The nucleosome remodeling and deacetylase complex promotes cell fate decisions throughout embryonic development. Its core enzymatic subunit, the SNF2-like ATPase and Helicase Mi2, is well conserved throughout the eukaryotic kingdom and can be found in multiple and highly homologous copies in all vertebrates and some invertebrates. However, the reasons for such duplications and their implications for embryonic development are unknown.C. elegans Mi2 homologues, LET-418 and CHD-3, which displayed redundant activities during early embryonic development. At the transcriptional level, these two Mi2 homologues redundantly repressed the expression of a large gene population. We found that LET-418 physically accumulated at TSS-proximal regions on transcriptionally active genomic targets involved in growth and development. Moreover, LET-418 acted redundantly with CHD-3 to block H3K4me3 deposition at these genes. Our study also revealed that LET-418 was partially responsible for recruiting Polycomb to chromatin and for promoting H3K27me3 deposition. Surprisingly, CHD-3 displayed opposite activities on Polycomb, as it was capable of moderating its LET-418-dependent recruitment and restricted the amount of H3K27me3 on the studied target genes.Here we studied the two Although closely homologous, LET-418 and CHD-3 showed both redundant and opposite functions in modulating the chromatin environment at developmental target genes. We identified the interplay between LET-418 and CHD-3 to finely tune the levels of histone marks at developmental target genes. More than just repressors, Mi2-containing complexes appear as subtle modulators of gene expression throughout development. The study of such molecular variations in vertebrate Mi2 counterparts might provide crucial insights to our understanding of the epigenetic control of early development.The online version of this article (doi:10.1186/s13072-016-0091-3) contains supplementary material, which is available to authorized users. A. thaliana expresses three Mi2 homologues, namely PICKLE (Pkl) and PICKLE-related proteins 1 and 2 (Pkr1 and Pkr2) [pkl and pkr2 show redundant functions in suppressing embryonic lethality and establishing cell identity [Drosophila dMi2 and dChd3 are structurally different, since dChd3 misses N- and C-terminal regions as well as one PHD finger and is incapable of replacing dMi2 functions [C. elegans LET-418 and CHD-3 proteins are 70 % identical and 86 % similar. Although chd-3 depleted worms show no obvious phenotype, and let-148 depletion leads to L1 arrest, double let-418; chd-3 depletion leads to embryonic lethality, suggesting a redundant function of let-418 and chd-3 in early development [let-418 and chd-3 act redundantly to promote proper differentiation of the vulval cell precursors P5.p and P7.p, implying that common functions of these proteins are required at later developmental stages and adulthood [Throughout embryonic development, gene expression must be dynamically regulated in a time- and cell-specific manner to promote proper lineage commitment, tissue formation, and growth. Epigenetic factors transiently modify the chromatin structure or composition to modulate gene expression in a rapid and reversible manner. The nucleosome remodeling and deacetylase complex (NuRD) appears as a central player during early embryogenesis. Its transcriptional repression activities are required to maintain pluripotency and to respond properly to developmental cues. NuRD is essential for early embryonic development , 2, for D member . The comD member ) and areD member , suggestD member . VertebrD member , 12. In neration . In the nd Pkr2) , and pklidentity . Drosophunctions . C. elegelopment . In addidulthood .C. elegans. We identified a gene subset redundantly and directly repressed by the two Mi2 homologues in early embryos. Surprisingly, we found that LET-418 and CHD-3 differentially regulated histone modifications at the promoter of these genes. While both proteins restrict H3K4 methylation, LET-418 is capable of recruiting the H3K27 methyltransferase Polycomb to target genes promoters and CHD-3 limits this recruitment. We propose a model for the fine-tuning of chromatin composition and structure by a well-regulated balance between two Mi2 homologues, which potentially could constitute a conserved mechanism in all eukaryotes retaining multiple Mi2 copies.To understand the reasons for maintaining multiple Mi2 copies in eukaryotic organisms, we decided to analyze the functions of both LET-418 and CHD-3 in C. elegans and D. melanogaster, many eukaryotic organisms express more than one Mi2 family member, most particularly in vertebrate species. Phylogenetic analysis performed on a selection of eukaryotic Mi2 homologues implies that they developed individually from a common ancestor and that duplications occurred independently within subkingdoms  and barrel clover (M. truncatula), but not in common rice (O. sativa) , suggesting that Mi2 duplications occurred early in the vertebrate kingdom; noticeably, CHD-5 seems to have derived from a common ancestor to CHD-3  Fig. a. Among ies Fig. a. In speted Fig. a. The obD-3 Fig. a. TherefC. elegans. Its two Mi2 proteins, CHD-3 and LET-418, share a high degree of sequence homology, with 71 % identity and 80 % similarity (BLASTP). Mi2 proteins are characterized by specific functional domains, including tandem N-terminal chromodomains and DNA binding (CHD) and plant homeo-domains (PHDs), and central SNF2-like ATPase and DEAD-like helicase domains  mutations with let-418 (RNAi) or with a temperature-sensitive (ts) let-418(n3536) allele, on C. elegans development   or by shifting chd-3(eh4); let-418(ts) mothers to restrictive temperature, showed a visible growth delay from the bean stage on, and a developmental arrest at the twofold stage  and chd-3(eh4) or double chd-3(eh4); let-418(RNAi) embryos were manually sorted and collected at the 24- and 100-cell stages, and compared to wild-type; control GFP(RNAi)-treated embryos in a transcriptome RNA sequencing (RNA-Seq) analysis  in single- and double-mutant embryos were investigated  and to undergo lineage establishment until the 100-cell stage . Importadatasets   background  analysis of LET-418 genome-wide binding sites was performed in duplicate embryonic extracts, using a fully functional LET-418::3xFLAG transgene expressed in a www.Ensembl.org) and were broad, with an average size of approximately 2000 bp , xol-1 (sex-determination gene), and ins-39 (insulin-like peptide). Quantitative RT-PCR analysis (qRT-PCR) was used to verify their redundant control by let-418 and chd-3 , and the validation is likely due the difference in the embryonic stages between the two experiments. ChIP-Seq trends were then validated by anti-FLAG ChIP followed by quantitative PCR (ChIP-qPCR) of mixed early embryonic populations on LET-418::3xFLAG in the let-418(s1617) null background  and its three core components mes-2,-3, and -6 [ric-3, asic-2, ins-39 and xol-1 in single or double let-418; chd-3 mutants, and compared them with control embryos  L4 hermaphrodites were fed for 24 h at 25 °C with HT115 bacteria expressing specific dsRNA against gfp(control) or let-418. Adult hermaphrodites were then dissected under a binocular. 2-cell stage embryos were isolated manually and incubated at 25 °C for 1 h (24-cell stage) or 3 h (100-cell stage) in M9 buffer. Approximately 200 embryos were collected for each sample. Total RNA was extracted with QIAzol  using 5 μg of linear polyacrylamide . Contaminating DNA was digested using DNase I (Qiagen), and RNA was purified using the RNeasy MinElute Cleanup kit (Qiagen). Quality and concentration of each sample were determined using the 2200 TapeStation and High Sensitivity RNA Screen Tape . cDNA library preparation and RNA sequencing were performed at the Genomics Technology Facility (GTF) in Lausanne (https://www.unil.ch/gtf). Amplified cDNA was prepared from total RNA using the Ovation RNA-seq V2 system , and cDNA libraries were built using the TruSeq mRNA sample prep kit . RNA sequencing  was performed on three biological replicates per sample with a HiSeq 2000 genome sequencer (Illumina). Four cDNA libraries were multiplexed per sequencing lane.Wild-type or http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were remapped onto the reference genome Ce10 (WBcel215/WS220) with Tophat2/Bowtie2 [Raw reads (SE 100 bp) were obtained from GTF (UniL) and their quality checked with FastQC.  for each comparison was converted to an excel sheet, and processed lists of deregulated genes are provided in the Additional file The DESeq  and EdgeChromatin immunoprecipitation (ChIP) and quantitative PCR were performed following standard protocols and are described in details in the Additional file http://htsstation.epfl.ch) [http://bbcf.epfl.ch/bbcflib/bbcflib_gfminer.html#bbcflib.gfminer.stream.annotate.getNearestFeature for more details. Splitting was required when several features are found for a peak. Scripts are available upon request. Genome-wide LET-418 binding profiles were visualized and aligned to the C. elegans genome version ce10 using the Integrated Genome Viewer (IGV) [Reads (PE 100 bp) were obtained from the UniBe sequencing facility (Illumina HiSeq 2500) and their quality checked with FastQC. Reads were loaded in the BBCF HTSstation (epfl.ch) . Within epfl.ch)  followedepfl.ch) . The outepfl.ch)  with theepfl.ch) . Additioepfl.ch)  and mergepfl.ch)  and the er (IGV) , 58. DatCellular compartment fractionation, immunoprecipitations (IPs) and western blotting analysis were performed as described elsewhere , 59 with


FLC expression by blocking FVE-mediated chromatin silencing of FLC, delaying flowering time.Arabidopsis inositol polyphosphate multikinase (AtIPK2β) functions in the activation of  FLC at the chromatin level. The atipk2β mutant flowers earlier, and AtIPK2β overexpressing plants exhibit late-flowering phenotypes. Quantitative reverse transcription-PCR (qRT-PCR) revealed that AtIPK2β promotes FLC expression. We performed chromatin immunoprecipitation-qPCR (ChIP-qPCR) assays and found that AtIPK2β binds to FLC chromatin. Further analysis showed that AtIPK2β interacts with FVE, a key repressor required for epigenetic silencing of FLC. qRT-PCR, ChIP-qPCR, and genetic analysis demonstrated that AtIPK2β is involved in FVE-mediated transcriptional regulation of FLC by repressing the accumulation of FVE on FLC. Moreover, we found that AtIPK2β associates with HDA6, an interaction partner of FVE mediating FLC chromatin silencing, and attenuates HDA6 accumulation at the FLC locus. Taken together, these findings suggest that AtIPK2β negatively regulates flowering time by blocking chromatin silencing of FLC.Timely flowering is critical for successful reproduction and seed yield in plants. A diverse range of regulators have been found to control flowering time in response to environmental and endogenous signals. Among these regulators, FLOWERING LOCUS C (FLC) acts as a central repressor of floral transition by blocking the expression of flowering integrator genes. Here, we report that Arabidopsis inositol polyphosphate multikinase (AtIPK2β) functions in flowering time control by mediating transcriptional regulation of  FLOWERING LOCUS C (FLC), which encodes a MADS-box transcription factor that blocks flowering by transcriptional repression of floral integrator genes including FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) , age, and autonomous pathways . Among t1 (SOC1) . FLC act factors . AlthougFLC. Active modifications at FLC chromatin, such as histone H3 acetylation (H3Ac), lysine-4 methylation (H3K4me2/H3K4me3), lysine-36 methylation (H3K36me2/H3K36me3), and histone H2B monoubiquitination (H2Bub1), induce gene expression, whereas repressive modifications, such as histone deacetylation, histone lysine-9 methylation, and histone H3 lysine-27 trimethylation (H3K27me3), result in silencing of FLC -like complex and histone deacetylase complexes, which silence FLC by depositing the repressive H3K27me3 mark or removing the active H3Ac mark at the FLC locus, and the FRIGIDA (FRI) complex, which activates FLC expression by accumulating the active H3Ac, H3K4me3, H3K36me2, and H3K36me3 marks on the chromatin  to generate inositol 1,4,5,6-tetrakisphosphate (IP4) and inositol 1,3,4,5,6-pentakisphosphate (IP5)  act as stimulators in modulating the activities of chromatin-remodeling complexes, such as INO80 and SWI/SNF, resulting in activation of target genes  complexes  play important roles in inositol phosphate metabolism and signal transduction. The conserved catalytic activities of IPMKs are phosphorylating inositol 1,4,5-triphosphate (IPte (IP5) . IPMKs aomplexes . In mamm defects . A recenFLC. AtIPK2β acts as a negative regulator of flowering time by promoting the expression of FLC. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis revealed that AtIPK2β mediates transcriptional regulation of FLC via binding to FLC chromatin. In addition, AtIPK2β interacts with FVE and represses the accumulation of FVE at the FLC locus. Gene expression, genetic, and ChIP-qPCR assays confirmed that AtIPK2β is involved in FVE-mediated transcriptional regulation of FLC by affecting chromatin modifications including histone H3K27 trimethylation and H3 deacetylation at FLC. Moreover, AtIPK2β associates with HDA6, an interaction partner of FVE, and attenuates the accumulation of HDA6 on FLC chromatin. In general, these results suggest that AtIPK2β functions as a repressor in flowering time control through promoting FLC expression at the chromatin level.In this study, we report the function of AtIPK2β in flowering time control via mediating the transcriptional regulation of Arabidopsis thaliana Columbia type (Col-0) background. The atipk2β-1 (SALK_025091) and atipk2β-2 (SALK_104995) mutant lines were described previously by flc-6 null mutant, obtained from the Arabidopsis Biological Resource Center, was originally described by fve T-DNA line (SALK_013789) was provided by Dr Ligeng Ma, and genotyping PCR was performed for identifying the T-DNA insertion. Double mutants of flc/atipk2β and fve/atipk2β were generated by crossing the fve and flc-6 lines, respectively, with atipk2β-2. The homozygous double mutants of F3 progenies were selected by genotyping PCR and used in this study. The primers used in identification of the mutants, with detailed sequences, are listed in JXB online.All the plants used in this study were atipk2β mutant were generated by introducing a construct encoding an AtIPK2β-Green Fluorescent Protein (GFP) fusion protein under the control of the native promoter into atipk2β-2, as described by AtIPK2β or FVE coding sequence was inserted into the pCAMBIA1302 vector after the CaMV 35S promoter region and followed by the coding sequence of GFP. Col-0 and the atipk2β-2 mutant plants were agro-transformed with the resulting vectors using the floral dip method  under long-day  or short-day  photoperiods. Seeds were stratified for 4–5 days at 4 °C on Murashige and Skoog (MS) agar plates before being transferred on to soil. Flowering time was measured by counting the number of rosette leaves and the number of days at bolting after germination. At least 10 plants were counted and averaged for each genotype.Plants were grown on soil in a controlled culture room at 22 °C with cold fluorescent light (100 μmol m−2 s−1) under the LD or SD photoperiod for 15 to 20 days before being harvested. RNA was extracted using a TRIzol (Molecular Research Center) method and treated with DNase I (Promega) before being transcribed into cDNA by M-MLV Reverse Transcriptase (Promega). Quantitative reverse transcription-PCR (qRT-PCR) was performed with gene-specific primers (listed in ACT2 and UBQ10) and then compared with the wild-type (WT) control plants. Data are presented as means±SD of at least two independent repeats with similar results.For analyzing gene expression levels, seeds were sterilized with ethanol and then stratified for 4–5 days at 4 °C on solid MS agar plates. The plates were kept in a controlled culture room at 23 °C with cold fluorescent light , and further on medium without Leu, Trp, His, and Ade (QDO). Plates were incubated at 30 °C for 3–5 days. The filter β-galactosidase assays were conducted according to the system protocol (Clontech).The pGBKT7 and pGADT7 vectors of the BD Matchmaker system (Clontech) and the yeast strain AH109 were used for yeast two-hybrid assays according to the manufacturer’s instructions. The full-length coding sequence of AtIPK2β or FVE was inserted between the CaMV 35S promoter and terminator regions and fused to the sequences encoding the N-terminal fragment or C-terminal region of Yellow Fluorescent Protein (YFP) to form the YN-AtIPK2β or YC-FVE fusion, respectively. The DNA fragments including the CaMV 35S promoter, coding sequence of YN-AtIPK2β or YC-FVE fusion, and the terminator regions were amplified by PCR and transformed into the protoplasts according to the method described previously by Arabidopsis protoplasts from rosette leaves of plants grown for 3 weeks under the LD photoperiod were isolated as described by Arabidopsis rosette leaves of indicated genotypes grown on MS agar plates for 15 days under the LD photoperiod were homogenized in extraction buffer containing 50 mM Tris-HCl (ph 7.5), 100 mM NaCl, 10% glycerol, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1× complete protease inhibitor (Roche). Total protein was extracted by centrifugation at 4 °C. The protein concentration was determined by using a Bradford assay kit . Western blot was performed by blotting protein samples on polyvinylidene fluoride membranes (Millipore) after being boiled and run on SDS-PAGE gels. Different primary antibodies were used for probing the blots. A polyclonal AtIPK2β antibody was raised in rabbits as described by Co-immunoprecipitation (Co-IP) assays were performed as described previously (ACTIN2 (ACTIN) and FUSCA3 (FUSCA) were used for normalization (ACTIN for H3Ac and FUSCA for H3K27me3) in the ChIP-qPCR assays to detect enrichments of H3Ac and H3K27md3 marks on FLC as described previously  exhibited similar or slightly delayed flowering compared with WT  were used for further analysis. The AtIPK2β overexpression lines exhibited delayed flowering , FLC, FT, and SOC1, which encode key flowering-time regulators and floral integrators, were analyzed in the atipk2β mutant and AtIPK2β overexpression plants grown under both LD and SD conditions. The AtIPK2β overexpression line (OE1) exhibiting a late-flowering phenotype  was detected in extracts from the transgenic plants  was detected with anti-AtIPK2β antibody in protein extracts from WT plants, but no such band was detected in the extracts from the atipk2β mutant  performed as described previously  was identified  and histone H3 deacetylation of FLC chromatin , which is a histone acetyl transferase, and mediates histone acetylation on the target loci . Here, whromatin . Since FFLC chromatin and represses FVE-mediated histone modification on FLC by attenuating the accumulation of FVE and HDA6 at the FLC locus, leading to transcriptional activation of FLC and delayed flowering. More widely, our study of the functional link between AtIPK2β and FVE-HDA6 will improve understanding of the FLC-dependent pathway in flowering time control. How AtIPK2β interacts with FVE and HDA6 to repress the accumulation of FVE and HDA6 on FLC chromatin will be the focus of future investigations.Taken together, our findings provide evidence for a regulatory mechanism by which AtIPK2β functions in flowering time control. AtIPK2β enriches on JXB online.Supplementary data are available at atipk2β mutants and complemented lines under the LD condition.Fig. S1. Analysis of flowering time in Fig. S2. Identification of FVE as a potential interactor of AtIPK2β by liquid chromatography coupled to tandem mass spectrometry.fve mutant.Fig. S3. Molecular characterization of the Table S1. Primer sequences used for genotyping PCR and qRT-PCR.Table S2. Primer sequences used for ChIP-qPCR.Supplementary Figures S1-S3 Tables S1-S2Click here for additional data file.


Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics. SoxF genes compromises arteriovenous specification, blood vascular integrity and lymphangiogenesis, and inhibits tumour growth and metastasis in animal models of cancer  of transcription factors (TFs) are key regulators of endothelial cell differentiation during development , and aref cancer . More ref cancer . PharmacTranscription factors often operate in mutually redundant families, thwarting conventional approaches to developing transcription factor-based therapies. Any attempt to develop pharmaceutically useful SOX18 inhibitors must overcome two obstacles — first, that SOX18 loss of function is compensated by the action of the remaining SOXF , and secSOX proteins activate individual target genes by recruiting specific interacting partners , but onlIn addition, we studied potential pairwise interactions of 6 well-known TFs able to regulate endothelial cell function , and the only identified SOX18 protein partner MEF2C . The welSm4 . We found that Sm4 significantly disrupted 6 out of the 12 validated SOX18 interactions  and ALPHA-Screen analysis further established that SOX18 and c-JUN could physically interact  to the TSS of s tested , signifyinteract  and 2. Wreatment . This isSm4 on SOX TF family members we focused on closely related SOXF and SOXE proteins. Sm4 did not affect the transcriptional activity of either SOX17 or SOX9 proteins at any tested concentration  in cell-based reporter assays  transgenic zebrafish reporter line, previously validated as a readout for the combined activity of Sox7 and Sox18 (Sm4 treatment significantly reduced SOX18-dependent egfp transcript levels (61%), similar to the effects of combined sox7/18 depletion using morpholino oligonucleotides (MO) (To investigate whether nd Sox18 . We treades (MO) . Importatg(Dll4in3:eGFP), which harbours a regulatory element located in the intron 3 of dll4 gene. The activity of this Dll4in3 enhancer does not fully recapitulate the endogenous dll4 expression  reporter line, and observed that continuous Sm4 treatment between 2 and 6 days post fertilization did not perturb the transcriptional activity of Sox9 or the process of chondrogenesis  with 1.5 pM Sm4 during the relevant developmental window, starting from 16 hpf . However, the blood circulation in the head is unaffected in Sm4-treated larvae, signifying that a short circulatory loop has not fully formed. This phenotype supports of the conclusion that Sox7 activity is only partially affected in presence of the small compound. Overall, these results are congruent with the genome-wide inhibitory effects observed in vitro, demonstrating that Sm4 selectively interfered with the transcriptional activity of Sox18 and SoxF-mediated vascular formation in vivo.Due to SoxF redundancy in arteriovenous specification, an A/V malformation phenotype is typically only observed in double loss of Sox7 and Sox18 function. Since l artery , resultil artery . In presSm4 in a therapeutically relevant setting, we next assessed its efficacy in a preclinical model of breast cancer. BALB/c mice were inoculated with highly metastatic 4T1.2 mammary carcinoma cells into the mammary fat pad, and three days were allowed for the engraftment of the tumor, after which treatment was initiated with either 25 mg/kg/day of Sm4, aspirin or vehicle PBS . To further investigate what could cause such an effect, the size of the tumors was monitored during the treatment, as well as the formation of spontaneous lung metastases. While the size of the primary tumor was unchanged by Sm4 treatment  in treated conditions, as well as the number of lymphatic endothelial cells   . This ly cancers  TogetherSm4 is an effective molecular strategy to interfere with the metastatic spread in a pre-clinical model of breast cancer.Induction of angio- and lymphangiogenesis is a hallmark of solid cancer, and is a critical step towards enabling tumor metastatic dissemination. Conventional approaches to target transcription factors have focused on interfering with oncogenes that are dysregulated to promote tumor cell transformation . Here, wAll data and statistical analysis in this study were generated from at least three independent experiments unless indicated otherwise. Technical replicates were included in every experiment to reduce background noise and detect technical anomalies. Samples of distinct experimental conditions were not exposed to any specific method of randomization, and groups were assessed under non-blinded conditions.SOX18 gene was synthesized (IDT) and the transfers to vectors was realized using Gateway PCR cloning.The genetically encoded tags used here are enhanced GFP (GFP), mCherry (Cherry) and cMyc (myc). The proteins were cloned into the following cell free expression Gateway destination vectors respectively: N-terminal GFP tagged (pCellFree_G03), N-terminal Cherry-cMyc (pCellFree_G07) and C-terminal Cherry-cMyc tagged (pCellFree_G08) .The OpenLeishmania tarentolae extract (LTE) was prepared as previously described (The translation competent escribed . ProteinmL (0.4 Mg) of Anti-cMyc coated Acceptor Beads in buffer B  were aliquoted into each well. This was followed by the addition of 2 pL of diluted sample and 2 pL of biotin labeled GFP-Nanotrap in buffer A. The plate was incubated for 45 min at RT. Afterward, 2 pL (0.4 pg) of Streptavidin coated Donor Beads diluted in buffer A, were added, followed by incubation in the dark for 45 min at RT. The ALPHA-Screen signal was obtained on an Envision Multilabel Plate Reader (PerkinElmer), using the manufacturer’s recommended settings . The resulting bell-shaped curve is an indication of a positive interaction, while a flat line reflects a lack of interaction between the proteins. The measurement of each protein pair was repeated a minimum of three times using separate plates. The Binding Index was calculated as:The ALPHA-Screen Assay was performed as previously described , using tneg is the lowest (background) signal level. The signals were normalized to the Iref signal obtained for the interaction of SOX18 with itself.For each experiment, I is the highest signal level (top of the hook effect curve) and ISm4 or DMSO alone . 100 pM Sm4 or DMSO was also added to buffer B. PPI disruption was calculated as: For PPI disruption assay, protein pairs expressed in LTE were incubated for 1 hr with 100 pM Sm4 was used (0.3–300 pM). Percentage of interaction was calculated as: SCR_007370) version 6.0 using 3-parameter nonlinear regression.For IC50 determination, the assay was identical but a dilution range of CVCL_0224) cultured at 37 °C, 5% CO2 in DMEM  with added FBS, sodium pyruvate, L-glutamine, penicillin, streptomycin, non-essential amino acids and HEPES (N-2-hydroxyethylpiperazine-N'—2-ethanesulfonic acid). COS-7 cells were transfected for 4–6 hr, and incubated for another 24 hr before lysis and luciferase assay . Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Australia (CC-2519A). HUVEC for ChIP-MS, ChIP-seq and RNA-seq analyses were transfection for 7 hr and incubated another 14 hr. During small molecule treatment, cells were grown in medium containing low serum (0.4% FBS). HUVECs were cultured at 37 °C, 5% CO2 in EGM-2 media supplemented according to the EGM-2 bullet kit instruction . Cells for were grown in 35 mm dishes to 80–90% confluency, and transfected with plasmid mouse pSG5 Sox18, plasmid pSG5 cMyc-Sox18, or plasmid cMyc using X-tremegene 9 DNA transfection reagent  according to the manufacturer’s instructions. All cell lines were tested negative for mycoplasma contamination.COS-7 cells were purchased from ATCC  on HUVECs overexpressing cMyc-tagged SOX18.ChIP experiments were performed as previously described . ImmunopFollowing IP, DNA amplification was performed using TruSeq ChIPseq kit , using 0.5 pM of the universal reverse PCR primer and the forward PCR primer containing the index sequence of choice in 50 pL 1 x NEBNext High-Fidelity PCR Master Mix . The number of PCR cycles ranged from 13 to 18, depending on the ChIP efficiency. The PCR product was purified using AMPure beads (1.8 vol) and eluted in 20 pL of resuspension buffer (Tris-Acetate 10 mM pH 8). The library was quantified using the KAPA library quantification kit for Illumina sequencing platforms  and 50 bp single end reads were sequenced on a HiSeq2500 following the manufacturer’s protocol. Illumina fastq files were mapped to the GRCh37/UCSC hg19 genome assembly using bowtie, and peaks were called using MACS version 2.1.0. using input. To avoid false positive peaks calling due to the cMyc epitope, ChIP-seq with the cMyc epitope only were performed in parallel to SOX18-cMyc ChIP-seq and peaks called in these experimental conditions were substracted to the peaks called in the SOX18-cMyc conditions.SCR_005807) was used to analyse the functional significance of cis-regulatory regions. ChIP-seq data are available in the ArrayExpress database  under accession number E-MTAB-4480 (SOX7) and E-MTAB-4481 (SOX18).Genomic Regions Enrichment of Annotations Tool . Reads were mapped to the hg19 reference human genome using STAR aligner , and onlSm4-treated and DMSO control in SOX18 over-expressing cells, and separated in up-regulated and down-regulated (DOWN) genes. The locations of their transcription start sites (TSS) were correlated to the locations of transcription factors binding events that are available from the ENCODE consortium (RRID: SCR_006793), and from the SOX18 and SOX7 ChIP-seq experiment we performed in this study. To ensure that the TSSs were independent, a TSS was allowed to only be assigned to 1 ChIP-seq peak. Transcripts with >2 fold absolute fold change (log2FC >1 or < —1) were included for distance to TSS analysis. The median distance between the TSSs and binding events was compared to the expected distance of a set of randomly selected genes to obtain the median ratio. The control set of genes was selected from the pool of genes expressed in HUVECs so that they had a similar distribution of expression levels. To ensure that no bias was introduced by potential co-regulation of genes by SOX18 and any other transcription factor analysed, we subtracted genes with SOX18 peaks from the analyses for other transcription factors. The reverse analysis was also performed, subtracting genes containing c-JUN peaks from the analysis for SOX18. RNA-seq data are available in the ArrayExpress database (https://www.ebi.ac.uk/biostudies/arrayexpress) under accession number E-MTAB-4511.Differentially expressed genes were identified between fi-actin for tg(Dll4in3: eGFP), ef1a for tg(—6.5kdrl:eGFP), chd5 for tg, RPL13 and GAPDH for HUVECs) were selected based on the stability of their expression throughout the set of experimental conditions, or chosen on grounds of their vascular expression to normalize to endothelial cell content. Primer efficiencies were calculated using LinRegPCR, and amplification data was analysed using ViiA7 software and the Q-gene PCR analysis template.Total RNA was extracted using RNeasy mini kit  according to the manufacturers protocol, including on column DNA digestion. cDNA was synthetised from 1 p,g of purified RNA using the high capacity cDNA reverse transcription kit . Amplification and quantitation of target cDNA was performed in technical triplicate of at least three biological replicates using the SYBR green  methods. Reactions were run in 10 pL in 384-well plates using the ViiA 7 Real-Time PCR system. Housekeeper genes (tg(—6.5kdrl:eGFP), tg and tg(Dll4in3:GFP) were previously described  in situ hybridization was performed as previously described  with prodab  and ephrphrinB2a . Yolk sasox7, sox18 (rbpj (tg (6.5kdrl:eGFP) and tg, and 0.125–0.15 pmol suboptimal concentrations for experiments performed with tg(Dll4in3:eGFP).All treatment with putative small molecule inhibitors, and corresponding control conditions, were performed in the presence of low concentration of DMSO (<1% v/v) to achieve reliable homogeneous solutions, and were prepared from 10 mM DMSO stock. For cell culture, small molecules were added to fresh media directly following transfection and cells were grown in this media until time-point of cell harvesting. For in vivo experiments involving zebrafish, compound treatment was initiated at the designated timepoints by replacing the media, and media +compound was refreshed daily for the duration of the experiment. PTU treatment (0.003%) was done in parallel with the small molecules to block pigment formation when necessary. Previously published and validated morpholino oligomers against 7, sox18  and rbpj18 .BALB/c wild-type (WT) were purchased from Walter and Eliza Hall Institute for Medical Research and used between the ages of 6 and 10 weeks. Mouse 4T1.2 mammary carcinoma cells were cultured in complete RPMI with 10% FBS in a 5% COescribed . BrieflyAB_2100038), ERG , PROX1  and Podoplanin  antibodies. Whole tumor sections were imaged by acquiring a series of images along the z-axis using a 10 x objective on a Zeiss LSM 710 confocal microscope. Subsequently, high-resolution images were captured using a 20 x objective on 3–4 separate regions from each tumor, to account for heterogeneity of the vascular density within the tumors and minimise bias. Raw image files with identical dimensions (1274.87 pm x 1274.87 pm x 89.05 pm) were loaded into Imaris , and processed using ’spots' function to count ERG or PROX1- positive nuclei and ’surface' to calculate volume or area of Endomucin or Podoplanin positive vessels. For each tumor (n=6), counts from the multiple regions were averaged and the data was plotted in Graphpad Prism 6.For quantitation of the vasculature in the tumors, fixed tissues were embedded in 4% agarose and sectioned all the way through at 300 pm on a Leica VT1000 S vibrating microtome. Sections were collected on glass slides and imaged for bright field analysis on the penetration of perfused vessels. Subsequently, immunofluorescent staining was performed on sections using anti-mouse Endomucin  and three reviewers, one of whom, Holger Gerhardt (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Gou Young Koh (Reviewer #3).Thank you for submitting your article "Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.General Assessment:The reviewers find your study provides interesting new insights into protein-protein interactions of Sox family member transcription factors and shows convincingly that such interactions can be targeted with small molecules to interfere with good selectivity with transcription in vitro and in vivo. The demonstration of in vivo efficacy of the small molecular inhibitor in both a zebrafish model using transcriptional reporters and in a mouse tumour model in which you find significantly reduced metastasis are particularly remarkable. Together with the comprehensive identification and validation of protein-protein interactions, these results provide a significant advance that the reviewers believe will be of interest to a wider scientific audience.Major Conclusions:1) Despite their similarities Sox family member transcription factors engage in distinct protein-protein interactions.2) Sox18 interacts directly with RBPjk and a number of other endothelial transcription factors in endothelial cells to regulate endothelial targets.3) Small molecule SM4 (identified in a screen reported elsewhere) shows good efficacy and selectivity to inhibit a subset of the identified Sox18 protein-protein interaction and suppresses target transcription in vivo.4) Targeting Sox18 PPI and thus its transcriptional activity is effective in vivo to reduce tumour angiogenesis and metastasis.Whilst the reviewers find your results supports these major conclusions, they find that the following points should be adequately addressed before publication:1) The claim of selectivity of SM4 against Sox18 deserves further investigation, in particular, to clarify if the potentially redundant activity of Sox17 in vascular development is also affected. Also, potential activity against Sox7 should be clarified. You may already have the required data on activity of SM4 against Sox17 and Sox7, in which case adding these to the figure and text, as well as commenting on their implications for the claimed selectivity of SM4 will be sufficient. In case this means you need to repeat experiments, we suggest to limit yourself to any that can be achieved reasonably within 2 months.Dll4int3 reporter and endogenous Dll4 gene expression effects of SM4 need to be clarified. Given that the Dll4int3 reporter does not contain all regulatory elements of the endogenous Dll4 gene, it could be that SM4 is more effective against the reporter and other elements still drive endogenous Dll4. As you currently don't comment on the discrepancy in the text, it is difficult for us to recommend precisely how you should address this issue. We suggest to carefully compare the response of endogenous Dll4 expression similarly to the dose response you find for the Dll4 reporter. Given the dynamic nature of Dll4 expression, it could also be a timing issue, and thus related to the half-life of the drug? A possible experiment in vivo could be to show in situ hybridisation for Dll4 in WT fish treated with SM4. As Dll4 levels have a major impact on blood vessel formation also in tumour angiogenesis, and your in vivo mouse experiments show effects on tumour angiogenesis, we feel this needs to be clear in order to understand the action of SM4. This should be possible to address in two months. In case experimental data do not provide a clear answer, please address this issue carefully in text and Discussion.2) The discrepancy between the effects on the 3) The mechanism of reduced tumour angiogenesis and metastasis should be adequately discussed and ideally experimentally clarified. Given that the route of metastasis is likely through lymphatics and that Sox18 also regulates lymphangiogenesis, we feel you should consider this as potential mechanism. As you have the expertise in mouse lymphatic analysis from previous studies, we hope you will be able to provide experiments that show whether or not SM4 interferes with tumour lymphangiogenesis through blocking Sox18 function. It will not be necessary to show that this is the definitive cause for changes in metastasis, but we feel that without analysing lymphatics, this work is incomplete. Given that you use a xenograft model, it should be feasible to perform these experiments within the 2 months of revision period.We hope you will find these comments useful and clear to allow efficient revisions of the essential points. For your information, we also append the full set of reviews below.Reviewer #1:This interesting report by Overman et al. presents the functional characterisation of a pharmacological compound targeting the interaction of the transcription factor SOX18 with other proteins. This particular compound, Sm4, was isolated through a high-throughput screen for selective SOX18 inhibitors described fully in an accompanying manuscript that will be published elsewhere. The current manuscript used successive screening by first CHIP MS and then Α screen to map protein+protein interactions of Sox18, and then study in detail the selectivity and inhibitory capacity of the compound Sm4 on SOX18 activity both in vivo and in vitro. The in vivo used the zebrafish model in which Sox18 transcriptional regulation of known vascular genes was investigated in reporter lines and by QPCR. Finally, the authors used a mouse orthotopic mammary tumour model to test efficacy in tumour angiogenesis and metastasis in vivo.Overall, the work convincingly demonstrates that the approach taken is valid to identify key protein protein interactions of transcription factors, and that targeting these interactions can be achieved with efficacy and some selectivity in vivo.The work is well written and illustrated, and the major claims appear supported by the data. Having said that, there are a number of issues that should be addressed to further clarify the claim of the specificity of Sm4's inhibition of SOX18 direct protein interactions and its role in inhibiting Sox18-mediated vascular formation in vivo. Critically, the authors test specificity and efficacy of Sm4 in vivo first in the Dll4int3 reporter line, but endogenous Dll4 seems unaffected. This is left uncommented and raises questions regarding the mechanism at play.Furthermore, whereas the arteriovenous development deficit is clearly demonstrated in fish, the rest of the vascular changes in fish and in mouse tumours don´t match with what would be expected if Sox18 and RBPJ activity was reduced in blood endothelium. Furthermore, whilst these vascular changes are well demonstrated in the tumours, the actual reason for reduced lung metastasis is not addressed. Surprisingly, despite having originally identified Sox18 as key regulator of lymphatic development, and given that metastatic spread from mammary tumours is largely driven by lymphatics, the authors did not look at and comment on tumour associated lymphatic changes and the potential effects of Sm4.Given that the work primarily aims to establish the approach to target PPI for transcriptional regulation in vivo, the latter point may not necessarily have to be addressed experimentally, but should at least be discussed. More pertinent to the overall claims are the remaining questions on specificity.Please find further detailed comments below:1) Given the redundancy of Sox18 and Sox17 in the context of vascular development, the authors should include OCT4 interaction in their functional analysis of the effects of SM4 on both SOX17/OCT4 (reported interaction) and SOX18/OCT4.2) – Could the authors describe what the arbitrary threshold used to define interaction in Figure 1C is? Were there any physiological or chemical reasons used in defining it?3) – Could the authors comment about the significant proximity between c-FOS and EZH2 binding sites and the transcriptional start site of up-regulated genes following Sm4 treatment? Are their transcriptional activities significantly affected by Sm4 treatment?4) The authors describe that "[…] we calculated the distance between the transcription start site (TSS) of a gene and a TF binding event, as a proxy for the likelihood of direct transcriptional regulation". But later mention that, in regard to this analysis, "The results indicate that Sm4 affected genes are dysregulated through a direct effect on SOX18 transcriptional activity". Given the 'proxy' nature of this experiment, I believe the claim could be turned down to better reflect the fact that this is a rather indirect indication of specificity.5) Could the authors provide evidence that Sm4 does not affect SOX18 DNA-binding role? This particular piece of data is required to show that Sm4 role on Sox18 role in vivo indeed relies on affecting SOX18 interaction with other proteins rather that in its ability to bind to DNA.6) Does Sm4 affect the transcriptional activity of Sox7? In Figure 3, could the authors please include a combined treatment with MO-sox18/Sm4 and MO-sox7/Sm4 to further demonstrate Sm4 specificity within SoxF factors. In addition, the authors should also include a MO-sox7/Mo-rbpj/Sm4 and MO-sox18/MO-rbpj/Sm4 controls in Figure 3C.7) As mentioned in the overall assessment, does Sm4 treatment affect the pre-existing lymphatic vasculature in area surrounding the tumour? In addition, is neo-lymphangiogenesis affected?Reviewer #2:In this paper, the authors describe a specific inhibitor of the transcription factor SOX18, a known regulator of vascular development. Using a combination of in vitro/ChIP-seq/in vivo experiment, they build a nice scientific approach to develop an anti-angiogenic drug able to inhibit tumor development. They start from an elegant experiment of ChIP-MS to isolate nuclear factors interacting with SOX18 presumably on chromatin. They test also interaction of SOX18 with several transcription factors obtained from the ChIP-MS to validate the protein-protein interaction and also to measure the effect of a small molecule sm4, describe in another manuscript, on these interactions. A ChIP-seq experiment coupled to RNA-seq shows, using a good bio-informatics approach, the specificity at the genomic level of Sm4 on the SOX18 transcriptional activity. The authors also predict a possible relationship of SOX18 with C-Jun.The second part of the manuscript, presents the in vivo validation of action of Sm4 on the genetic pathway controlled by SOX18 followed by experiments in mouse showing the inhibition of tumor vascularization by Sm4. To my knowledge, it is one of the first studies showing the development of a drug able to block specifically the activity of a SOX protein.Reviewer #3:Although the roles of SoxF members including Sox18 are crucial for formation and maintenance of vascular system, exactly how they form the protein complex for their transcriptional activities remains poorly elucidated. The present work may be the first report unveiling the entity of the proteins that physically interact with Sox18 and the interaction between SoxF members. Thus, this work provides a significant scientific advance by deciphering the mechanism of transcriptional regulation which are critical for vascular morphogenesis. Some additional in vivo data would improve the integrity of the manuscript for publication.tg(-6.5kdrl:eGFP) and tg(Dll4in3:eGFP)), in which arteriovenous specification defects were assessed. All these zebrafish systems are known to be modulated by the cooperation of Sox7 and Sox18 rather than by Sox18 alone. Therefore, the authors should be cautious to claim the effect of Sm4 is dependent solely on Sox18 inhibition based on these complicated in vivo systems. This potential complexity should be discussed.1) The authors demonstrated that a natural compound Sm4 effectively inhibits the transcriptional activity of Sox18 in molecular and cellular levels. Moreover, the inhibitory effect of Sm4 was verified in vivo by using zebrafish reporter lines (2) Sm4 appears to have a potential as an oral therapeutic agent to treat cancer as they have shown that Sm4 treatment reduced lung metastasis in a mouse orthotopic mammary tumor model. Reduced tumor angiogenesis is suggested as a working mechanism for the reduced tumor metastasis in this work. On the other hand, vascular destabilization is well-known to induce tumor metastasis. Additional analysis of tumor vessels integrity may provide clues as to how the vascular changes induced by Sm4 administration are associated with decreased metastasis.3) The authors showed Sox18 expression in tumor vessels to rationalize the inhibitory effect of Sm4 on tumor angiogenesis. It has been previously reported that tumor angiogenesis/lymphangiogenesis was suppressed in Sox18-knockout mice (JNCI 2006); thus, pharmacological blockade of Sox18 can indeed reduce tumor angiogenesis. However, there is a possibility that Sm4 can inhibit tumor angiogenesis and tumor growth by affecting molecules other than Sox18. This possibility could be tested by administering Sm4 to Sox18-knockout mice bearing tumors, since the effect of Sm4 on Sox18 will be abolished in this mouse model. […] Whilst the reviewers find your results supports these major conclusions, they find that the following points should be adequately addressed before publication:(1) The claim of selectivity of SM4 against Sox18 deserves further investigation, in particular, to clarify if the potentially redundant activity of Sox17 in vascular development is also affected. Also, potential activity against Sox7 should be clarified. You may already have the required data on activity of SM4 against Sox17 and Sox7, in which case adding these to the figure and text, as well as commenting on their implications for the claimed selectivity of SM4 will be sufficient. In case this means you need to repeat experiments, we suggest to limit yourself to any that can be achieved reasonably within 2 months.We acknowledge that selectivity is a crucial consideration for an inhibitor to be of use as a chemical probe or therapeutic. Therefore, we have broken down the matter regarding selectivity into three key aspects: PPI disruption, inhibition of transcriptional actvity, and SOXF loss of function phenocopy in vascular development.PPI disruption selectivityTo further investigate the selectivity of Sm4 between all three SOXF family members  we have performed additional PPI analyses by ALPHAScreen, focusing on the recruitment of RBPJ , MEF2C  and the SOX17 protein partner OCT4 .50 19.6 μM, Figure 1—figure supplement 1F) and partially disrupts SOX7-RBPJ interaction. In addition, as suggested by reviewer #1, we have investigated the effects of Sm4 on SOXF-OCT4 protein-protein interaction. Results show that Sm4 has the potential to disrupt SOX17-OCT4 interaction but not SOX18-OCT4 or SOX7-OCT4.Figure 1—figure supplement 1 (new): This in vitro protein-protein interaction analysis revealed that SOX18 has the capacity to selectively form a hetero-dimer with SOX7 and RBPJ whereas SOX17 does not interact with other SOXF proteins, nor does it interact with RBPJ or MEF2C. The interaction with RBPJ is conserved between SOX18 and SOX7 (left panel). Sm4 has the ability to interfere with SOX7-SOX18 heterodimer formation  suggest that Sm4 selectivity is mostly towards SOX18 but has some potential to interfere with SOX7- or SOX17-dependent PPI (in the high micro-molar range [Sm4] 50-100 μM). Our observations indicate that this inhibitory effect predominantly occurs in the scenario where SOX18 and the other SOXF protein share a particular interactor.Of note the interaction between SOX17 and OCT4 (POU5F1) is not relevant to endothelial cell biology since this transcription factor is not expressed by endothelial cells as shown by transcripts analysis from FANTOM5 database , and froIt is often challenging for small molecules to achieve selective targeting between closely related proteins, such as those within the SOXF group of transcription factors . The small molecule that we describe acts in such a way that SOX18-dependent protein complexes are disrupted. Each SOXF protein has its distinct ‘primary’ function mediated by specific sets of PPIs, which bestows opportunities for selective inhibition. However, these 3 TFs also do have certain PPIs in common, which could explain their overlaping function in the context of rescue mechanism. Redundancy has been shown for SOX7, 17 and 18 protein that can act interchangeably to rescue the loss of function of one another within the F-group . The fact that Sm4 has the potential to inhibit a subset of SOX7, SOX17 or SOX18–dependent PPIs is an advantage to prevent any potential redundancy mechanism.The claims regarding the specific targeting of SOX18 by Sm4 has been reworded more carefully in light of these new data (fifth paragraph of the main text). The additional PPI analysis has been added as new Figure 1—figure supplement 2.Off-target profilingTo include a wider analysis of Sm4 specificity towards SOX18, we have performed an unbiased off target investigation using a CEREP/Eurofins/ Panlabs profiling panel. This is shown by a new data set we have now included in the revised version of our companion manuscript . Proteins tested on this panel are representative of various biological processes, such as: GPCRs, kinases, nuclear receptors, HDACs, sirtuins and membrane receptors. CEREP uses as a cut off 50% inhibition to flag any potential off target effect. This panel analysis with Sm4at 10 μM did not flag any non-specific binding out of 36 protein tested.Selectivity on transcriptional interferenceSOX9 and SOX17 as negative controls throughout the study. We show that Sm4 does not perturb:To assess other SOX proteins’ activity that could be potentially affected by Sm4 we have included SOX9 homodimer formation SOX9 transcriptional activity in cell-based assay in vitro SOX9-induced Col2a1 transactivation in zebrafish larvae SOX17-induced ECE1 transactivation .The analysis of ChiPseq/RNAseq data sets  further demonstrates that Sm4 is specific to SOX18 interference amongst endothelial TFs, including SOX7.Selectivity on vascular development- phenotypic outputsox7/18 double morphants/knockout  and the ALPHAScreen data, demonstrates the ability of Sm4 to block Sox18 activity in vivo. Since Sm4 has the ability to interfere partially with Sox7/RBPJ interaction, we further investigated whether Sm4 could directly interfere with Sox7 function in zebrafish. For this approach, we used as a readout the phenotypic outcome of the sox7 KO zebrafish line  and compared it to Sm4-induced phenotype.The current data set based on two SoxF reporter assays in zebrafish , combined with the partial phenocopy of sox7 genetic disruption in zebrafish is a short circulatory loop in the head with no circulation in the trunk and tortuous lateral dorsal artery (LDA). In presence of Sm4, we observe a partial phenocopy of Sox7 loss of function characterized by a mild vascular defect in the LDA  strongly suggest that Sm4 has no conspicuous effect on other SOX TFs. Interference  with developmental transcription factors at the stages we investigated would results in severe defects, while we observe that Sm4-treated zebrafish larvae develop normally, with the exception of the phenotype associated with perturbed SOXF function .2) The discrepancy between the effects on the Dll4int3 reporter and endogenous Dll4 gene expression effects of SM4 need to be clarified. Given that the Dll4int3 reporter does not contain all regulatory elements of the endogenous Dll4 gene, it could be that SM4 is more effective against the reporter and other elements still drive endogenous Dll4. As you currently don't comment on the discrepancy in the text, it is difficult for us to recommend precisely how you should address this issue. We suggest to carefully compare the response of endogenous Dll4 expression similarly to the dose response you find for the Dll4 reporter. Given the dynamic nature of Dll4 expression, it could also be a timing issue, and thus related to the half-life of the drug? A possible experiment in vivo could be to show in situ hybridisation for Dll4 in WT fish treated with SM4. As Dll4 levels have a major impact on blood vessel formation also in tumour angiogenesis, and your in vivo mouse experiments show effects on tumour angiogenesis, we feel this needs to be clear in order to understand the action of SM4. This should be possible to address in two months. In case experimental data do not provide a clear answer, please address this issue carefully in text and Discussion.Dll4int3 synthetic reporter line versus endogenous dll4 expression. It is often the case that synthetic enhancers – containing only a discrete number of regulatory elements – are more responsive to a subset of regulators. For example, the synthetic -6.5kdrl promoter that we use the assess SoxF activity is highly responsive to Sox7 and Sox18 activity, while the endogenous gene kdrl (flk1) is not . We also observe this for this synthetic promoter fragment using both Morpholino approaches and Sm4-treatment .We agree with the interpretation regarding the discrepancy in terms of activity comparing dll4 solely relies on SoxF activity. It has been shown that another dll4 enhancer Dll4int12 is also required to drive proper expression of this gene in endothelial cells . This enhancer relies on Ets and Rbpj combinatorial mode of action and it is unknown whether SoxF are at play to modulate this particular regulatory element.Work by Sacilotto et al. demonstrated that deletion of the Sox binding motif in this Dll4int3 enhancer fragment leads to a loss of transcriptional activation, which demonstrates that this transgene is dependent on SoxF activity . This does not mean that the endogenous regulation of Dll4in3 activation, and correspondingly, we show in our study that Sm4 treatment slightly affects this transgene in zebrafish . A more profound effect was observed when Sm4-treatment was combined with rbpj morpholino injections. Overall, we do not claim that Sox18 is a master regulator of dll4 expression, nor do we suggest that Sm4 is a chemical regulator of dll4 transcription. Instead, we utilize Dll4int3 synthetic enhancer activity as a readout for on-target Sox18 inhibition. To make this clear to the reader, we have adjusted the text accordingly (tenth paragraph of the main text).In the context of vascular development in the zebrafish and mouse, Sacilotto et al. showed that individual loss of SoxF proteins or Rbpj has little effect on Sm4 on dll4 endogenous transcriptional activation we have performed in situ hybridization on DMSO ctrl and Sm4 treated zebrafish larvae, as suggested , which also show a very mild reduction in the overall dll4 transcript levels.This analysis shows that the effect of Sm4 on the endogenous 3) The mechanism of reduced tumour angiogenesis and metastasis should be adequately discussed and ideally experimentally clarified. Given that the route of metastasis is likely through lymphatics and that Sox18 also regulates lymphangiogenesis, we feel you should consider this as potential mechanism. As you have the expertise in mouse lymphatic analysis from previous studies, we hope you will be able to provide experiments that show whether or not SM4 interferes with tumour lymphangiogenesis through blocking Sox18 function. It will not be necessary to show that this is the definitive cause for changes in metastasis, but we feel that without analysing lymphatics, this work is incomplete. Given that you use a xenograft model, it should be feasible to perform these experiments within the 2 months of revision period.We greatly appreciate this suggestion, given the reported role of Sox18 in lymphangiogenesis and the contribution of lymphatic vasculature to the malignancy of many types of solid tumour. To address this request, we have quantified the tumour-induced lymphangiogenic response in absence or presence of Sm4. Immunofluorescence staining of 4T1.2 tumour sections was performed for lymphatic specific markers PROX1 and Podoplanin along with the blood vascular marker endomucin. Results show that both lymphatic vessel density and number of lymphatic endothelial cells is reduced in presence of Sm4 treatment. This lack of lymphatic outgrowth in presence of the small molecule is likely to contribute to the decrease in lung metastasis and improved disease latency. This result is now included in the manuscript as new Figure 4—figure supplement 3 and in the main text (fifteenth paragraph).


HP-25 gene is repressed in hibernating chipmunks under the control of endogenous circannual rhythms. However, the molecular mechanisms that differentially regulate the HP-25 gene during the nonhibernation and hibernation seasons are unknown. Here, we show that the hibernation-associated HP-25 expression is regulated epigenetically. Chromatin immunoprecipitation analyses revealed that significantly less hepatocyte nuclear receptor HNF-4 bound to the HP-25 gene promoter in the liver of hibernating chipmunks compared to nonhibernating chipmunks. Concurrently in the hibernating chipmunks, coactivators were dissociated from the promoter, and active transcription histone marks on the HP-25 gene promoter were lost. On the other hand, small heterodimer partner (SHP) expression was upregulated in the liver of hibernating chipmunks. Overexpressing SHP in primary hepatocytes prepared from nonhibernating chipmunks caused HNF-4 to dissociate from the HP-25 gene promoter, and reduced the HP-25 mRNA level. These results suggest that hibernation-related HP-25 expression is epigenetically regulated by the binding of HNF-4 to the HP-25 promoter, and that this binding might be modulated by SHP in hibernating chipmunks.The chipmunk hibernation-related protein 25 (HP-25) is involved in the circannual control of hibernation in the brain. The liver-specific expression of the  Of the many physiological seasonal adaptations that occur in animals in response to the physical world, hibernation is one of the most extreme. Mammalian hibernation is a unique circannual physiological adaptation that allows life to be sustained at extremely low body temperatures, which can fall below 10 °C or even 5 °C. During hibernation, heart and breathing rates drop, and the metabolic rate falls to only a few percent of the euthermic level, resulting in a considerable conservation of energyTamias asiaticus (chipmunk) hibernation-related protein 25 is a component in a 140-kDa complex, consisting of HP-20, HP-25, HP-27, and HP-55, that decreases drastically in the blood during hibernation2et al. showed that the onset of hibernation is marked by an increase in the HP-20c protein complex in the brain; this complex is composed of HP-20, HP-25, and HP-27The HP-25 gene, which is expressed specifically in the liver, is downregulated in hibernating chipmunksHP-25 mRNA level in the liver fluctuates in parallel with levels of the 140-kDa complex in the blood. Since the levels of the 140-kDa complex fluctuate independently of changes in body temperatureHP-25 mRNA expression in the liver is thought to be controlled by endogenous circannual rhythms, rather than by changes in body temperature or by metabolic inhibition at low body temperatures. Thus, elucidating the mechanism of such hibernation-associated endogenous circannual rhythms will shed light on the molecular mechanisms of mammalian hibernation.The in vitro studies revealed that hepatocyte nuclear factor 4 (HNF-4) activates HP-25 transcription7611Our previous HP-25 expression in chipmunks. We first analyzed whether the hibernation-associated loss of HP-25 expression is regulated at the transcriptional level. Subsequently, we focused on the HNF-4-regulated transcriptional activity and on hibernation-associated changes in epigenetic marks on the HP-25 gene promoter region. Our results suggest that HNF-4 and its coactivators bind the HP-25 promoter region to epigenetically regulate the HP-25 transcription changes associated with circannual hibernation.In this study, we investigated the mechanism of the hibernation-associated loss of HP-25 gene is downregulated in the liver of hibernating chipmunks, based on Northern analysisHP-25 primary transcripts; this approach to determining a gene’s transcriptional state is an alternative to the nuclear run-on assayHP-25 primary transcripts was compared by RT-PCR using a pair of primers, one located in exon 1 and the other in intron 1 , which is a helix-loop-helix transcription factor5HP-25 gene is not expressed [HP-25 mRNA was detected only in the liver, but not in the kidney, by northern analysisOur previous HP-25 gene  to determine whether HNF-4, USF-1, and USF-2 were involved in the liver-specific transcription of the -25 gene . In the -25 gene . In the  by ChIP . While td kidney . The resifically , consisthipmunks , where thipmunks . In the hipmunks . On the hipmunks . These fHP-25 gene transcription, we first compared the total amount of histone H3 acetylated at K9 and K14 and trimethylated at K4 in the liver between nonhibernating and hibernating chipmunks by immunoblotting  4 and the acetylation at K9 and K14, are generally involved in gene activation, whereas the lack of histone acetylation is a hallmark of gene repression171819blotting . The tot by ChIP . Histonein genes . Considein genes , these hHP-25 gene promoter, we used ChIP to analyze the histone acetyltransferases (HATs), histone methyltransferases (HMTs), and histone deacetylases (HDACs) bound to the HP-25 promoter region  in the liver of nonhibernating chipmunks.To investigate the involvement of HATs and HMT(s) in activating the 4 singly  or in co4 singly . Althougcription . CBP’s eHP-25 transcription was probably repressed in hibernating chipmunks due to reduced levels of HNF-4 bound to the promoter , and active transcription histone marks are removed from the promoter region  genes are expressed specifically in the liver, circulating levels of HP-25 and HP-27 in the blood and cerebrospinal fluid change seasonally as is the case with the chipmunk HPs49HP-25 gene promoter (data not shown), transcription of the cow HP-25 gene may be regulated by a general nuclear receptor HNF-4 as the chipmunk HP-25 gene. Although it has not been established that cow HPs oscillate in an endogenously generated circannual manner51HP-25 transcription and HNF-4’s binding to the HP-25 promoter poses an interesting challenge. Based on our results, elucidating the upstream factors that control the epigenetic regulation of HP-25 transcription will help to identify the circannual signals from the circannual pacemaker common to hibernators and nonhibernators. Also, comparative analyses of the pathways affecting the HP-25 gene transcriptional regulation between hibernators and nonhibernators, together with functional analysis of the HP-25 protein, may lead to identification of hibernation signals.The r region . Since tAll recombinant DNA experiments in this study strictly followed a protocol and guidelines approved by the Institutional Recombinant DNA Experiment Safety Committee of Kitasato University.Tamias asiaticus) purchased from Pet Easy Space (Osaka) were individually housed and offered standard rodent chow and water ad libitum. The chipmunks were kept at 23 °C with a 12 h:12 h light:dark period during the nonhibernation season (April–September) and at 5 °C in darkness during the hibernation season (October–March). Nonhibernating chipmunks were summer-active animals sampled in June or July. Hibernating chipmunks were sampled approximately three months after first immergence into torpor. The topor bouts usually last 5–6 days. At the sampling time, these hibernating chipmunks had been hibernating for 3–4 days: they were torpid, and their body surface temperatures were under 10 °C measured by an infrared thermometer. All animals were deeply anesthetized with isoflurane prior to sacrifice. Tissues were immediately excised, frozen in liquid nitrogen, and stored at −80 °C until use. All of the protocols were in accordance with the guidelines of the Institutional Animal Care and Use Committee of Kitasato University, and all experimental procedures were approved by the same committee.Male chipmunks  was treated with DNase I using the TURBO DNA-free kit (Ambion). The reverse transcription of total RNA and subsequent PCR were performed using the mRNA Selective PCR Kit (Takara) to amplify the Nuclear extracts were prepared from the liver, kidney, heart, and lung of nonhibernating and hibernating chipmunksHP-25 gene primer sets, CM25-230F and CM25 + 79 R, and CM25-166F and CM25-24R, can amplify the regions encompassing the USF-1, USF-2, and HNF-4 binding sites. The PCR amplimers were confirmed to be a single band of the correct size by agarose gel electrophoresis. ChIP-qPCR data were normalized by the % input method and were shown as the fold increase over the value in the nonhibernating chipmunk liver. Results are expressed as means ± SEM of the indicated number of independent determinations.Chromatin immunoprecipitation (ChIP) was performed as described previously4 cells in a 24-well plate. After 24 h, the cells were transfected with 400 ng of a firefly luciferase promoter-reporter plasmid, and with 10 ng of the Renilla luciferase internal control plasmid pRL-SV40 (Promega), using TransIT-LT1 reagent (Mirus). Where denoted, the cells were cotransfected with 100 ng of the mouse CBP expression construct pcDNA3/mCBP, 100 ng of the mouse SETD1A expression construct pcDNA3/mSETD1A, 5 ng of the chipmunk HNF-4 expression construct pcDNA3/cmHNF-4, and/or 100 ng of the chipmunk SHP expression construct pcDNA3/FLAG-cmSHP. After 24 h, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Where denoted, curcumin (Sigma) was added at the indicated concentrations 12 h after transfection, and the luciferase activity was measured after another 12 h.The promoter-reporter plasmid construction of pCM25G-260/luc was described previously2O, mounted on a slide, and observed under the microscope.HeLa cells were grown on uncoated glass coverslips and transfected with pcDNA3/cmHNF-4 and pcDNA3/FLAG-cmSHP using Polyethylenimine Max . At 24 h after transfection, the cells were fixed with 10% Formaldehyde Neutral Buffer Solution  for 10 min, mounted on glass slides, and observed with a BZ-8100 fluorescence microscope (Keyence) or an LSM510 confocal laser-scanning microscope (Zeiss). To detect HNF-4 and FLAG-SHP, the cells were incubated with an anti-HNF-4 antibody and anti-DYKDDDDK antibody overnight at 4 °C, washed three times with 0.5% BSA in PBS, and incubated with Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Thermo Scientific) for 1 h at room temperature. To detect nuclei using a fluorescence microscope, the cells were washed twice with PBS and stained with 1 μg/ml Hoechst 33258 (Thermo Scientific) solution for 10 min at room temperature. The cells were then washed with Hg for 30 min at 4 °C. The supernatants were collected and incubated with Ab-Capcher (ProteNova) and IgG. The pre-cleared samples were centrifuged, and the supernatants were collected. The pre-cleared supernatants were incubated with an anti-HNF-4 antibody, anti-FLAG M5 antibody (Sigma Aldrich), or pre-immune IgG (used as a negative control) at 4 °C overnight. Immunoprecipitated samples were then captured by Ab-Capcher. The Ab-Capcher beads were washed three times with wash buffer , and the immunoprecipitated proteins were eluted with SDS-PAGE loading buffer. The eluates were analyzed by immunoblotting using an anti-HNF-4 or anti-DYKDDDDK antibody.HeLa cells were plated on a 10-cm dish, and transfected with pcDNA3/cmHNF-4 and pcDNA3/FLAG-cmSHP using Polyethylenimine Max . At 48 h after transfection, the cells were lysed in RIPA buffer  for 30 min at 4 °C. The lysates were centrifuged at 12,000 x t-test.Total RNA was obtained from chipmunk livers and primary hepatocytes using ISOGEN II (Nippon Gene). First-strand cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara) with random primers following the manufacturer’s instructions; qPCR was performed with four samples in technical triplicate using the SYBR Green Realtime PCR Master Mix (Toyobo) and a Rotor-Gene Q apparatus (Qiagen). The following PCR primers were used: cmSHP + 490 F, 5′-CTGGGCCCCAAAGAGTATGC-3′, and cmSHP + 572 R, 5′-ATGTGGGAGGAGGCATGAAG-3′; cmHP25 + 607 F, 5′-AAGCCAATGACAGCTACAAACAT-3′, and cmHP25 + 799 R, 5′-TTCAGAATTACAAGAGGGATGGA-3′; cmALB + 502 F, 5′-AGAAAATAACCTGAGGCTTTTGG-3′, and cmALB + 675 R, 5′-TTCATAGCCTCAATCTTTGGTGT-3′; cmACTB + 226 F, 5′-GTCACCAACTGGGACGACAT-3′, and cmACTB + 401 R, 5′-ACATACATGGCTGGGGTGT-3′. Beta-actin (ACTB) was used as an endogenous control to normalize gene expression. Relative mRNA expression levels were presented as means ± SEM. Statistical differences were analyzed by Student’s Primary hepatocytes of nonhibernating chipmunks were obtained by the conventional collagenase method with minor modifications. Following liver perfusion with collagenase type IV (Worthington), the dispersed cells were filtered successively through 500-μm and 125-μm stainless steel sieves, and washed with DMEM four times. In all experiments, the hepatocytes were more than 80% viable as judged by trypan blue exclusion. After centrifugation, the cell pellet was resuspended in Williams’ medium E supplemented with 10% FCS, 1 mM insulin, 1 mM dexamethasone, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.5 mg/ml fungizone, and 0.1 mg/ml kanamycin, and the hepatocytes were plated on collagen-coated BioCoat dishes (Corning). On the next day, pcDNA3/FLAG-cmSHP or pcDNA3/FLAG were transfected into the hepatocytes using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. The hepatocytes were cultured for 2 additional days, and then whole-cell extracts and total RNA were prepared.t-test , Tukey-Kramer Multiple Comparison test, one-way ANOVA, or two-way ANOVA were used to compare the difference from the control group. When the interaction was significant at p < 0.05 by one-way ANOVA or two-way ANOVA, post hoc multiple comparison was made using Tukey-Kramer test. Two-way ANOVA was also used to analyze the interaction between treatments.Student’s Accession codes: The accession number for the chipmunk SHP sequence reported in this paper is Genbank: LC144633.How to cite this article: Tsukamoto, D. et al. HNF-4 participates in the hibernation-associated transcriptional regulation of the chipmunk hibernation-related protein gene. Sci. Rep.7, 44279; doi: 10.1038/srep44279 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development.The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as  The process of progenitors undergoing cell fate decisions to determine specific cellular identity and tissue patterning is fundamental in the field of developmental biology. Neural development is an excellent model system to study cell fate processes to understand how neural restricted progenitors differentiate into diverse types of neurons through complex interactions of transcriptional activators and repressors. Multiple regulatory design principles involving combinations of transcription factors have emerged through studies from vertebrate and invertebrate model systems that have significant repercussions in determining and maintaining cell identity . During The dorsal spinal cord functions as an essential center for integration of somatosensory information . ApproprPRDM13 is a transcriptional repressor present in progenitor domains of the dorsal but not the ventral neural tube from hindbrain regions to the spinal cord  Figure . It is aPrdm13 mutant mouse strains. In contrast to the previously reported Prdm13 mutant deleting exons 2 and 3  of PRDM13 ΔZF)  . This ZFdm13ΔZF) . The phend PTF1A . PRDM13-nd PTF1A  do not dnd PTF1A . A thirdm13∆115) . Surpris protein . Westerndm13∆115 . This muPrdm13 was identified as an important specification gene in the dorsal neural tube required for the balance of inhibitory  interneurons and excitatory  interneurons in the chick neural tube  and an increase in dI3/5 (TLX1/3+) in both the GFP/GFPPrdm13 and ΔZF/ΔZFPrdm13 neural tubes was seen at this stage. These phenotypes are identical to the Ptf1a null mouse  (for review see ). Prdm13ral tube . Overexping PAX2 . Supportulations . A complll mouse   in the Prdm13 mutants that is not seen with the loss of Ptf1a , a marker of excitatory neurons, is detected in the lateral region of the domain lacking Gad1  in dorsal neural tube cells in the presence and absence of PRDM13. RNA-seq was performed on GFPal tubes . 837 gengulation .Tlx3 , and used only those sites identified in at least two experiments. We identified 220 PRDM13 binding sites largely located at distal sites relative to the transcription start sites of genes. These targets included a known target of PRDM13, Tlx3 . The gen of Tlx3 . This reenhancer .De novo motif analysis failed to identify a novel binding motif unique to PRDM13, but PRDM13 bound sites were enriched for motifs for known neural TFs such as E-box (bHLH) (40%), Rfx (17%), Pdx (HD) (25%) and Sox (30%) motifs . Indeed,GFPPrdm13 neural tubes  whereas only six are decreased   .Prdm13 mutants. For example, PRDM13 binds the Tlx3 enhancer and restricts its expression at E10.5/E11.5 (eTlx3). Consistently, Tlx3 is elevated 17-fold, while Pax2 is reduced 6.3-fold in the absence of Prdm13  ( control . To dete control . Becausecription  these dacription  where th (Ptf1a) .Ptf1a transcription is regulated through a 2.3 kb autoregulatory enhancer (e2.3Ptf1a)    . PTF1A b.3Ptf1a) . One posr region .Ptf1a autoregulatory enhancer using the chick electroporation assay and a GFP reporter under the control of the 2.3 kb Ptf1a autoregulatory enhancer (e2.3kb-Ptf1a::GFP) , and HEB (Tcf12) (Ptf1a transcription by interrupting PTF1A auto-regulation through binding with PTF1A to the Ptf1a autoregulatory enhancer (We next tested the ability of PRDM13 to regulate transcription through the 1a::GFP) . As was activity . In contmpletely . Finallympletely . Becausempletely   . We propenhancer .Prdm13 and Ptf1a loss of function phenotypes in the second wave of neurogenesis in the dorsal neural tube ). The latter finding is striking because these ventral specification factors are restricted to the ventral neural tube. Three factors with the highest fold increase in the Prdm13 mutant are Olig1, Olig2 and Prdm12 , as well as a number of neural related factors misregulated upon loss of PRDM13 . Notablyd Prdm12 . We usedOlig1 and Olig2 show striking ectopic dorsal expression within GFP/GFPPrdm13 neural tubes  in the ventral spinal cord at these early stages of neurogenesis, and it is required for specification of motor neurons  Figure . Upon loal tubes . This ab mutants . Co-labe mutants . Notably mutants , factorsOlig1 and Olig2, we identified three genomic regions surrounding the Olig genes bound by PRDM13 from ChIP-seq  and is required for generation of V1 interneurons  survived to adulthood and exhibited a retinal phenotype  for function  activity dependent on the PR domain of the protein . Those PDecades of work have gone into defining progenitor domains and resulting neuronal populations in the developing spinal cord mainly by combinatorial TF expression of HD and bHLH factors . RepressPrdm13 mutants they are aberrantly present dorsally, altering the identity of these neurons. Taken together, PRDM13 is a transcriptional repressor essential in the dorsal spinal cord development to modulate the transcriptional activity of neural bHLH factors to generate precise neuronal identities.Here we provide evidence for an additional variation of these models whereby the transcriptional repressor is broadly expressed and it limits inappropriate gene expression driven by progenitor-specific activators, the bHLH factors. PRDM13 is identified as a key transcriptional repressor of a whole battery of genes, not just those in domains adjacent to the domain giving rise to the dorsal inhibitory neurons, but also factors normally restricted to the ventral neural tube and required for neuronal identity in that region  Figure . PRDM13 Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Jane E. Johnson (jane.johnson@utsouthwestern.edu).GFPPrdm13 mutant and the fusionGFPPrdm13 mouse model were developed using zinc-finger nuclease technology (Prdm13 gene (CACCAGCGTGAACGCTGActgctGCATCCCGGCTGGCT) were purchased from Sigma-Aldrich  and delivered by pronuclear injection into fertilized mouse eggs along with a donor plasmid. For GFPPrdm13 mutant mice, the donor plasmid encoded GFP followed by a stop codon, and contained two homology regions each 750 bp in length to allow for homologous recombination of the insert into the Prdm13 locus (fusionGFPPrdm13 mouse that was used for ChIP-seq. Homozygous fusionGFPPrdm13 mice live to adulthood and show no changes in PAX2 or TLX1/3 expression at E10.5 (data not shown). Mice were genotyped by PCR with 5’-GCTGCTCCTGGTTCTGTCA-3’, 5’-CCTTTTCTCTGCTGCTCGTC-3’ and 5’-GCTGGAGTACAACTACAACAGCCA-3’ .The chnology . mRNA en13 locus . Out of Δ115Prdm13 mutant line is a 115 bp deletion generated within the first exon of Prdm13 upon injection of the ZFN mRNA . Homozygous Δ115/ Δ115Prdm13 mice live to adulthood, and show no changes in PAX2 or TLX1/3 expression at E10.5. PCR genotyping used primers 5’-GCTGCTCCTGGTTCTGTCA-3’ and 5’-CCTTTTCTCTGCTGCTCGTC-3’  (The  198 bp) .ΔZFPrdm13 mutant mouse line was generated by pronuclear injection of CAS9 mRNA and two sgRNA targeting the 3’ end of Prdm13 . The CREPtf1a mouse line replaces the coding sequence for Ptf1a with that for Cre recombinase  and cryosectioned at 20 µm (E10.5-E11.5) and 30 µm (E12.5-E16.5) focusing on sections from the upper limb level.20/1X loading dye and injected into the lumen of the closed neural tube at stages HH13-15 along with either a pMiWIII-Myc epitope tagged vector serving as an electroporation control or a TF containing expression vector. The injected embryos were then electroporated with 5 pulses of 25 mV each for 50 msec with intervals of 100 msec. Embryos were harvested 48 hr later at stages HH22-23, fixed with 4% paraformaldehyde for 45 min, and processed for cryosectioning and immunofluorescence.For in ovo chick electroporation assays, fertilized White Leghorn eggs were obtained from the Texas A and M Poultry Department  and incubated for 48 hr at 37⁰C. The supercoiled reporter plasmids were diluted to 1.5 mg/ml in HImmunofluorescence was performed using the following antibodies: guinea pig anti-PRDM13 , mouse Prdm13 and Ptf1a (Prdm12 (Gad1 and Vglut2 (Slc17a6) . The chick Prdm12 probe was generated starting from a pBSK+ cPrdm12 cDNA (EST BU233582). Probes for Ccbe1 and Mfng were generated by PCR from mouse neural tube cDNA. Imaging was performed with a Hamamatsu Nanozoomer 2.0HT digital slide scanner.In situ hybridization was performed as previously described . Probes nd Ptf1a , mouse P  , mouse Od Phox2b , mouse Ob , e2Prdm12 , e1Olig , e2Olig , e3Olig . E1Olig-EboxMut, e2Olig-PTF1Mut and e2Olig-allEboxMut were subcloned using gBlocks with E-box mutations . KpnI/SpeI restriction sites were introduced on each end for cloning adjacent to the β-globin minimal promoter in the reporter cassette. The Ptf1a 2.3 kb enhancer reporters are as described   with pMiWIII  expressi in pCIG , and toto acids) . The HEKGFPPrdm13 embryos either heterozygous or homozygous for GFP were individually placed into cold DMEM/F12 and dissociated in 0.25% trypsin for 15 min at 37 degrees C. Trypsin activity was quenched with 2% fetal bovine serum, and GFP positive cells were purified from the resulting single cell suspension by fluorescence activated cell sorting (FACS). Total RNA from FACS isolated cells was extracted and purified with Mini RNA Isolation Kit . Genotypes were determined and RNA from 9 homozygous and 7 heterozygous embryos were combined, mRNA was purified, reverse transcribed and amplified for sequencing with Illumina’s mRNA-Seq kit  . Gene exfusionGFPPrdm13 embryos were used for GFP ChIP. ChIP was performed as previously described (PTF1A and ASCL1 ChIP-Seq were previously published  (GSE5584escribed . Notablyescribed . LibrariSequence reads for each sample were mapped to the mm10 genome assembly by using Bowtie2 (v.2.2.6) . Mapped enh::GFP expression was measured by ImageJ on the electroporated side of the neural tube with the background levels measured from the non-electroporated side subtracted. Significant differences between control and experimental samples in each case were calculated using a two-tailed two-sample unequal variance (homoscedastic) Student’s t-test in Microsoft Excel. *p<0.05, **p<0.01, ***p<0.001, ns = not significant. Error bars indicate SEM. The enrichment of PRDM13 ChIP targets in the list of genes that showed significant expression change in mutant samples relative to controls was calculated using the hypergeometric test.In sections from mouse embryos with cells labeled by immunofluorescence  and 3, qRNA-Seq and ChIP-Seq data generated here are available on the GEO database (GSE90938). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Diogo S Castro (Reviewer #1); John LR Rubenstein (Reviewer #2).Thank you for submitting your article "Repression by PRDM13 is critical for generating precision in neuronal identity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submissionThe described study utilizes newly generated null mutations in Prdm13 to investigate the function of this transcriptional regulator in the developing dorsal spinal cord. Resulting data supports previous findings that Prdm13 suppresses the fate of excitatory neurons in inhibitory lineages. By a combination of developmental and genomic assays, the authors demonstrate that the recruitment of PRDM13 to bHLH bound enhancers inhibits the expression of ventral transcription factors in the dorsal neural tube. While consistent with current models of neural tube patterning this study provides significant mechanistic insight into the Prdm13-mediated gene regulatory network that regulates neurogenesis in the dorsal spinal cord.While a carefully performed study, interpretation of the data would be further strengthened by additional analysis which must be adequately addressed in a revised manuscript.1) Prdm13 ChIP-seq peaks do not show enrichment for a specific motif but instead for several sequences recognized by various neural transcription factors, suggesting Prdm13 is indirectly recruited to DNA. Thus, it become even more important to check the specificity of the Prdm13 antibody. This should be done by validating a series of peaks (associated with the various DNA motifis) in chromatin from wild type and Prdm13 null embryos.2) In 3) Although the authors conclude that "these findings highlight the function of PRDM13 in repressing bHLH transcriptional activators" which implicitly includes Ascl1, the manuscript does not provide an example of a validated Ascl1/Prdm13 target. The authors should rewrite the conclusion accordingly, so that it is not misleading. While a carefully performed study, interpretation of the data would be further strengthened by additional analysis which must be adequately addressed in a revised manuscript.We thank the reviewers for their time and suggestions for improving this study. As you will see below, in performing additional analysis in response to the reviewer’s comments we have significantly strengthened our findings and thus, support for our conclusions.1) Prdm13 ChIP-seq peaks do not show enrichment for a specific motif but instead for several sequences recognized by various neural transcription factors, suggesting Prdm13 is indirectly recruited to DNA. Thus, it become even more important to check the specificity of the Prdm13 antibody. This should be done by validating a series of peaks (associated with the various DNA motifis) in chromatin from wild type and Prdm13 null embryos.Prdm13 mutant neural tubes. Instead we offer a different set of experiments that we believe serves a similar purpose and provides a high confident set of PRDM13 target genes. We now identify PRDM13 target genes based on 3 PRDM13 ChIP-seq data sets that were generated with 3 independent strategies. These include: 1) ChIP-seq data from the original submission that is from E11.5 neural tube tissue using a lab produced antibody generated against the terminal ZnF domain of mouse PRDM13. 2) ChIP-seq data from E11.5 neural tube tissue using an independently produced antibody generated against full-length mouse PRDM13. Here, telencephalon, a PRDM13 negative tissue, was used to control for non-specific binding of the antibody. 3) We generated a mutant mouse strain with a GFP fused in-frame N-terminal to the PR domain of PRDM13. Mice homozygous for this GFP-PRDM13 fusion are indistinguishable from wildtype. ChIP-seq data from E11.5 neural tube tissue from these animals using an antibody directed against GFP was generated.We agree it is important to strengthen the data identifying PRDM13 bound sites in the genome and provide some validation for these sites. While we would have liked to provide data from the suggested experiment, we have been unable to obtain efficient enough ChIP enrichment from the limited source material provided by Ptf1a, Olig2, and Prdm12 were all confirmed as PRDM13 targets providing the validation requested by the Reviewers. We show the genome browser tracks for all three ChIP-seq experiments in supplementary data associated with This exercise had a substantial positive impact on the support for the conclusions reported. We restricted our analysis to PRDM13 bound sites that were called as peaks in at least 2 of the 3 ChIP-seq datasets. This limited the number of PRDM13 target genes identified. The genes highlighted in the study including Prdm13 mutants relative to controls were identified as PRDM13 targets , whereas only 6 of 356 genes that decreased in the Prdm13 mutants were identified as targets . These findings support the role of PRDM13 as a repressor  In Olig1/2. We did this for e1Olig and e2Olig rather than e3Olig for reasons described below, and because e3Olig was not responsive to activation by PTF1A or ASCL1. These experiments demonstrate that the E-boxes are required for e1Olig and e2Olig enhancer activity. In the process of performing these experiments we corrected an error in the data reported in the original manuscript. The e1Olig sequence originally tested was not the correct sequence (consistent with its lack of activity in the reporter assay). In the current revised manuscript, we corrected this error. None of the 3 Olig enhancers direct reporter expression restricted in the endogenous Olig domains. Nevertheless, e1Olig and e2Olig require the Ebox motifs within the enhancers for their activity  Although the authors conclude that "these findings highlight the function of PRDM13 in repressing bHLH transcriptional activators" which implicitly includes Ascl1, the manuscript does not provide an example of a validated Ascl1/Prdm13 target. The authors should rewrite the conclusion accordingly, so that it is not misleading.Tlx3 previously in Chang et al., 2013 and this is described and referenced where relevant. In the current study, we provide additional support for those conclusions showing the increase in Tlx3 in the Prdm13 mutant mice. In the revised manuscript, we now point out that although this current study does not show an example of a validated ASCL1/PRDM13 target, this was shown in Chang et al., 2013 for regulation of TLX3 at the site highlighted in We disagree that this statement in the abstract is misleading. In this manuscript, we explicitly provide evidence for PRDM13 repressing multiple bHLH activators including PTF1A, NEUROG1, and NEUROG2. We don’t explicitly show a validated ASCL1/PRDM13 target here, but we have demonstrated this for