to the detection of 392,396,275 and 449,828,472 Hi-C contact pairs, after combining inter-chromosomal and intra-chromosomal contacts for replicates 1 and 2, respectively , rather than individual loop anchors, were analyzed and counted, depending on whether loops were fully embedded within a hierarchical TAD-like domain, or whether one or both loop anchors were located within 2Kbps flanking domain boundaries. Results are shown in Table Approximately 37% of all loops detected from Hi-C replicates 1 and 2 either co-located with multiple Level 0 domains  or did not overlap with any domain. At least 60% of the remaining loops as shown on Tables Sequence coverage peaks from ATAC-Seq libraries are generally seen as a proxy for transcription factor binding sites and gene regulatory elements in genomic DNA . SequencCo-location analysis of ATAC-Seq NF peaks with chromatin loops initially was performed using whole replicate 1 and 2 Hi-C loop datasets and assessed, based on the following criteria. As many chromatin loop anchors were shared between multiple loops, in a significant number of cases, a peak could align to an anchor for one loop and a loop interior for another loop. Therefore, peak overlaps to loop regions were determined first based on their potential overlap to at least one loop anchor. If no overlap was detected, peaks were assessed based on their potential overlaps to loop interiors, then to genomic regions located outside of chromatin loops. Using this approach, up to 13,026 peaks, out of 19,532, overlapped primarily with anchors, while up to 4649 peaks overlapped primarily with loop interiors  in B73 leaf whole tissue  were alTo assess whether ultra-deep sequencing of Hi-C libraries captured chromatin loops carrying distinct functions, loops mapping to hierarchical TAD-like domains  showed that, for 71% of anchors harboring genes, at least one of those genes was expressed  co-locate with a conserved set of chromatin loop anchors. Chromatin loops co-locate with hierarchical TAD-like domains and sub-domains, where chromatin interactions may occur in a series of nested hierarchical sub-domains contained within larger compartment domains. Based on their co-location, loops can be divided into specific categories, each reflecting apparent variable overlap frequencies with open chromatin regions, expressed genes and public eQTL associated traits. This suggests the possibility of distinct loop types, and, possibly, distinct molecular functions, although no apparent differences in gene expression was observed between genes mapping to various loop types and regions. A vast majority of the chromatin loops detected via Hi-C share anchors with other loops, including high-resolution interactions detected in the same genotype (B73), but with more accurate techniques (HiChIP and ChIA-PET). A subset of these anchors overlaps with one or more expressed genes and open chromatin regions. Many are shared between multiple loops, including some that are fully embedded within larger ones. Taken together, these data indicate that loop formation may be linked to gene transcription and regulation through highly dynamic three-dimensional mechanisms reminiscent of enhancer/promoter interaction complexes described elsewhere  and invoInterestingly, while most of the detected active genes and open chromatin regions co-locate with chromatin loop anchors, a large portion of anchors, conversely, do not overlap with any genes or ATAC-seq peaks. It is likely that, due to technical constraints, the 19,532 consensus NF peaks used here represent a conservative estimate of the total peak counts. Therefore, many of the “empty” loop anchors listed here may harbor putative open chromatin regions that were undetected in the present study.When combined with the observation that repeat content is denser in loop interiors than in the flanking loop anchors, the results shown here are in line with a phase separation model of chromatin dynamics in maize . In thisThe results shown here indicate that the scope of the functional maize genome may be narrowed down to a restricted number of loop anchors. Data suggest that these regions anchor regulatory complexes interacting with a majority of those genes being expressed in maize, through three-dimensional interactions hypothesized to be consistent with a phase separation model of chromatin dynamics. While further investigation will be required to confirm this model, the various sequencing datasets generated for this study will facilitate the systematic discovery of new motifs, such as enhancers and eQTL variants, making them prime candidates for future functional genomics studies through their direct association with the transcription of specific gene sets. In addition, the current datasets may be used to facilitate prediction modeling for breeding and genomic selection applications, through the ranking of specific variants in loop anchors and of their predicted functional impact in genotypes of interest.Whole maize B73 v04 leaf tissue, grown in a greenhouse, was collected and stored at − 80 °C after grinding. Plants were divided into four biological replicates (four plants per replicate). The same replicates were used for gene expression profiling (RNA-Seq), accessible chromatin (ATAC-Seq), and three-dimensional chromatin profiling (Hi-C).Grinded tissue from two biological replicates (replicates 1 and 2), kept at − 80 °C and each containing v04 leaves from four plants, were used for preparing two Hi-C replicate libraries. Libraries were constructed with the Dovetail Hi-C Library preparation Kit (Dovetail), according to the manufacturer’s instructions, including protocol modifications recommended by Dovetail for plant tissue. Since tissues had previously been grinded (in liquid nitrogen) before long-term storage at − 80 °C, 2 ml 1X PBS and 81 μl 37% formaldehyde were added directly to 250 mg of frozen and grinded tissue in a 15-ml tube then incubated at room temperature for 15 min, prior to completing the protocol.Total RNA was extracted from grinded v04 leaf tissue previously stored at − 80 °C using the RNeasy 96 kit (Qiagen). Four biological replicates, each containing v04 leaves from four plants, were used for total RNA extraction and subsequent poly(A) RNA library construction. Poly (A) RNA-Seq libraries were built using the TruSeq stranded mRNA library preparation kit (Illumina) according to the manufacturer’s instructions.Grinded tissue from three biological replicates, kept at − 80 °C and each containing v04 leaves from four plants, were used for nuclei extraction and ATAC-Seq library preparation. Prior to library construction, 1 L of 1X Nuclei Isolation Buffer (NIB) solution  was prepared and sterilized by filtration. Approximately 1 g of frozen leaf tissue was mixed with 25 ml NIBM (0.001% beta-mercapto ethanol in 1X NIB) and incubated on ice for 15 min. After filtering through a 100 μm filter, the pellet was washed with 15 ml NIBM and filtered again with a 40 μm filter. 2 ml of NIBT (10% Triton X-100 in 1X NIB) were added to the 40 ml solution and incubated on ice for 15 min. After centrifugation at 2400 g for 15 min at 6 °C, the pellet was resuspended on ice and 25-35 ml of 1X NIB were added. After another centrifugation at 2400 g for 15 min at 6 °C, the supernatant was removed, and the nuclei pellet was kept on ice. Nuclei were counted after mixing 20 μl Trypan Blue with 10 μl 1X NIB and 10 μl nuclei suspension and loading 10 μl of the resulting mix on a hemocytometer. After counting, 40,000 nuclei were mixed with 1X Tagment DNA (TD) Buffer (Illumina) and 2.5 μl Tagment DNA Enzyme 1 (TDE1) (Illumina) in a 50 μl reaction. After incubation at 37 °C for 30 min, mixing up and down a few times, the tagmented DNA was cleaned-up in 15 μl EB buffer with a MinElute PCR Purification Kit column (Qiagen) and stored at − 20 °C. PCR reaction then was performed after mixing 15 μl of tagmented DNA with 2.5 μl each of 25 μM i5 (AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC) and i7 (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG) PCR primers (where N indicates the presence of an 8-bp barcode) in 1X Q5 HotStart Master Mix (NEB), and incubating 5 min at 72 °C, 30s at 98 °C, followed by 5 cycles of: 10s at 98 °C; 30s at 63 °C; 1 min at 72 °C, and hold at 4 °C. After this initial PCR, 5 μl of amplified DNA was quantified via qPCR (using the same PCR primers) to determine the optimum number of PCR cycles for the remaining 45 μl before over-amplifying the sample (characterized by ¼ of the maximum fluorescent intensity on the qPCR plot). Typically, PCR reactions were completed after adding 6–8 amplification cycles. The DNA then was cleaned-up with a MinElute column and resuspended in 15 μl EB buffer (Qiagen).Sequencing of the two Hi-C biological replicates (replicates 1 and 2) was performed on Illumina HiSeq 2500 and NovaSeq 6000 systems, at 2x150bps. Filtering of the raw read data and mapping to the B73 AGPv4 reference genome sequence were performed using the Juicer package . FrequenFor repeat density analysis, repeat elements for the B73 AGPv4 reference genome sequence were obtained from MaizeGDB. The manner in which these elements overlap the loops predicted from the replicates 1 and 2 data sets was examined. Each loop was treated as the union of two anchor regions and an interior region. These three components were compared to the set of repeat elements using “bedtools -coverage”  which reFor eQTL analysis, 61,188 eQTL records  provideGramene gene models for build 4.0 of B73 were obtained from the MaizeGDB repository, specifically from the GFF3 file Zm-B73-REFERENCE-GRAMENE-4.0_Zm00001d.2.gff3. Feature types “gene,” “lincRNA_gene,” “miRNA_gene,” and “tRNA_gene” were selected, yielding 44,474 genes. Of these, 38,847 were reported to be associated with chromosomes 1–10 and represent the set used for genic analyses.RNA-Seq libraries derived from all four biological replicates were sequenced on an Illumina HiSeq 2500 system at 1x50bps (single 3′ end sequenced), targeting ~20MM single reads per replicate. Read sequences were trimmed based on quality scores and those matching B73 mitochondrial, chloroplast, and rRNA sequences were filtered with FACS  pseudoalSequencing of three ATAC-Seq biological replicates was performed on an Illumina NovaSeq 6000 system at 2x50bps. After removing contaminant adapter and organellar sequences, reads were mapped to the AGPv4 reference genome. After filtering for proper read pair alignments and removing duplicated reads, resulting BAM files were separated based on the distance between read pairs into “NF” (“nucleosome-free”) regions and “MN” (“multi-nucleosome”) regions. Reads mapping to specific regions known to exhibit high coverage depth  were removed using “bedtools -intersect” . NF and ATAC-Seq NF consensus peaks were classified in relation to their proximity to a particular expressing gene. The location of each expressing gene was compared to the set of 19,532 NF consensus peaks on chromosomes 1–10. For each, the closest peak and its distance in bp was determined using “bedtools -closest” [Peaks were mapped to chromatin loops using BEDTools . PrioritAdditional file 1. Hi-C replicate 1 TAD-like hierarchical domain coordinates. (seqid) Chromosomal assignment for domain and sub-domain; (start0) (end0) Coordinates (in bps) of start and end, respectively, of domain or sub-domain; (level) TAD-like domains are denoted as level 0, while sub-domains are denoted as level 1 or higher.Additional file 2. Hi-C replicate 2 TAD-like hierarchical domain coordinates. Categories are the same as in Additional File Additional file 3. Hi-C replicate 1 chromatin loop coordinates. Coordinates (in bps) and chromosome assignments are shown for both anchors forming a loop. (chr1) Chromosome assignment of first anchor;  Coordinates in bps of first anchor; (chr2) Chromosome assignment of second anchor;  Coordinates in bp of second anchor. All coordinates are against the maize B73 AGPv4 assembly.Additional file 4. Hi-C replicate 2 chromatin loop coordinates. Coordinates (in bps) and chromosome assignments are shown for both anchors forming a loop. (chr1) Chromosome assignment of first anchor;  Coordinates in bps of first anchor; (chr2) Chromosome assignment of second anchor;  Coordinates in bp of second anchor. All coordinates are against the maize B73 AGPv4 assembly.Additional file 5. ATAC-Seq libraries. Sequencing yields and peak counts are shown for all three biological replicates. All reads were aligned against the maize B73 AGPv4 assembly. Consensus peaks (NF) were used for the study.Additional file 6. ATAC-Seq consensus NF peak coordinates. Coordinates (in bps) and chromosome assignments are shown for all 19,532 consensus NF peaks. (chr) Chromosome assignment;  Coordinates in bps of consensus NF peak borders. All coordinates are against the maize B73 AGPv4 assembly. Additional file 7. gene expression data. RNA-Seq were aligned to the maize AGPv4 reference genome and assigned to Gramene gene models for build 4.0 of B73 from the MaizeGDB repository. Genome coordinates and TPM counts for each gene transcripts are shown.Additional file 8. overlap analysis of Hi-C replicate 1 and 2 chromatin loop anchors against expressed genes and ATAC-Seq consensus peaks. See Additional File Additional file 9. replicate 1 chromatin loop anchor annotation and counts for each feature listed below. (seqid) Chromosomal assignment for loop anchor; (start) (end) Coordinates (in bps) of start and end, respectively, of chromatin loop anchor; (locus) name and loop assignment of anchor; (bare) presence or absence of any of the features listed below on anchor . The following features are annotated: (ef) (uf) expressed (ef) or unexpressed (uf) gene with nearest open chromatin peak >2Kbps away from gene; (en3) (un3) expressed (en3) or unexpressed (un3) gene with nearest open chromatin peak <2Kbps downstream from gene; (en5) (un5) expressed (en5) or unexpressed (un5) gene with nearest open chromatin peak <2Kbps upstream from gene; (eo) (uo) expressed (eo) or unexpressed (uo) gene with nearest open chromatin peak overlapping with gene; (mp) open chromatin peak overlapping an anchor; (mpfe) open chromatin peak overlapping an anchor but with the nearest expressed gene >2Kbps away from the peak.


In higher eukaryotes, enhancers determine the activation of developmental gene transcription in specific cell types and stages of embryogenesis. Enhancers transform the signals produced by various transcription factors within a given cell, activating the transcription of the targeted genes. Often, developmental genes can be associated with dozens of enhancers, some of which are located at large distances from the promoters that they regulate. Currently, the mechanisms underlying specific distance interactions between enhancers and promoters remain poorly understood. This review briefly describes the properties of enhancers and discusses the mechanisms of distance interactions and potential proteins involved in this process. In higher eukaryotes, the regulation of gene expression is complicated as a consequence of cell differentiation during embryonic development ,2,3. CelEnhancers were first described nearly 40 years ago when 72-bp tandem sequences from the SV40 virus were found to enhance gene expression when placed at large distances from the promoter and in any orientation relative to the regulated gene . Two yeaEnhancers are regions of DNA, typically 100 to 1000 bp in size, that contain TF-binding sites that stimulate the initiation and elongation of transcription from promoters ,9,10. InDuring transcriptional activation, enhancers are usually located in close proximity to gene promoters ,11. HereEnhancers consist of combinations of short, degenerate sites, 6–12 bp in length, that are recognized by DNA-binding TFs, which determine enhancer activity . The comRecently, super-enhancers have been identified, representing a special class of regulatory elements, characterized by large sizes, sometimes reaching tens of thousands of bp, with a high degree of TF and co-activator enrichment ,13. SupeDuring the activation of transcription , enhanceThe H3K27ac and H3K4me1 modifications of histone H3 are thought to reduce the stability of nucleosomes . PRC1 anA highly conserved mediator complex, consisting of 25 subunits in yeast and 30 subunits in humans, plays a primary role in the enhancer-mediated activation of transcription . The subDrosophila genome, approximately 20% of enhancers are located at a distance of 50–100 kb from promoters associated with them, and often between them there can be other genes that have their own regulatory systems . T. T90]. TDrosophila expresses two homologs of the YY1 protein, PHO and PHOL, which are involved in the recruitment of Polycomb proteins . This study used a 660-bp PRE, found in the regulatory bxd region of the homeotic Ubx gene of the bithorax locus (bxd PRE). Typically, pairing two PRE-containing transgenes results in the increased repression of the reporter, which is associated with an increase in the recruitment efficiency of PcG complexes [gypsy insulator resulted in the significant repression of reporter genes [gypsy insulators can facilitate functional interaction between the enhancer and the promoter located in different transgenes inserted into the genome at distances reaching 1–2 megabases [The first work on this topic  tested tomplexes . The comer genes , despiteegabases . bithorax complex. The Mcp boundary separates the regulatory domains of the homeotic abd-A and Abd-B genes and consists of an insulator, which is flanked on both sides by Polycomb-dependent silencers [Mcp were inserted in different regions of the third chromosome. The results indicated that combinations of transgenes located at a distance of several megabases cause an increase of marker gene repression that assume the physically interaction between them. In another work, the bxd PRE, in combination with the 210-bp core of the Mcp insulator, was able to support repression between two transgenes located at super-long-distances [Mcp insulators for both tested transgenes [A similar study was performed with the insulators from the ilencers . Transgeistances . The co-ansgenes ,131,132.Fab-7 boundary, which separates the domains of the Abd-B regulatory region in the bithorax complex [Fab-7 boundary that included insulator and an adjacent PRE located on X-chromosome functionally interacted with endogenous Fab-7 [Fab-7 insulators was also able to support super-long-distance interactions between transgenes in the Drosophila genome [Super-long-distance interactions can be also be maintained by the  complex . A 3.6-kus Fab-7 . The paia genome .Neighbor of Homie (Nhomie) and Homing insulator at eve (Homie), were identified at the 16-kb boundaries of the regulatory region of the even-skipped (eve) gene [Nhomie and Homie interact with each other and can also maintain super-long-distance interactions between the transgene and the endogenous eve locus, which allows endogenous enhancers to activate the reporter gene promoter in the transgene [Two insulators, ve) gene ,135,136.P-transposon containing either an insulator, or a promoter region, is inserted with high frequency into the genomic region where this regulatory element is located. For example, the P-transposon that contains the promoter region of engrailed or linotte was predominantly inserted (20–30%) into the corresponding genes [P-transposon containing the Fub boundary, which organizes TADs that separate the regulatory regions of the Ubx and abd-A genes in the bithorax complex [P-transposon and directs the integration of the P transposon into the region of the corresponding gene.The interaction specificity between identical regulatory elements is also manifested in the “homing” phenomenon, in which the ng genes ,138. Sim complex . The “hogypsy insulator consists of 12 Su(Hw) binding sites [gypsy retrotransposon is found only in heterochromatin [gypsy insulator is unique due to a large number of Su(Hw) binding sites. According to ModEncode, the homie insulator contains binding sites for the Drosophila homolog of CTCF (dCTCF), Su(Hw), and GAF proteins [Mcp boundary contains binding sites for dCTCF and Pita [Fub boundary contains binding sites for CTCF, Su(Hw), and Pita [Fab-7 boundary consists of three DNase 1 hypersensitivity sites (HS) [In all of these examples, super-long-distance interactions are supported by a pair of identical regulatory elements that are unique to the genome. The ng sites ,141,142.hromatin . On the hromatin . Thus, tproteins . The Mcpand Pita . The Fuband Pita ,146,147.and Pita ,149,150.tes (HS) ,152,153.tes (HS) , whereastes (HS) . LBC is tes (HS) ,155. In Drosophila proteins containing clusters of C2H2 domains (ZAD-C2H2) [in vivo, showed that the presence of ZADs determined the ability of these proteins to support distance interactions [In transgenic model systems, the pairing between two copies of four or five binding sites of dCTCF, Su(Hw), or Pita was able to bring the yeast GAL4-dependent activator region and the reporter gene promoter in close proximity, resulting in the transcription of the reporter gene ,157,158.AD-C2H2) ,161. An AD-C2H2) ,164,165.ractions ,158. Thugypsy insulator [gypsy insulators. Most likely, CP190 and Mod(mdg4) are involved in stabilizing pre-formed specific distance interactions. The C2H2 proteins, including Pita, dCTCF, and Su(Hw), interact with BTB-containing proteins , such as Mod(mdg4) and CP190 ,167,168.nsulator ,177,178.nsulator ,180,181,According available data the well described C2H2 proteins bind predominantly to promoter and insulator/boundary elements ,165,181.Drosophila CTCF protein is not key in the formation of TADs and does not have intense colocolization with the cohesin complex on chromatin [Drosophila are predominantly located in the regions of housekeeping gene clusters, which are actively transcribed.It is now believed that the formation of chromatin architecture in mammals and Drosophila occurs using different mechanisms. This conclusion is based on the observation that the hromatin ,183,184.Drosophila nerve cells [Drosophila, the cohesin complex is predominantly located in the region of active promoters and enhancers, which is consistent with its potential role in the organization of TADs and the formation of interactions between enhancers and promoters [Nipped-B gene that regulates the binding of the cohesin complex to chromatin affect the distance interactions between enhancer and promoter at the cut locus [Drosophila homolog called Chip, which is also involved in maintaining distant enhancer—promoter interactions [Drosophila than it currently seems. Probably in the near future it will be possible to create a unified model of distance interactions in higher eukaryotes. However, it has recently been shown that dCTCF is located at the TAD boundaries in ve cells . In Drosromoters ,187. Mutut locus . The mamractions ,190. A dractions , which rractions . Thus, tDrosophila C2H2 proteins will facilitate the assessment of the true contributions of this class of proteins to the organization of the chromosomal architecture. In addition, the use of gene editing methods, such as the CRISPR/Cas9, will make it possible to assess the role of each identified C2H2 protein in maintaining distance interactions using model regulatory systems. There is more and more experimental evidence that in mammals other C2H2 proteins can participate in the formation of distance interactions and interact with CTCF in this process. The identification of binding sites for currently uncharacterized human and


HMX1 are linked to an ocular defect termed Oculo-auricular syndrome of Schorderet-Munier-Franceschetti (MIM #612109). We identified UHRF1 as a target of HMX1 during development. UHRF1 and its partner proteins actively regulate chromatin modifications and cellular proliferation. Luciferase assays and in situ hybridization analyses showed that HMX1 exerts a transcriptional inhibitory effect on UHRF1 and a modification of its expression pattern. Overexpression of hmx1 in hsp70-hmx1 zebrafish increased uhrf1 expression in the cranial region, while mutations in the hmx1 dimerization domains reduced uhrf1 expression. Moreover, the expression level of uhrf1 and its partner dnmt1 was increased in the eye field in response to hmx1 overexpression. These results indicate that hmx1 regulates uhrf1 expression and, potentially through regulating the expression of factors involved in DNA methylation, contribute to the development of the craniofacial region of zebrafish.H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development as it is widely expressed in the eye, peripheral ganglia and branchial arches. Mutations in  Common to vertebrates, during early embryonic development, the neural crest cells, a transient migratory cell type, migrate from the dorsal neural tube to different regions of the body and contribute to the shaping of the developing embryo . CranialHMX1). HMX1 belonging to a homeobox (HMX) family of transcription factors containing HMX2 and HMX3, presents a phylogenetically conserved 60-amino acid homeobox domain [The development of the mammalian eye also requires the participation of two other embryonic tissue sources. The neural ectoderm gives rise to the neural retina, the pigmented epithelium and the optic stalk while cornea and lens originate from the surface ectoderm . MorphogHmx1 is involved in the development of sensory neurons; it is highly expressed in the neural retina and peripheral ganglia as well as in the branchial arches [hmx1 is broadly expressed at 10-somite stage in the eye field, and from 24 hours post fertilization (hpf) is gradually restricted to the nasal side of the retina, before being limited to the nasal part of the inner nuclear layer. In the developing lens, hmx1 is transiently expressed within a time window ranging from 24 to 72 hpf [In mouse, l arches ; moreovel arches . In zebro 72 hpf .HMX1 as well as the other eye field transcription factors are not yet fully understood and altered molecular signaling during early eye development may result in ocular malformation [HMX1 [The molecular mechanisms regulating ormation . Schordeon [HMX1 . Microphon [HMX1 –14.dmbo” and “misplaced ears” exhibiting microphthalmia and ear and cranial malformations were reported. “Dmbo” mice showed malformations of the squamous temporal bone and hyperplasia of the gonial bone and failed to develop somatosensory neurons in the geniculate ganglion [hmx1 deficient embryos exhibited increased apoptosis in eye and brain in addition to a delayed withdrawal of retinal progenitors from cell cycle [Two mutant mouse models called “ganglion , 15. In ll cycle .HMX1 in transcriptional regulation. Amendt et al. showed that HMX1 preferentially binds to a consensus sequence 5’-CAAGTG-3’ and acts as a transcriptional regulator [HMX1 potential target genes [UHRF1).Despite the advances allowed by the use of several animal models, still not much is known about the role of egulator . Boullinet genes  and idenUHRF1, which contains a methyl DNA binding domain (SRA) binds preferentially to hemimethylated CpG sites and collaborates with DNA methyltransferase proteins DNMTs [uhrf1 and its partner dnmt1 is initiated at early embryonic stages where it is guiding the pre-gastrula development [uhrf1 is involved in both the regulation of intestinal development and the hepatic growth in zebrafish embryos [uhrf1 and dnmt1 showed altered lens development and maintenance [Uhrf1 conditional knockout mice exhibit shortened long bones as a result of altered chondrocyte differentiation and proliferation [DNA methylation is essential for epigenetic modulation of gene expression during mammalian development and has to be kept during cell division. The DNA daughter strand, after replication is methylated in accordance with the hemimethylated parent strand . UHRF1, ns DNMTs . Changesns DNMTs . Uhrf1 helopment , while a embryos , 23. Zebntenance , while lferation .UHRF1 human promoter region and showed that HMX1 inhibits UHRF1 expression in in vitro experiments. In situ hybridization in hmx1 transgenic and mutant zebrafish showed that uhrf1 expression is modulated in the hindbrain, eye region and branchial arches. Moreover, changes in uhrf1 and dnmt1 transcript levels in the eye field in heat shock transgenic zebrafish, indicate a possible involvement of hmx1 in regulating essential factors involved in DNA methylation.In this study we identified UHRF1 carrying all the HMX1-binding sites identified was subcloned into the luciferase reporter pGL3-basic vector  to produce the pGL3-Uhrf1 reporter construct for transfection in mammalian cells. The following primers were used: UHRF1-F 5’-TGCGAACGGACTTGGACTTA-3’ and UHRF1-R 5-CCTCCAAACCCTGGACCCT-3’. Human HMX1 and mutant HMX1 carrying deletions of the conserved domains SD1, SD2 and homeodomain were previously generated [A 1000-bp DNA amplicon around the transcription start site (TSS) of enerated . All DNAHuman embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose with stable glutamine , supplemented with 10% FBS , 100 U/ml penicillin and 100 μ/ml streptomycin . Transfection occurred at approximately 50% confluence using the Calcium Phosphate method . The DNA ratio between pGL3 and pcDNA3 was held constant across transfections, which were repeated a minimum of 5 times.48 hours post transfection the cells were harvest and firefly luciferase activity was measured using the DualGlo Luciferase Assay system  on the Multiplate Reader Synergy HT  with KC4 software. The β-gal reporter under the control of a CMV promoter was used for normalization and transfections with stable β-gal values between the different conditions were considered.Average of the different experiments was expressed as mean ± sem.; either Student’s t-test, or Anova one way were used to express significance of difference between two groups. Significance was set at 0.05.All animal procedures were carried out in accordance with the policies established by the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals and were approved by the Veterinary Service of the State of Valais (Switzerland). Zebrafish (Danio rerio) were maintained in a 14/10-h light/dark cycle; embryos were kept at 28.5°C in E3 medium . All embin situ hybridization underwent eye dissection procedure for RNA extraction and qRT-PCR analysis. Heat shock activation was confirmed by RT-PCR.The Tg(Hsp70-HMX1) transgenic line was previously generated and reported . Embryoshmx1 mutant was generated using zinc-finger nuclease (ZFN)-mediated mutagenesis. We identified zinc-finger nuclease (ZFN) target sites that were exclusively comprised within exon 2 of Hmx1 (NM_001113526.1). Several zinc-finger arrays were designed against the target sites; cloning and ZFN mRNA synthesis followed standard protocols [Hmx1mut10 and Hmx1mut150 were selected and separately raised to generate stable independent mutant lines. All experiments were carried with wildtype and mutant zebrafish collected at F6 and F7 generations.Zebrafish rotocols ; mRNA wauhrf1 (NM_213077.1) with the following primers: uhrf1-F 5’-AGGATGGTCACACCATATTTG-3’ and uhrf1-R 5’-AGTTCACCATCACAATCATTCC-3’. In vitro transcription with the Roche RNA Labeling Kits  was performed according to the manufacturer’s protocol. One-color whole-mount in situ hybridization was performed according to standard protocols [DIG-Labeled RNA probes were synthetized for rotocols . Washinguhrf1 expression, wildtype and hmx1 knockout embryos at 24 hpf and 5 dpf were euthanized. For whole body transcript analysis at 24 hpf and 5 dpf, 60 embryos obtained from different breeding were pooled together. For eye transcript analysis, both eyes were isolated from 20 embryos at 5 dpf and pooled together. All experiments were repeated three times.For the analysis of ™ Multiple Temperature Reverse Transcriptase kit  according the manufacturer’s protocol. cDNA was generated  and Real time PCR (FastStart SYBR Green Master Roche) was performed using uhrf1-F 5’-TCCAGGAGTCCAAGAGAGGAA-3’ and uhrf1-R 5’-TCTGCTGAACACAGTTCGGG-3’; dnmt1-F 5’-TTACTTTGGGCAAGAGGAGAGC-3’, dnmt1-R 5’-GACACCACACCGTTGTCTCT-3’; hmx1-F 5’-CGAAACCTCCAGGAG TCCAAG-3’, hmx1-R 5’-CGGGTCTTTTTCTTTCGGGC-3’.First-strand cDNA synthesis was performed using the AffinityScript–ΔΔCt method; relative values were normalized with β-actin.Gene expression change was determined using the 2UHRF1 promoter region using the Eukaryotic Promoter Database (https://epd.epfl.ch). In human, mouse and zebrafish genomes, inspection of a region ranging in size from −1500 to +100 nucleotides (nt) around the TSS showed the presence of both the canonical sequence 5’-CAAGTG-3’ and the minimal core motif 5’-CAAG-3’ with a lower affinity. The human region of interest contained one CAAGTG and three minimal CAAG motifs. In mouse, we observed two CAAGTG motifs and seven CAAG motifs, while in zebrafish one CAAGTG and three CAAG motifs were observed  and two domains SD1 and SD2, located 3´ to the HD [UHRF1 transcriptional activity as observed in luciferase assays  and SD1 were implicated in the dimerization of HMX1. Mutations that removed the dimerization domains of Hmx1 inhibited Hmx1 activity on potential targets [hmx1. Several mutations were screened and two mutants were selected. Hmx1mut150 zebrafish carried a deletion of 150 nucleotides, replaced with an insertion of 21 base pairs while hmx1mut10 presented a deletion of 10 base pairs. The deletions were confirmed by electrophoresis and sequencing analysis. Using ExPasy translation tool, we verified that the mutations disrupted the encoding sequence of the homeodomain and the downstream region including SD1 and SD2 domains in exon2 . targets . We usedin exon2 . The HmxHmx1mut10 and Hmx1mut150, we performed a morphometric analysis of the developing eye. Three parameters were considered: diameter of the eye, lens size and its ratio. By tracking wildtype eye growth, starting at 1 dpf and reaching 3 dpf stage, we observed a continuous growth in size  is caused by defects associated with mutations in the n factor . Hmx1 haelopment  as well elopment , 31. Whias Soho1 .in situ hybridization revealed a transient hmx1 expression in the developing lens between 24 and 72 hpf. Using a morpholino-based inhibition of hmx1 translation, Boisset et al. observed microphthalmia and delayed retinogenesis at 3 dpf. At 5 dpf, retinal and lens formation recovered but microphthalmia persisted [hmx4 expression causes severe neural defects ranging from failure of the neural tube closure to the narrowing of the eye field [Hmx1 binding sequence 5’-CAAGTG-3’ and the developed predictive promoter model are important tools to identify potential downstream targets and exploring the morphogenetic aspects in which Hmx1 could be involved.In zebrafish, ersisted . Morpholye field . The disUhrf1 expression is crucial for proper embryonic development as during oocyte growth stage Uhrf1 contributes to CG methylation [uhrf1 is highly expressed in proliferative tissues including the tectum, retina and branchial arches. At 4 and 5 dpf, uhrf1 persists in liver bud and gut [Uhrf1 deficiency is reported to alter the epigenetic pattern of several target genes and consequently induce transcriptomic changes resulting in developmental abnormalities. Deletion of maternal Uhrf1 causes developmental defects in preimplantation embryos [uhrf1 expression results in embryonic lethality [uhrf1 and dnmt1 presented defects in lens formation and microphthalmia [Uhrf1 deficiency in mouse model increased the expression of several genes including Hspb1, a gene involved in chondrocyte differentiation [hylation  while be and gut . Uhrf1 d embryos , while aethality . Knockouhthalmia , while cntiation  and suppntiation .HMX1 as part of a complex gene network regulating maxilla and mandibles formation. As a consequence of a perturbed HMX1 activity short mandibular rami, asymmetry of the jaws and altered premaxilla were observed.Recently, the phenotype of OAS has been extended by the description of additional patients , 13. TheHmx1 and Uhrf1 presented similar altered phenotypes, suggesting that a functional interaction might be established between the two genes; we therefore investigated the relationship between HMX1 and UHRF1  and sense (B-D) probes. Uhrf1 expression was detected with the sense probe in the cranial region, eye and gut of wildtype zebrafish at 5 dpf. (B) uhrf1 expression at 5 dpf in wildtype and hmx1mut10 zebrafish in the cranial region. (C) uhrf1 expression at 5 dpf in wildtype and hmx1mut150 zebrafish in cranial and ventral regions. Bar, A 100 μm. White arrow head; gut.(A) (TIF)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.