Arabidopsis, 79% of genes contain introns and more than 60% of intron-containing genes undergo alternative splicing (AS), which ostensibly is considered to increase protein diversity as one of the intrinsic mechanisms for fitness to the varying environment or the internal developmental program. In addition, recent findings have prevailed in terms of overlooked intron functions. Here, we review recent progress in the underlying mechanisms of intron function, in particular by focusing on unique features of the first intron that is located in close proximity to the transcription start site. The distinct deposition of epigenetic marks and nucleosome density on the first intronic DNA sequence, the impact of the first intron on determining the transcription start site and elongation of its own expression , translation control in 5′-UTR, and the new mechanism of the trans-acting function of the first intron in regulating gene expression at the post-transcriptional level are summarized.Most of the transcribed genes in eukaryotic cells are interrupted by intervening sequences called introns that are co-transcriptionally removed from nascent messenger RNA through the process of splicing. In  More than four decades ago, we underwent a paradigm shift in gene organization in higher organisms and caught a glimpse of the molecular processes in manufacturing messenger RNA from heterogeneous nuclear RNA (hnRNA). In 1977, electron microscopy images visualizing DNA:RNA hybrids annealed between adenovirus 2 hexon mRNA and restriction endonuclease cleavage fragments of viral DNA were the smoking gun proof demonstrating that cytosolic mRNA is matured through the removal of intervening sequences from nascent mRNA, called splicing . He alsoArabidopsis revealed that both 5′- and 3′-splice sites (5′-SSs and 3′-SSs) are preferentially enriched in nucleosomes, nucleosomal DNA is more highly methylated, and nucleosomes are enriched in exons [met1-2 null allele, whose methyl-CG level is globally lost, are affected, suggesting the involvement of DNA methylation in AS, but the contribution is relatively minor [In humans, the first intronic DNA sequence is the longest and most conserved, and exhibits the highest density of regulatory chromatin marks when compared to the remaining intronic sequences ,18. DNA in exons . Interesin exons . Repressin exons . In humain exons . Recent in exons ,26 In huin exons  IR in plin exons . In ricely minor .in trans [HID1) encodes ncRNA that forms a protein complex in the nucleus to bind to the PIF3 first intronic sequence to repress PIF3 expression at the transcriptional level [When intronic regions are larger, regulatory elements are likely to be embedded within them to regulate their gene expression ,31 A welin trans . On the in trans  or are tal level . This waFLC) first intron contains two RY  cis-elements (TGCATG) that are recognized by the B3 DNA binding domain of VAL1 (VIVIPAROUS1/ABI3-LIKE factor 1), a transcriptional repressor capable of repressing FLC expression by modifying the H3K27me3 level in the FLC intronic region and decreasing H3 acetylation in the first exon by recruiting plant homeodomain-polycomb repressive complex 2 (PHD-PRC2) [The FLOWERING LOCUS C (HD-PRC2) .As described above, exonic and intronic DNA exhibit distinct nucleosome occupancy/chromatin structure, as well as DNA and histone depositions, all of which could define RNA polymerase II (Pol II) elongation speed. Due to high GC content and concentrated nucleosomes in the exonic region, elongation speed is slower than that in the intronic region , top 3838,39 As Throughout the whole processes of transcription, Pol II interacts with various proteins  via its carboxy-terminal domain (Pol II-CTD) in post-translational modification, especially in a phosphorylation-dependent manner . It is wArabidopsis, cap-binding proteins, CBP20 and CBP80, and its interacting partner SERRATE (SE) modulate the first intron AS by affecting the 5′-SS of the first intron [Nuclear cap-binding complex (CBC) consists of two subunits, CBP20 and CBP80, which bind to the cap structure of transcripts and affect splicing . From tht intron ,52,53ASCO-lncRNA) highjacks AS regulators by interacting with NUCLEAR SPECKLE RNA-BINDING PROTEIN (NSR), resulting in IR of NSR-target transcripts [Arabidopsis and rice genomes target intronic sequences [Non-protein-coding RNAs are shown to modulate AS, especially IR in plants. AS COMPETITOR LONG NONCODING RNA  in maize [ADH1 construct [It is widely recognized that the presence of intron stimulates mRNA accumulation . Back inin maize , and theonstruct ,56 This The majority of mammalian promoters initiate transcription on both sides in opposite directions, called divergent transcription ,58,59 whADH1 [GAP1 [UBI1 [SODCC2 (cytosolic superoxide dismutase) [Arabidopsis PAT1 (phosphoribosylanthranilate transferase 1) [UBQ) genes [MHX [Arabidopsis and rice were carried out, and a word-based discriminator called the IMEter was developed [Arabidopsis and TCGATC in rice [Arabidopsis have been further revealed. Contrary to IME observed in yeast cells [UBQ10 first intron not only increases mRNA accumulation, but also affects tissue specificity when the reporter gene containing the UBQ10 first intron is under the control of different promoters [UBQ10 intron renders tissue specificity over promoter sequence. Likewise, the first intron of PROFILIN2 (PRF2), encoding a small actin-binding protein expressed in vegetative organs, is capable of boosting mRNA accumulation and confers vegetative tissue expression when driven by reproductive tissue-specific PRF5 promoter [Investigations into plant IME have been extensively carried out by Rose’s group ,66. For ADH1 , SHRUNKEADH1 ,68 GAP1 H1 [GAP1 , UBI1 [7P1 [UBI1 , rice SOsmutase) , Arabidoerase 1) , UBIQUITQ) genes , and MHXnes [MHX ,75 have eveloped . Consequ in rice ,77,78. H in rice . The abist cells ,62, splist cells ,80 The Uromoters , suggestpromoter .UBQ10 first intron determines the transcription start site (TSS) from where it locates  is the major AS in plants ,89, but GGT1) contains a TGTGATTTG sequence that is highly similar to IME motif and enhances leaf-specific expression at the transcriptional level by enriching Pol II abundance [ZIF2 (encoding ZINC-INDUCED FACILITATOR 2) is shown to provide a stable stem-loop structure that enables more efficient translation when compared to the spliced variant, in which the stable structure is lost by splicing [HAC1 represses translation [When AS of the first intron within 5′-UTR takes place, the impact on gene expression will be quite the opposite. A positive effect of the first intron retention on boosting mRNA accumulation was shown by Laxa et al.: the first intron within 5′-UTR of Glu:glyoxylate aminotransferase 1 . In ediction , and higediction . The preediction ,102,103.ediction  and -indediction ,106.ASV1, one of the splicing variants containing longer 5′-UTR of PHYTOENE SYNTHASE (PSY) mRNA, acting as a riboswitch-like module to repress translation and AS of the first intron is the key to modulating retrograde signaling [Arabidopsis [CRK10), whose expression is downregulated by cytokinin application, is likely to contain an adenine aptamer-related sequence in its first intronic sequence [Riboswitches are regulatory elements in mRNA, predominantly found in the noncoding region, that act as metabolite sensors by selectively binding to ligands and regulate transcription or translation without the need for protein factors . It is pignaling . Similarbidopsis . Having sequence . In bothSlyWRKY75 contains the first intron downstream of ATG, and its expression is regulated by miR1127-3p, which is complementary to the first intron sequence of SlyWRKY75. Under noninfected conditions, the first intron is retained, whereas upon Botrytis cinerea pathogen challenge, the overall SlyWRKY75 transcript level is elevated, which is in sharp contrast to compromised miR1127-3p expression. Interestingly, pathogen challenges facilitate splicing of the retained first intron that remained unspliced under noninfected conditions [miR1127-3p binds to SlyWRKY75 pre-mRNA in the nucleus to inhibit the splicing of target intronic region under noninfected conditions.IR that takes place in introns positioned after the ATG affects protein in its sequence, function, subcellular localization, or IR mRNAs is subjected to degradation by the NMD ,114. IR,nditions . These rFLC, a central repressor in the flowering transition [CUV) encoding DEAD-Box RNA-dependent ATPase PRP16 in facilitating the splicing of genes involved in auxin biosynthesis, transport, perception, and signal transduction [At-RS31 has been shown to be regulated under retrograde signaling control [LEF) encoding PORR domain-containing RNA-binding protein [Pathogen infection  is not tansition . Tsugekisduction . AS of t control . Light/d control , suggest protein . Nuclear protein . Accordi protein , which aArabidopsis weak mutant allele, dbr1-2, defective in RNA debranching enzyme 1 (DBR1) accumulates lariat RNAs that act as a decoy by competing with the dicing complex to inhibit genome-wide miRNA processing [In eukaryote cells, transcribing intronic regions and correctly finding junctions to cut and subsequently combine both ends are energy-consuming and laborious. Indeed, introns are junk on a good day but can be treasures on a bad day in the case of budding yeast . In yeasocessing . CollectInformation on defined epigenetic marks deposited on the first intronic DNA sequence and the higher sequence conservation within the longest first introns in all domains of life suggests that the first intronic DNA sequence is more distinguishable than the other introns. The underlying mechanisms of promoter-proximal introns to boost mRNA accumulation and determine TSS from downstream, a phenomenon called intron-mediated enhancement (IME), appear different from those of the initiation of DNA sequence-specific transcription by transcription factors or conventional enhancers. The biggest breakthrough for a better understanding of IME in plants was the discovery of the consensus IME motif enriched in introns capable of boosting mRNA accumulation . HoweverWhen pondering the underlying mechanism for stimulating introns to determine TSS from the gene body in a relatively short range (up to about 1000 bp) , IME mot


Regulated expression of genetic elements that either encode polypeptides or various types of functional RNA is a fundamental goal for gene therapy. Inducible expression may be preferred over constitutive promoters to allow clinician-based control of gene expression. Existing Tet-On systems represent one of the tightest rheostats for control of gene expression in mammals. However, basal expression in absence of tetracycline compromises the widespread application of Tet-controlled systems in gene therapy. We demonstrate that the order of P2A-linked genes of interest was critical for maximal response and tightness of a chimeric antigen receptor (CAR)-based construct. The introduction of G72V mutation in the activation region of the TetR component of the rtTA further improved the fold response. Although the G72V mutation resulted in a removal of a cryptic splice site within rtTA, additional removal of this splice site led to only a modest improvement in the fold-response. Selective removal of key promoter elements  confirmed the suitability of the minimal CMV promoter and its downstream sequences for supporting inducible expression. The results demonstrate marked improvement of the rtTA based Tet-On system in Sleeping Beauty for applications such as CAR T cell therapy. Unlike prokaryotes, mammalian genetic control is not usually mediated by single or oligo-component regulators, but rather by multiple transcription factors that bind to both promoters, as well as often distant enhancer regions located on different chromosomes. Moreover, both promoters and enhancers may be regulated by epigenetic control mechanisms, and the site of transgene insertion in the genome influences the response profile of transgenes4. Tet-On systems utilise a mutant TetR component that binds to tetracycline response elements (TRE) in the presence of tetracycline, or its stable analogue doxycycline5. To activate transcription, fusion of the herpes-simplex VP16 transcriptional activator to the C-terminus of the mutant TetR, recruits generalised transcription factors, as well as RNAP II to initiate gene transcription. Modified tetracycline-inducible systems represent the most widely used inducible system in eukaryotic systems, from yeast to human cells5. The potential exists for drug inducible systems to be used in cell-based immunotherapy to control the expression of genes or other sequences of interest (GOI). Although 103 to 106-fold induction of gene expression with tetracycline-based control systems has been reported, basal expression in the absence of inducer can result in undesirable GOI expression5. In vivo use would be compromised by such leakiness, particularly if the GOI was involved in T cell survival, or resistance to apoptosis. Unfortunately, compared to Tet-Off systems, Tet-On systems are less sensitive to tetracycline and generally exhibit a higher level of basal expression in the absence of induction6. On the other hand, Tet-Off systems are slow to respond to withdrawal of tetracycline and this may be compounded by sequestration of tetracycline in vivo, especially within bones7.Inducible-gene expression is one of the most sought-after elements of synthetic gene regulation systems. Engineering mammalian cells to express proteins or RNA in an inducible fashion offers opportunities for the development of safe cellular-based therapies to treat a wide spectrum of inborn and acquired diseases. Compared to prokaryote genetic systems, the development of tight, inducible gene expression in eukaryote cells has been challenging8. Compared to retroviral-based insertions into transcriptional units and their regulatory regions, SB vectors insert almost randomly into TA-sites throughout the genome. This property minimises deleterious integrations and helps maintain constitutive or inducible gene expression10. SB-based vectors carry a GOI along with optional markers or selection elements flanked by inverted terminal repeats (ITRs)12. Although the transposase has been re-engineered to enhance activity13, a lower-activity SB-transposase is preferred for human clinical trials to minimise the incidence of multiple genome integrations. Along with piggy bac transposase systems, SB transposase systems have been used in CAR T cell therapy trials for B cell malignancies15. To expand the utility of SB-based vectors to express a CAR together with additional GOI under drug-control, we revisited the SB-based Tet-On system, through: (1) alterations in the placement of genes within the P2A-linked CAR cassette, (2) the introduction of a G72V mutation in rtTA-M2 – previously only described for yeast Tet-On control16 (3) the placement of rtTA under auto-regulatory control, (4) the removal of cryptic splicing sites, and (5) modifications of the proximal promoter. To test the induction of the Tet-On system, we expressed myeloid leukaemia cell differentiation (Mcl-1) as a GOI involved in T cell survival and resistance to apoptosis.The Sleeping Beauty (SB) transposon system was developed from extinct Salmonid transposons awoken after 10 million years of inactivity through consensus-based correction of accumulated mutationsWe reasoned that placing a codon-optimised rtTA-M2 gene proximal to the RPBSA promoter pSBtet-1) should result in robust rtTA-M2 expression and therefore tight control of inducible gene expression  or two (pSBtet-3) additional GOI. However, inducibility of the GOI was still poor  missense mutation in rtTA mitigated basal gene expression in the absence of an inducer in S. cerevisiae clones. Because of the similarity of transcriptional machinery amongst eukaryote cells, we reasoned that this approach may give similar results in human cells. The G72V mutation was next introduced into pSBtet-2 and pSBtet-3 to create the pSBtet-4 and pSBtet-5 constructs  sequence, to allow expression of GOI-P2A-G72VrtTA under TCE promoter  in the TetR sequence-M2 Fig. . Because-M2 Fig. . We ther23. Indeed, positions 320 and 367 have low score and confidence which represent strength and the probable occurrence of a splice site, respectively  markedly decreased the tightness of the TCE promoter Fig. B, and alThe only core promoter element found upstream of the TATA box is B recognition element (BREu). As shown in Fig. 31, (2) mutation of rtTA to increase binding to doxycycline or DNA26, (3) autoregulatory systems32, (4) removing a cryptic splice sites in the rtTA coding region20, and (5) alteration of the core promoter elements within the proximal region of the TCE promoter 27. We revisited these strategies for use in the SB-based Tet-On system in a human cell line for future investigation in CAR T cell therapy.Several strategies have been proposed to reduce the leakiness and enhance the inducibility of Tet-On systems, with only some tested in human cells. Such approaches include: (1) increased expression of rtTA using a strong promoter and codon optimisationS. cerevisiae16. Future studies may explore the use of a G72P instead of G72V in our system as a candidate amino acid at position G72, though G72P appeared to result in a small loss in sensitivity, as compared to G72V16. It is interesting that independent efforts into the rtTA structure have resulted in distinct amino acid changes in different studies, but with similar outcomes. For example, mutations introduced into the rtTA-M2 gene used here are present in distinct positions, as compared to the original four mutations in rtTA20. Moreover, introducing sensitivity enhancing (SE) mutations26  could further increase the sensitivity to doxycycline, without increasing the background, as demonstrated in yeast16.The introduction of a single mutation G72V, gave the optimal induction results at both mRNA and protein level, as reported in 19 or an IRES sequence32. However, our constitutive expression of G72V-rtTA gave tighter expression, but was less sensitive to doxycycline compared to the autoregulatory system. The autoregulatory system may be preferred for controllable expression of a toxic rtTA or toxic GOI in mammalian protein production35.Autoregulatory systems have recently generated interest, with both the rtTA and GOI transcribed by a single TCE promoter, using either a bi-directional promoter30 Removing these splice sites reduced the basal expression and further increased the maximal expression. Unfortunately, using the combination of the G72V mutation with all splice sites removed  created a non-responsive system. It appears likely that the combination of the E71Q and G72V mutations disrupted the turn between two critical α-helixes 4 and 5.Next, our analysis found evidence of cryptic splice sites within an rtTA, a sequence that was previously optimised for mammalian expression by Urlinger et al.31. For example, Urlinger et al. modified the S. cerevisiae-developed rtTA-M2 sequence to remove potential hairpin, splice, and endonuclease sites, as well as codon optimising the sequence for use within mammalian systems21.It is also noteworthy that different programs identified other possible splice sites that needed to be investigated Table . At leas27. Removal of the BREu element increased maximal expression, but did not markedly affect the tightness of the TCE. The deletion of the BRE site might enhance the elongation and / or reduce the TFIIB-rtTA sequestration. BRE plays a role in the preinitiation complex (PIC), leading to the dissociation of TFIIB from the promoter which is necessary for RNA polymerase II to initiate the elongation step38. Hence, interrupting the BRE-TFIIB interaction may enhance transcription via the enhancement of elongation39. Alternatively, direct sequestration VP16 on TFIIB has been reported41 that may act to reduce VP16-mediated transcriptional activation.In the proximal region of the TCE promoter, we confirmed that the TATA box was essential for the function of the TCE promoterCollectively, our results demonstrate marked improvements to the rtTA-M2 based Tet-On system in a Sleeping Beauty system through the yeast-optimised G72V mutation. The results especially highlight the necessity to investigate the placement of individual GOI and rtTA within an expression cassette. The use of the clinically relevant CAR cassette within this setting offer the possibility to enhance adoptive cell therapy though drug-inducible expression of cell-survival and memory enhancing genes, or death switches to conditionally ablate CAR T cells following the onset of cytokine release syndrome.27 between the TRE and the minimal CMV promoter  with Bsu36I and BstBI sites was cloned into pSBtet to remove GFP-P2A-rtTA-P2A-puromycin to create pSBtet-MCS. The codon optimised rtTA-M2 gene and FRP5 scFv Her2-CAR43 were synthesised as gene blocks (IDT Singapore) and cloned into pSBtet MCS to create pSBtet-1. Other plasmids were generated by splicing by overlap extension (SOE) PCR to fuse the original rtTA2S-M2 (rtTA)21, GFP and Her2CAR in different combinations as illustrated in Fig. The Tet-On SB (pSBtet-GP) contains the tetracycline-inducible pTIGHT promoter upstream of two asymmetric SfiI sites for cloning genes of interest (GOI), with a downstream RPBSA promoter driving GFP-P2A-rtTA-P2A-puromycin. pSBtet-GP and the SB-transposase vector (pCMV(CAT)T7-SB100) were purchased from Addgene. The pTIGHT promoter is a derivative of (Ptet-14) with shorter spacer (16–17 bp) sequences44 and PROMO46 programs. The transcriptional start site (TSS) was predicted as reported previously for the minimal CMV promoter47. Screening of rtTA for cryptic splice sites was carried out using Alternative Splice Site Predictor (ASSP) software and Human Splicing Finder (HSF)48. The protein structure of TetR and the prediction of secondary structure were obtained from Protein Data Bank (PBD).Analysis of the TCE proximal promoter for core promoter elements, including the initiation repeats , TATA box, B recognition element (BRE) site and downstream promoter element (DPE), was carried out using YAPP Eukaryotic Core promoter predictor. TF binding sites were predicted using AliBaba 2.12. One day prior to transfection, HEK293 cells were cultured in a 24 well plate at 2 × 105 cells/ mL. A ratio of 5:1 (transfer plasmid: transposase) was used to stably transfect HEK293 cell line using Lipofectamine 3,000 (Thermo Fisher) and the medium was replaced at 24 h post transfection. For induction of the TCE promoter, at 72 h post transfection, cells from each well were detached and divided into four wells in a 96-well plate. Two wells were cultured with DMEM containing 5 µg / mL of doxycycline (Sigma), while control wells were maintained with only DMEM, for additional 24 h. Doxycycline-induced and control values for each construct are derived from each independent transfection to eliminate the possibility of different transfection efficiencies between Dox + and Dox- wells. Then, cells were proceed either with qPCR or luciferase assays. For Fig. The human embryonic kidney 293  cell line was cultured in high glucose Dulbecco’s Modified Essential medium (DMEM) supplemented with tetracycline-free 10% foetal bovine serum , Pen-Strep (100 U/mL penicillin and 100 µg/mL streptomycin) (Gibco) at 37 °C with 5% CO−ΔΔct) method was used to analyse the relative expression level of cop-Mcl1, by normalising to β-actin. Primers used for the qPCR reactions were: Mcl1-Fwd: GCA GAA TTG TGA CAC TGA TAA G, Mcl1-Rev: TTT TGT TCT AAC CAA TAC ATC G, β-actin-Fwd: CTT CCT TCC TGG GCA TG, β-actin-Rev: GTC TTT GCG GAT GTC CAC.Total RNA was extracted using NucleoSpin RNA Plus kit  and cDNA prepared using PrimeScript RT Reagent Kit . QPCR was carried out using Luna Universal qPCR Master Mix (NEB) in a ViiA 6 Real-Time PCR . The comparative CT (25 cells per 100 µL in a 96 well plate. Firefly Luc One-Step Glow assay working solution (100 µL) was added to each well. Cells were incubated at room temperature for one hour before reading with a Varioskan LUX multimode microplate reader . Luciferase data was presented either as relative luminescence units (RLU) or fold change. Fold change was calculated with the following formula:Luciferase assays were carried out using Pierce Firefly Luc one-step glow assay kit (ThermoFisher #16197) with cells at 10All data are presented as mean ± standard deviation (SD) and pooled from three independent experiments. Statistical analysis was performed by two-tailed t-test or one-way ANOVA test with Bonferroni post-test correction in GraphPad prism (version 8). The P values of ≤ 0.05 were considered statistically significant . .Supplementary Information.


Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome. Between 20–25 000 protein coding genes have been identified in the human and mouse genomes that give rise to ∼200 000 transcripts in tissue- and developmental stage-specific combinations . These tEpigenetic modifications of histone tail residues and cytosine bases can influence AS  and APA Mcts2 is an imprinted, monoexonic gene that has resulted from the retrotransposition of Mcts1 into the fourth intron of the H13 gene, an event that occurred over 90 million years ago  that overlaps with the promoter of Mcts2 and undergoes DNA methylation in the female germline , which undergo 3′UTR-APA downstream of the iCGI using the canonical polyadenylation sites of the H13 gene to generate full length transcripts. In contrast, on the paternal allele, the iCGI is unmethylated and transcriptionally active  that undergo intron retention and IPA upstream of the iCGI  are generally unmethylated  but whenechanism . The hypechanism . Conversechanism . Importaechanism .Mcts2/H13 model locus and more broadly. We also sought to determine the influence of intragenic transcription and DNA methylation on host gene pre-mRNA processing. This was achieved through the identification of over 4000 host genes harbouring an iCGI in the mouse genome. The activity of these iCGIs has been found to be tissue- and developmental stage-specific and, for the first time, we demonstrate that the abundance of host gene transcripts terminating upstream of iCGIs is closely correlated with the level of iCGI transcription. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide, this in turn provides a means to enable spatiotemporal diversification of both the transcriptome and proteome.Here, we sought to investigate H3K36me3 and transcription as mediators in the generation of alternative transcripts at the 2 in DMEM high glucose  supplemented with 10% FBS  and 1× penicillin–streptomycin . Cells were harvested using 1× trypsin-EDTA .NIH/3T3 cells were cultured at 37°C and 5% CO2 in a well of a six-well plate. After 24 h, 3.75 μl Lipofectamine 3000  was diluted in 125 μl Opti-MEM . Setd2-specific or Scrambled siRNAs  were diluted in 125 μl Opti-MEM to a final concentration of 10 nM. Diluted Lipofectamine 3000 and siRNAs were mixed and incubated for 20 min at room temperature. The siRNA–lipid complex was added to the cells and incubated for 48 h. Transfections were carried out in triplicate and gene expression was assayed by RT-qPCR.On day one, NIH/3T3 cells were seeded at 15 625 cells/cm4 cells/μl in loading buffer  SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol, 100 mM DTT) at 98°C for 5 min. Proteins were separated on 4–12% SDS-PAGE and transferred onto PVDF membrane. Successful transfer of proteins was confirmed by Ponceau S staining. The membranes were blocked in 5% (w/v) TBS-T BSA, incubated with the appropriate primary and secondary antibodies . Membrane-bound genomic DNA was digested with RNase-Free DNase Set . First strand cDNA was synthesised using a ProtoScript II First Strand cDNA Synthesis Kit  using oligo d(T)RT-qPCR was performed on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) in a 10 μl reaction including 1 μl cDNA, 1× TaqMan Gene Expression Master Mix  and appropriate TaqMan probes .−ΔCt method  and KAPA Library Quantification Kit . All samples were sequenced on one Illumina HiSeq 2500 lane.After RNA extraction, RNA integrity was measured with 2200 TapeStation (Agilent) using high sensitivity RNA ScreenTape and reagents . TruSeq Stranded mRNA Library Prep  and TruSeq RNA Single Indexes Set A  were used to prepare two Scrambled and two Raw data in the format of FastQ files were subject to quality control using FastQC (0.11.5) . AdapterAll mRNA-seq datasets were aligned to the GRCm38 reference genome by HISAT2 (2.1.0)  and qualGenomic DNA (gDNA) was extracted form pelleted cells using DNeasy Blood and Tissue Kit . 500 ng of gDNA were bisulfite converted using EZ DNA Methylation-Gold Kit .Nested PCR was performed with appropriate primers  using OnAfter colony PCR, amplicons were subject to enzymatic clean-up with ExoSAP-IT PCR Product Cleanup Reagent  and sequenced with T7 and SP6 primers using a BigDye Terminator v3.1 Cycle Sequencing Kit  in a 3730xl DNA Analyzer (Applied Biosystems).Sanger sequencing results were uploaded to BISMA  and the et al.  were calculated using the following formula:U) and across (A) the iCGI calculated across available conditions (i). ρ values obtained with this formula were used to generate density plots in Figure This correlation coefficient measures the linear association between the transcriptional activity of the iCGI and the ratio of host gene expression upstream  were downloaded from ENCODE datasets. Percentage of CpG methylation at iCGIs (±5kb) was taken into account. The heatmap was generated using the package EnrichedHeatmap  in R andH3K36me3 and H3K4me3 mouse E14.5 brain ChIP-seq data from the ENCODE project  were utiIn mammals, SETD2 is a histone methyltransferase that can interact with the C-terminal domain of phosphorylated RNA Pol II and deposit H3K36me3 along the body of actively transcribed genes with increasingly greater occupancy towards their 3′ ends . PreviouSetd2-specific siRNAs. The efficiency of the knockdown was confirmed by RT-qPCR, which showed an ∼80% reduction in Setd2 mRNA levels in knockdown (KD) samples compared to wild type (WT) samples  ≤–1 and 346 (8.11%) of the upregulated transcripts showed a log2(fold change) ≥+1  displayed changes in intron retention and in all three cases this type of alternative splicing event was less frequent in the KD than in the WT  ≥+1 . Transcrnge) ≥+1 . Gene onnge) ≥+1 . Furthernge) ≥+1 ,55, modinge) ≥+1 . GO analnge) ≥+1 . SETD2 hnge) ≥+1  and to ange) ≥+1 . Therefon the WT . Additioutations ,29. Impoutations  and contMcts2/H13  allele at the introns that are retained in the paternally expressed (CAST/Ei) H13d and H13e transcripts  mRNA levels measure 3′UTR-APA, whereas H13d and H13e expression are a proxy for intron retention and IPA . H13 isoforms terminating downstream of the iCGI also decreased by 28% . This decrease was expected since H13a-c constitute the vast majority of H13 transcripts . Expression from the iCGI was minimally affected  and its DNA methylation profile was not altered  were calculated between upstream:across ratios and the number of RNA-seq reads mapping to the iCGIs themselves.The l stages . 4033 hod Figure . Additioupper). The null distributions are centred around zero and have a moderate range, indicating that this approach has a sufficient level of specificity and that extreme ρ values are unlikely to occur by chance. The distributions originating from iCGI/host gene pairs are skewed to the right  iCGI/host gene pairs scored above this threshold. The sensitivity of this approach was validated by the detection of the imprinted pair Nap1l5/Herc3 (ρ = 0.94) among the significant loci. The promoter of Nap1l5 is an iCGI that, when transcriptionally active, leads to Herc3 IPA  Figure , upper. t Figure , showingerc3 IPA . The samerc3 IPA  and resuobs/CpGexp) and CpG density of the CGIs identified in this study. As previously shown (obs/CpGexp ratio and CpG density when compared to other CGIs (left). In order to determine whether these features could be important in the apparent correlation between iCGI expression and upstream:across host gene transcription ratios, their distribution was interrogated in the context of iCGIs/host gene pairs. Thus, we observed no difference between iCGIs whose expression significantly correlates with upstream:across host gene transcription ratios (ρ > 0.59) and other iCGIs (ρ ≤ 0.59) (right). This indicates that these features have probably no involvement in the regulation of iCGIs expression and in their influence on host gene transcription.Using publicly available data , we detely shown , iCGIs pher CGIs , left. I ≤ 0.59) , right. Mcts2/H13 locus is subject to genomic imprinting, we have shown through this analysis that the majority of the identified iCGI/host gene pairs (over 4000) are not imprinted, suggesting that iCGI influence on host gene polyadenylation is widespread and conserved across at least two mammalian species.Although the model The transcriptional activity at iCGIs from iCGI/host gene pairs with ρ > 0.59 was compared across all tissues, developmental stages and cell lines. Both mouse and human datasets showed striking tissue-specific iCGI activity with iCGIs often being highly expressed in a single tissue or cell line Figure  and D. Hupstream:across ratios were selected for further analysis. For each iCGI, two tissues were assayed that exhibited the highest and lowest measured expression levels based on RNA-seq data. For five of the iCGIs, the gene expression measured by RT-qPCR recapitulated the gene expression patterns detected in the RNA-seq datasets . However, lower expression levels observed in heart, testis and cerebellum could contribute to the discrepancy detected between the RNA-seq and RT-qPCR results .In order to establish a role for DNA methylation in the regulation of iCGIs transcriptional activity in mouse, ten candidate iCGI/host gene pairs that showed the highest correlation between iCGI transcription and sed here . The ENCHoxa, DNA methylation correlated with gene expression . For the other four, no correlation between iCGI expression and DNA methylation was detected  suggesting that DNA methylation may not the major regulatory factor involved. In order to validate our observations, publicly available genomewide bisulfite sequencing datasets from the ENCODE project were utilised. Although, not all the tissues in the bisulfite sequencing PCR experiments were available, data from brain, lung, heart and stomach were used. When examining the DNA methylation percentage of the iCGIs used for validation across these tissues, it was found that the majority of them are unmethylated (Dhrs3), suggesting that DNA methylation is not the only mechanism involved in the regulation of expression at iCGIs.The DNA methylation status of these candidate loci was assayed by PCR amplification of bisulfite-converted genomic DNA. At one of the concordant iCGI/host gene pairs, namely n Figure , right. d Figure , right sthylated . Variatiupper). H3K36me3 gradually accumulates downstream of highly expressed iCGIs before decreasing again upstream of host gene transcription termination sites (TTSs), resembling the deposition of H3K36me3 along the body of actively transcribing genes . H3K4me3 was enriched at all intronic iCGIs with a bias towards highly expressed iCGIs . Taken together, these findings suggest that the iCGIs tested may indeed be discrete tissue-specific promoters and that histone modifications could be important to regulate their expression.To determine whether these iCGIs are independent transcriptional units or a by-product of host gene transcription, ENCODE H3K36me3 and H3K4me3 ChIP-seq datasets from mouse E14.5 brain  were ints Figure , lower. SETD2 mutations are characterised by increased intron retention and altered transcription termination site usage (Epigenetic modifications are associated with transcription and pre-mRNA processing. In some cases, the deposition of one mark is dependent upon the presence or absence of another. The same marks can differentially influence transcription depending on genomic context. For instance, DNA hypermethylation at CGI-associated promoters negatively impacts transcription  but facite usage ,29.Setd2 KD and WT samples, indicating that H3K36me3 is not a master regulator of this type of pre-mRNA processing. Locus-specific experiments on the imprinted iCGI/host gene pair Mcts2/H13 are consistent with our genomewide conclusions since we did not detect changes in H13d transcript abundance, a proxy for intron retention, upon H3K36me3 depletion. These findings confirm the involvement of H3K36me3 in AS but differ from previous studies in human kidney tumours that associate SETD2 mutations with increased intron retention. This could be partially explained by the use of polyadenylated RNA rather than total RNA for the preparation of RNA-seq libraries. If H3K36me3 depletion does indeed lead to increased intron retention in the nucleus, there must be efficient downstream checkpoints that prevent the export of those transcripts into the cytoplasm. However, re-analysis of previous studies shows consistent percentages of bases aligning to intronic sequences across all datasets, suggesting that the use of improved bioinformatic tools could also be the basis of these discrepancies.We investigated the influence of H3K36me3 on intron retention and IPA in NIH/3T3 cells using siRNAs coupled with transcriptome analysis and locus-specific gene expression assays. We show that depletion of H3K36me3 leads to significant deregulation of 4266 transcripts and 136 AS events. Changes in intron retention are virtually absent between H13 transcripts are characterised by intron retention and IPA upstream of an actively transcribing iCGI, we interrogated the impact of transcription from iCGIs on host gene pre-mRNA processing more generally. We bioinformatically analyse 30 mouse tissues and developmental stages using RNA-seq datasets from the ENCODE project and identify 4033 iCGI/host gene pairs. For one in five pairs, iCGI activity is tissue- or developmental stage-specific and the level of host gene transcription upstream of the iCGI positively correlates with the level of iCGI transcription. We repeated the same analysis using ENCODE RNA-seq data from 18 human cell lines and find the same results, indicating that these observations are reproducible across two mammalian species. Additionally, we demonstrate that this effect is largely independent of DNA methylation for a small subset of iCGI/host gene pairs. Finally, we provide evidence that these iCGIs may be discrete tissue-specific promoters since they are enriched for H3K4me3 and depleted of H3K36me3, a chromatin profile typically associated with active gene promoters (Since paternal romoters .de novo DNA methylation machinery (Previous studies have shown that iCGIs become methylated and silenced when host gene transcription traverses them and triggers the recruitment of the achinery . Howeverachinery . These iachinery . Our worachinery . This isachinery . Using dhttp://rulai.cshl.edu/new_alt_exon_db2/HTML/score.html.The splice donor site score calculation tool is freely available at Setd2 knockdown RNA-seq data have been deposited under accession number GSE147077.gkaa556_Supplemental_FilesClick here for additional data file.


CCCTC binding factor (CTCF) is an important factor in the maintenance of chromatin–chromatin interactions, yet the mechanism regulating its binding to chromatin is unknown. We demonstrate that zinc finger protein 143 (ZNF143) is a key regulator for CTCF-bound promoter–enhancer loops. In the murine genome, a large percentage of CTCF and ZNF143 DNA binding motifs are distributed 37 bp apart in the convergent orientation. Furthermore, deletion of ZNF143 leads to loss of CTCF binding on promoter and enhancer regions associated with gene expression changes. CTCF-bound promoter–enhancer loops are also disrupted after excision of ZNF143. ZNF143-CTCF-bound promoter–enhancer loops regulate gene expression patterns essential for maintenance of murine hematopoietic stem and progenitor cell integrity. Our data suggest a common feature of gene regulation is that ZNF143 is a critical factor for CTCF-bound promoter–enhancer loops. CTCF mediates DNA–DNA interactions by forming dimers that directly bind to both sides of the DNA. Here the authors reveal that a CTCF known associated zinc finger, ZNF143, mediates gene transcription through CTCF–DNA binding to maintain murine hematopoietic stem and progenitor cell integrity. Different transcription factors (TFs) bind to these regulatory elements to regulate promoter–enhancer loops in a cell-type-specific manner. Although they play an essential role in control of gene expression, how TFs contribute to promoter–enhancer loops is not well understood.Gene transcription is commonly regulated by chromatin loops between promoters and regulatory elements such as enhancers and silencers9, several lines of evidence suggest that CCCTC binding factor (CTCF) plays a critical role in promoter–enhancer loop regulation as well. First, down-regulation of CTCF by either targeted degradation or RNAi decreases the intensity of enhancer–promoter loops, consistent with high enrichment of CTCF motifs within promoter regions10. Second, detection of CTCF in protein complexes enriched on active promoters by ChIP-MS supports that CTCF is involved in enhancer–promoter loops11.In addition to its essential role in maintaining Topologically Associating Domains (TADs)13, the regulatory mechanism controlling CTCF–DNA binding remains unknown. One hypothesis is that other factors such as cohesin play roles to mediate CTCF–DNA binding. Although multiple studies indicate that CTCF and cohesin interact with each other to contribute to chromatin structure maintenance15, a recent crystallographic structural study demonstrated that a specific CTCF mutation abolished CTCF–cohesin interaction, and further affected cohesin localization on CTCF sites, whereas CTCF binding on its sites remained unchanged16. This study indicates that CTCF recruits cohesin to its binding site, but CTCF–cohesin interaction does not regulate CTCF–DNA binding. So, to date it is not known which factor(s), if any, mediate CTCF–DNA binding to regulate chromatin interactions and gene expression.While it has been well-established that CTCF mediates DNA–DNA interactions by forming dimers that directly bind to both sides of the DNAXenopus laevis functioning as a sequence specific transcriptional activator of both Pol II and Pol III18. Later on, studies reveal that as a co-activator, ZNF143 might be a key regulator in T lymphoblastic leukemia (TLL) through its association with Notch120. Despite bioinformatic analysis suggesting that, similar to cohesin, ZNF143 co-localizes with CTCF22, and knocking down ZNF143 affects chromatin interaction at individual loci22, how CTCF and ZNF143 regulate the function of each other remains unknown. Building upon our previous work identify ZNF143 as an important regulator of Cebpa expression23, in this study, we used a hematopoietic model system to investigate whether ZNF143 mediates gene transcription through CTCF in a general manner.The zinc finger protein 143 (ZNF143/ZFP143), was first discovered as selenocysteine tRNA gene transcription activating factor (STAF) in 23, whereas most commercial anti-ZNF143 antibodies recognize both ZNF143 and ZNF76 (data not shown), we first generated a specific anti-ZNF143 rabbit polyclonal antibody which only recognizes ZNF143, and not ZNF76, in both Western-blot and IP  was confirmed at the level of both RNA and protein , share over 60% protein sequence identity and bind in vitro to the same DNA sequenceZnf143 depletion, dramatic loss of the CTCF ChIP-seq signal was detected in regions where both ZNF143 and CTCF were enriched in wild type cells, without affecting CTCF mRNA or protein levels  convergent on opposite strands (convergent); 2) divergent on opposite strands (divergent); 3) both on forward strand (F-F); 4) both on reverse strand (R-R). After analyzing 10,544 pairs of ZNF143 and CTCF motifs located 37 bp away from each other, we observed 99.9%  formed the convergent orientation  , we confirmed the decrease of interaction frequency between the Cebpa promoter and the 37 kb Cebpa enhancer after deletion of ZNF143  Fig. , the Ceb143 Fig. . In agreted Fig. , indicatZnf143 f/f and Znf143 −/− HSPCs . Consistent results obtained using both embryonic and adult hematopoietic cells demonstrated that long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), multi-potent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), granulocyte progenitor cells (GMPs), and megakaryocyte-erythroid progenitor cells (MEPs) were all dramatically reduced after Znf143 excision  chimerism over 4 months. In agreement with the results obtained with fetal liver cell transplantation  with 300 µg poly I:C for three consecutive days. Then mice were euthanized at day 8 after the first injection to assess deletion. All mice were housed in a sterile barrier facility within the Comparative Medicine facility at the National University of Singapore under housing condition of 22 °C temperature, 50% humidity and a 12:12 light/dark cycle. All mice experiments performed in this study were approved by Institutional Animal Care and Use Committee of National University of Singapore. In this study, 8- to 12-week-old male mice were used bone marrow harvesting or transplantation; 12- to 16-week-old female mice were used for time mating. See “Methods” for more information.C57BL/6J, All antibodies and primers used in this study were listed in supplementary data Znf143 f/+ x Vav1-iCre+ male mice were mated with Znf143 f/f female mice in the evening on day 0. Embryos were collected at E14.5-day stage for fetal liver isolation and genotyping. Fetal livers were kept in PBS at 4 °C until genotyping of each embryo was completed. Fetal livers from Znf143 f/+ x Vav1-iCre- (Znf143 f/+) and Znf143 f/f x Vav1-iCre+ (Znf143 −/−) embryos were dissociated into single cells by passing through a 40 µm BD strainer with a 1 ml syringe plunger in PBS plus 2% FBS and 2 mM EDTA.For fetal liver isolation, 2 euthanasia, mice were dissected for vertebrae, femur, tibia, and hip collection. Bones were crushed using a pestle with ice-cold PBS and then bone marrow cells suspension were filtered through a 70 µm BD cell strainer. Red blood cells were removed with RBC lysis buffer treatment. After filtering, a 20 µl cell suspension was diluted into a 2 ml solution for white blood cell (WBC) counts using a NIHOKODEN auto blood cell counter under Pre-dilute 20 µl mode. The remaining cells were kept on ice for other experiments.After COFor flow cytometry analysis, 1 million WBCs or Fetal liver cells were stained for 15 min as indicated in Supplementary Data For FACS sorting, mature blood cells were stained with biotin-labeled anti-lineage surface marker antibodies as indicated in Supplementary Data Lineage depleted cells were stained as previously described for 15 min. Then cells were washed once and were resuspended to a final cell concentration of 40 M/ml for FACS on an Arial I/II. Post-sort was performed for double checking of cell populations and for cell number counting. The gating strategy for different populations is provided in Supplementary Fig. FlowJo was used for data plot of flow cytometry and FACS.Znf143 f/+ x Vav1-iCre- or Znf143 f/f x Vav1-iCre+ embryos) or RBC lysis buffer treated BMCs  were mixed with two hundred thousand RBC lysis buffer treated BMCs isolated from CD45.1 congenic mice to a final cell concentration of ten million per ml. Recipient CD45.1 congenic mice were lethally irradiated (9.5 Gy) using a γ-irradiator, followed by injecting one million mixed cells per mouse. Every four weeks after injection, 100 µl peripheral blood (PB) was collected from each mouse to check for chimerism. To examine chimerism formation, PB was lysed with 1 ml RBC lysis buffer for 5 min at room temperature. After washing with PBS, cells were stained with antibody mixtures as indicated in Supplementary Data Eight hundred thousand fetal liver cells  were pooled together and were sequenced using a Nextseq high output 150 cycle v2 kit in the Illumina Nextseq 500 platform to obtain at least 50 million 76 bp paired-end reads per RNA library. Illumina bcl2fastq2 Conversion Software (v2.20) was used for Next Generation Sequencing library demultiplexing. Fastqc software was used for data quality check.The HSC SLAM population (LSK+CD150+CD48−) was sorted into PBS plus FBS and EDTA. After post-sort to confirm the sorted population and to count cell number, cells were spun down for RNA extraction following trizol-chloroform-ethanol extraction with glycogen or glycoblue precipitation. RNA samples with RNA integrity number (RIN) score higher than 8.0 were selected for RNA-seq library construction using SMART Seq v4 Ultra Low Input RNA kit from Takara and Nextera XT DNA library prep kit from Illumina, with PAGE-gel purification for final library size selection. For next-generation sequencing (NGS), 6 libraries  were washed with PBS once and then lysed in 1 ml IP buffer  on ice for 10 min with occasionally inverting. After spinning down at 13,200 rpm for 10 min at 4 °C, the supernatant was transferred into a new tube containing antibody pre-bound dynabeads protein beads for overnight incubation at 4 °C ; 50 µl of the supernatant was stored at -20 °C as input. The next day, after washing beads for five times with IP buffer, loading dye was added to the beads and input for boiling at 97 °C for 10 min, followed by Western-blot analysis.23. Briefly, after post-sort to confirm the sorted population and to count the cell numbers, HSPCs (lineage- c-kit+) were resuspended in PBS to a concentration of 1 M per ml. Cells were fixed with 1% formaldehyde for 15 min at RT with rotation. Then formaldehyde was neutralized by adding 2.5 M glycine to a final concentration of 0.125 M and rotating for 5 min at RT. After washing with PBS, cells were lysed with ChIP SDS lysis buffer , and then stored at -80 °C until further processing. Nuclei were collected by spinning down at 13,000 rpm for 10 min. The nuclear pellet was resuspended in IP solution , and fresh proteinase inhibitor) for sonication using either a Bioruptor or Covaris to obtain 200 bp to 500 bp DNA fragments. After spinning down to remove debris, sonicated chromatin was pre-cleared by adding 30 µl washed dynabeads protein A/G and rotated at 4 °C for 2 h. Pre-cleared chromatin was incubated with antibody pre-bound dynabeads protein A/G overnight at 4 °C  antibody, #9733BC, Cell Signaling Technology; anti-histone H3 (acetyl K27), #ab4729, Abcam). Next day, magnetic beads were washed through the following steps: buffer 1  two times; buffer 2  two times; buffer 3  two times; TE buffer one time. After reverse crosslinking and purification of DNA, qPCR or library construction was performed accordingly.ChIP was performed as described previouslyFor library construction, a Thruplex DNA seq kit from Rubicon Genomics was used following the manufacturer’s instructions. TBE PAGE-gel size selection was performed for the final library size selection to obtain 250 bp to 500 bp ChIP-seq libraries. Pooled libraries at 10 nM concentration were sequenced in the Illumina Nextseq 500 platform. Illumina bcl2fastq2 Conversion Software (v2.20) was used for Next Generation Sequencing library demultiplexing. The Fastqc software was used for data quality check.ZNF143 ChIP was performed as described above until the TE buffer washing step. Then immunocomplexes were eluted by incubating with 50 µl 10 mM DTT for 30 min at RT. After diluting the elution with 1 ml IP buffer, 20 µl CTCF antibody or 5 µg normal rabbit IgG  pre-bound protein A dynabeads were added for overnight rotation at 4 °C, followed by standard ChIP procedure.® Accel-NGS® 2S plus DNA library kit following the low input procedure instructions.HSPCs (lineage- Sca-1+ c-kit+) were used for in situ HiC libraries construction using Arima-HiC kit and Swift biosciences48. In brief, HSPCs (lineage- c-kit+) were crosslinked in 1% formaldehyde for 15 min at RT with rotating. Then formaldehyde was neutralized by adding 2.5 M glycine to a final concentration of 0.125 M and rotating for 5 min at RT. After washing in PBS, cells were resuspended in 1 ml lysis buffer  and lysed with a douncer on ice. After spinning down at 2000 rpm for 10 min at 4 °C, nuclei were washed with 1X CutSmart buffer from NEB, followed by resuspending in 1.8 ml nuclease free (NF) H2O. After splitting into 4 tubes containing 60 µl of 10X CutSmart buffer and 15 µl of 10% SDS buffer, samples were incubated at 37 °C 1 h with shaking, followed by adding 75 µl of 20% Triton X-100 to each tube for 1-h incubation at 37 °C with shaking. From each tube 20 µl samples were taken out and were combined as “undigested” samples and stored at -20 °C. HindIII-HF was added into each tube (700 U per tube) for overnight digestion at 37 °C with shaking. The next day after confirming a digestion efficiency over 80%, 50 µg digested DNA were taken out into a new tube and the volume adjusted to 600 µl with NF H2O. After adding 80 µl 10% SDS, samples were incubated at 65 °C for 25 min. Heat inactivated chromatin was added into ligation mix  and the volume adjusted to 50 ml with NF H2O to a final concentration of 1 ng/µl, followed by 1 h incubation at 37 °C with rotation at 30 RPM. After adding 990 U T4 DNA ligase (Thermo Scientific), samples were incubated at 16 °C overnight with rotation at 15 RPM. The next day, 132 µl of proteinase K (Ambion) were added into each sample, followed by 65 °C incubation overnight. The next day, after adding 132 µl of RNase A (PureLink), samples were incubated at 37 °C for an hour, followed by phenol: chloroform DNA purification. Chromatin-chromatin interactions between the Cebpa promoter and −37 kb enhancer region and between the Cebpa promoter and −18 kb upstream region were examined by qPCR. The −76 kb upstream region having no interaction with Cebpa promoter was selected as negative control.3C was performed as described previouslyGraphPad Prism unless further indicated in the data analysis section.All dot plots and survival curves are drawn with TrimGalore, paired-end reads were aligned to mouse genome GRCm38 (mm10) using STAR49. After calculating raw gene expression (raw reads count) by FeatureCounts50, normalized profiling results were obtained with DESeq2 for differential expression analysis51. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA pipeline from the Broad Institute53. For visualization on the UCSC genome browser or IGV, bam files obtained from STAR were first converted into bedgraph format using BEDTools54, then converted into bigwig files with UCSC bedGraphtoBigWig program.After trimming of adaptor sequences with TrimGalore, paired-end reads were aligned to mouse genome GRCm38 (mm10) using bowtie2 with output in sam file format55. SAMtools was used (1) to convert sam file to bam file, (2) to remove both low mapping quality score reads (mapq score less than 20) and PCR duplicates56. ChIP-seq peaks were called by MACS2 with a false discovery rate (FDR) less than 0.001, followed by peak annotation with HOMER58. The resulting alignments were extended to 150 bp and then converted into signals by BEDTools in bedGraph format54. We adjusted the average coverage of each ChIP-seq experiment to 1. Finally, the resulting files were converted to bigwig format by bedGraphToBigWig for visualization and generating heatmaps59. Correlation of replicates was assessed with the Deeptools multiBigwigSummary BED-file function60.After trimming of adaptor sequences by MACS2 were used for de novo motif scanning using HOMER findMotifsGenome function with default settings58. Then the discovered CTCF and ZNF143 motifs were used for whole genome scanning using HOMER scanMotifGenomeWide function with output of bed files. Distances between CTCF and ZNF143 motifs in genome-wide were determined by BEDTools. R package ggplot was applied for result visualization in Rstudio.To discover CTCF and ZNF143 motifs in the mouse genome, bed files generated by HOMER to the online program Regulatory Sequence Analysis Tools (RSAT) with permute-matrix function24. Then distances between CTCF and mutant ZNF143 motifs were discovered by BEDTools and were visualized by R in Rstudio.ZNF143 mutant motifs were generated by loading ZNF143 motif obtained from HOMER, and then each pair of motifs were sub-grouped as described above to get the final count by R in Rstudio.As ZNF143 and CTCF motifs are not palindromic and could locate on either the forward or reverse strand of DNA, a pair consisting of ZNF143 and the nearest CTCF motif have four potential orientations as illustrated in Figure Active promoters were identified by H3K27ac peaks within 1000 bp to a nearest transcription start site (TSS). Active enhancers were identified by H3K27ac peaks that located outside of promoter regions.HOMER was used to make tag directories of individual ChIP-seq bam files, followed by calculating ChIP-seq signal coverage using HOMER annotatePeaks function58. The output was visualized as a mean plot using the R package in Rstudio.For calculating the average of ChIP-seq signals on a promoter, enhancer, or nearby CTCF motifs within loops anchor regions, Deeptools or ngsplot was used to generate heatmap of ChIP-seq on different regions61. First, desired bed files indicating such regions were generated as described in this manuscript. Then bigwig files from ChIP-seq data were used to generate a matrix on input regions by Deeptools computeMatrix function, followed by Deeptools plotHeatmap function to draw heatmap. Deeptools was also used for calculating genome-wide correlations among ChIP-seq signals in bigWig files with bin size of 5000 bp.ngsplot, bam files from ChIP-seq data and desired bed files were used for heatmap generation with ngs.plot.r function.For Juicer tools suite (v1.9.9_jcuda.0.8) following the instruction7. The Knight-Ruiz balanced observed/expected interaction frequencies were calculated at 2.5 Mb, 1 Mb, 500 kb, 250 kb, 100 kb, 50 kb, 25 kb, and 5 kb resolution. The matrices were visualized in Juicebox (v1.9.8). The eigenvector values were calculated at 1 Mb resolution using Juicer tools eigenvector function. The PC1 values were calculated using HOMER following its HiC analysis tutorial62.HiC contact matrices were generated using the 28. Dense contact matrices for each chromosome were extracted from.hic files using Juicer tools dump function returning the observed contacts without normalization at 25 kb resolution. The resulting matrices were transformed into the HiC-PRO format63 using a custom R script. The insulation score was calculated from these HiC-PRO contact matrices using the the matrix2insulation.pl function in HiC-PRO28. The resulting insulation score per chromosome was aggregated into a single bedGraph and then transformed to bigwig using the bedGraphToBigWig function from UCSC together with the chromosome size information for mm10 in UCSC. Profiles of genome-wide insulation score around TADs boundaries were visualized by Deeptools.Insulation scores were calculated as describedJuicer tools Arrowhead algorithm at 25 kb resolution. Loops were called using Juicer tools Hiccups function at 5 kb resolution. Loops intensities were calculated by Juicer tools dump function with input of.hic files generated by Juicer tools. Aggregate peak analysis on subgroup loops was performed using Juicer tools APA function, and the result is visualized with R package in Rstudio. To discover CTCF motifs on loop anchors, bedpe files containing loops information were examined by Juicer tools MotifFinder function.TADs were called using the Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Reporting Summary


Xenopus embryos. Foxd4l1.1 directly bound within the promoter of endogenous neural repressor ventx1.1 and inhibited ventx1.1 transcription. Foxd4l1.1 also physically interacted with Xbra in the nucleus and inhibited Xbra-induced ventx1.1 transcription. In addition, Foxd4l1.1 also reduced nuclear localization of Smad1 to inhibit Smad1-mediated ventx1.1 transcription. Foxd4l1.1 reduced the direct binding of Xbra and Smad1 on ventx1.1 promoter regions to block Xbra/Smad1-induced synergistic activation of ventx1.1 transcription. Collectively, Foxd4l1.1 negatively regulates transcription of a neural repressor ventx1.1 by multiple mechanisms in its exclusively occupied territory of neuroectoderm, and thus leading to primary neurogenesis. In conjunction with the results of our previous findings that ventx1.1 directly represses foxd4l1.1, the reciprocal repression of ventx1.1 and foxd4l1.1 is significant in at least in part specifying the mechanism for the non-neural versus neural ectoderm fate determination in Xenopus embryos.Neuroectoderm formation is the first step in development of a proper nervous system for vertebrates. The developmental decision to form a non-neural ectoderm versus a neural one involves the regulation of BMP signaling, first reported many decades ago. However, the precise regulatory mechanism by which this is accomplished has not been fully elucidated, particularly for transcriptional regulation of certain key transcription factors. BMP4 inhibition is a required step in eliciting neuroectoderm from ectoderm and Foxd4l1.1 is one of the earliest neural genes highly expressed in the neuroectoderm and conserved across vertebrates, including humans. In this work, we focused on how Foxd4l1.1 downregulates the neural repressive pathway. Foxd4l1.1 inhibited BMP4/Smad1 signaling and triggered neuroectoderm formation in animal cap explants of  In this regard, inhibition of BMP4 signaling by various BMP4-antagonists such as the dominant-negative BMP receptor (DNBR), noggin and chordin cause dorso-anteriorization and neuroectoderm formation of embryos8. These observations have raised the possibility that the neuroectoderm formation is a default pathway instead of a previously proposed inductive event by instructive signals emanating from the organizer10 and for non-neural ectoderm formation, there are instructive signals that include BMP411 with the BMP4/Smad1 axis leading to a negative regulation of the neuroectoderm11. In support of this, BMP target transcription factors including Gata1b, Msx1 and Ventx1.1 have been shown to be repressive transcription factors (TFs) of neural ectoderm15. Despite these findings, although the inhibition of BMP signals for eliciting the neuroectoderm has been well documented at the extracellular level, the pathway and its target genes in the neuroectoderm remain undefined, particularly at the transcription level.Central to vertebrate embryogenesis, during the gastrula stage of embryogenesis, neuroectoderm develops from the naive ectoderm, giving rise to the neural plate, neural tube, and the neural crest. It is known that the vertebrate neuroectoderm develops in response to the dorsal mesoderm (the Spemann organizer)16. Ventx1.1 is a homeobox transcription repressor which inhibits the expression of organizer-specific and neural genes, including gsc, chordin, noggin, ncam, ngnr, zic3 and foxd4l1.118. Dorsal expression of ventx1.1 results in the headless phenotype and neural inhibition in Xenopus embryos18. FGF signaling is involved in germ layer specification and anterior–posterior patterning of neural tissues in vertebrates. Specifically, FGF signaling leads to mesodermal formation through activation of an autocatalytic loop of FGF/Ras/Xbra/AP1 in Xenopus20. Studies have also revealed the opposing roles of FGF signaling that regulate neuroectoderm formation during gastrula24. FGF/MAPK activation was shown to inhibit BMP4/Smad1 signaling and promote neuroectoderm formation by phosphorylating the linker region of Smad1, resulting in the cytoplasmic retention of Smad124. On the other hand, signaling by both BMP4/Smad1 and FGF/Xbra had a synergistic role in activating ventx1.1 transcription to inhibit neuroectoderm formation16. The details of inhibitory gene regulatory network and transcriptional mechanism of the neural repressor gene ventx1.1 in neuroectoderm region await elucidation.BMP4/Smad1 is known to interact with FGF/Xbra to induce the expression of an endogenous neural repressor transcription factor (TF) Ventx1.127. The primary neuroectoderm expresses various transcription factors that may inhibit it from becoming the epidermis28. Foxd4l1.1 of the forkhead box (fox) family of transcription factors  is reported as one of the earliest neural genes and is evolutionally conserved and expressed in neuroectoderm across vertebrates including humans31. Foxd4l1.1 is known to actively participate in various developmental events, such as immature neuroectoderm fate maintenance, neural plate formation and neural differentiation. It has been shown that the ectopic expression of DNBR stimulates foxd4l1.1 mRNA expression level in animal cap explants34. Further, transition of ectoderm-neuroectoderm is regulated by fine-tuning of several transcription factors such as foxd4l1.1, zic2/3, sox1-3/d and xiro1-335. Foxd4l1.1 has been documented to increase the expression of neural specific genes, including sox2/3, geminin, n-tubulin, ncam, and neuroD in a dose-dependent manner, leading to neural differentiation36. Also, ectopic expression of foxd4l1.1 inhibits BMP4/Smad1 signaling and leads to neural differentiation and neuroectoderm formation34.Once the neural ectoderm is formed and neural transcription factors (nTFs) are expressed, some of the earliest expressed nTFs need to prevent cells from reverting to a non-neural fatefoxd4l1.1 inhibits the promoter activity of ventx1.1 and promotes primary neurogenesis in Xenopus embryos15. A study documented that foxd4l1.1 and its engrailed (repressor) conjugated construct (EnRfoxd4l1.1) induce the expression of neural markers such as xngnr1, n-tubulin, geminin and xnr3 in animal cap explants37. However, the molecular details for foxd4l1.1 in ectoderm-neuroectodem specification and foxd4l1.1-mediated BMP4/Smad1 inhibition, leading to neuroectoderm formation, remain to be elaborated.We have previously shown that ventx1.1 and inhibit neuroectoderm formation in Xenopus embryos18. On the other hand, the neuroectoderm region may require a neuroectoderm specific repressor in order to inhibit ventx1.1 expression. In this work, we found that foxd4l1.1 and engrailed-foxd4l1.1 (EnRfoxd4l1.1) negatively regulated BMP4/Smad1 signaling and inhibited ventx1.1 expression. It is also shown that Foxd4l1.1 directly binds to the proximal region of endogenous ventx1.1 promoter and inhibits ventx1.1 transcription during neuroectoderm formation. Foxd4l1.1 also inhibits the direct binding of Xbra-Smad1 to block the synergistic activation of ventx1.1 transcription. This study suggests that neuroectoderm specific repressor Foxd4l1.1 inhibits expression of the neural repressive transcription factor ventx1.1 to commit and maintain the neuroectoderm fate, obviating mesoderm commitment during germ-layer specification of Xenopus embryos.In the present study, we focused on the mechanisms of reciprocally exclusive germ-layer  specification in early vertebrate embryogenesis. In the mesoderm region, both BMP/Smad1 and FGF/Xbra synergistically upregulate transcription of neural repressor Foxd4l1.1 is an early neural transcription factor that contains three distinct domains, each displaying different activities. The three domains are (i) the N-terminal activation domain (the “acidic blob”), (ii) the winged-helix domain (the DNA binding domain) and (iii) the C-terminal repressor domain (Region-II and P/A/Q)37. We examined wild type foxd4l1.1 and Drosophila repressor domain (engrailed repressor domain) fused-foxd4l1.1 (EnRfoxd4l1.1) on whether they inhibit ventx1.1 expression in animal cap explants. EnRfoxd4l1.1 is the same as EnRfoxd5a reported by Sullivan et al.37 RT-PCR results indicated that both foxd4l1.1 and EnRfoxd4l1.1 suppressed expression of ventral genes, including bmp4, ventx1.1 (PV.1), and ventx1.2 (Xvent1) at stage 11  was increased by foxd4l1.1 and EnRfoxd4l1.1  (FRE) within the ventx1.1 promoter, ventx1.1 (− 2481) promoter construct was injected with and without foxd4l1.1 (wild-type) construct at the one-cell stage at various dosages (1–4 ng) and the reporter gene activity assay was performed. Increasing levels of foxd4l1.1 reduced the relative promoter activity of ventx1.1 (2481) at up to 2 to 3.5-fold in a dose-dependent manner  was then examined. Ventx1.1 (− 2481) promoter construct was co-injected with and without the aforementioned repressor form of foxd4l1.1  at the one-cell stage and the reporter gene activity was measured at stage 11. EnRfoxd4l1.1 remarkably decreased the relative promoter activity as well as the relative mRNA levels of ventx1.1 at up to eightfold compared with those without EnRfoxd4l1.1  more efficiently inhibited the relative promoter activity of ventx1.1 compared to those of wild type foxd4l1.1  for Foxd4l1.1 to inhibit ventx1.1 transcription. To identify the location of FRE(s) within the ventx1.1 promoter region, we generated and examined serially-deleted promoter constructs of ventx1.1 with and without EnRfoxd4l1.1  promoter still contained the putative consensus FRE. A previous study indicated putative human fork-head proteins binding consensus sequences  within the promoter region of Fox family-targeted genes38, and ventx1.1 (− 157) promoter region also contains one of these putative FREs, ATAAAA, referred to as FRE1 (− 82 to − 76 region of ventx1.1 promoter). To determine whether the putative consensus FRE1 actually functions as a Foxd4l1.1 response element within the ventx1.1 promoter region, we mutated the sequence (ATAAAAA to AGCAAAA) within the ventx1.1 (− 157) promoter construct as shown in Fig. ventx1.1 (− 157) mFRE1 and the unaltered ventx1.1 (− 157) constructs were then examined in the same assay. As shown, foxd4l1.1-mediated reduction of ventx1.1 (− 157) was reversed in the FRE1-mutated ventx1.1 (− 157) mFRE1  promoter contains the cis-acting FRE1 for Foxd4l1.1, which is required in inhibition of ventx1.1 transcription. We next asked whether Foxd4l1.1 directly bound within the proximal region of ventx1.1 promoter. HA-foxd4l1.1 mRNA was injected at the one-cell stage and a ChIP-PCR assay was performed at stage 11 in Xenopus whole embryos. The results showed that Foxd4l1.1 directly binds within the proximal region of endogenous ventx1.1 promoter  promoter construct (previously designated as ventx1.1 (− 103))15. XbRE (Xbra) cooperates synergistically with BRE  was located within the ventx1.1 (− 157) in close proximity to XbRE  was co-injected with and without xbra and EnRfoxd4l1.1, in combination or separately. EnRfoxd4l1.1 abolished xbra-induced relative promoter activity of ventx1.1 (− 157)  also locates within the x1.1 − 10)15. XbREx1.1 − 10)15. XbREx1.1 − 10)15. XbREx1.1 − 10)15. XbREEnRfoxd4l1.1 inhibited expression of BMP4 and its target genes, resulting in the neuroectoderm formation in the animal cap explants 34. In Xenopus, the expression domains of szl and bmp4 overlap and it was shown that szl expression is strongly dependent on BMP435. Therefore, we examined whether foxd4l1.1 indeed inhibits Smad1 activity and Smad1-induced transcription activation of ventx1.1. To examine the Smad1 activity, 3BRE  reporter gene construct was injected with and without EnRfoxd4l1.1 at the one-cell stage and collected the injected embryos at stage 11 for reporter gene assays. EnRfoxd4l1.1 significantly decreased the relative reporter activity of 3BRE construct compared to that of 3BRE alone  (BMP4 inhibition dependent) or the increase of MAPK-mediated linker region phosphorylation (pSer-206) (FGF dependent)40, we examined the phosphorylation status of Smad1 to test on which pathway mediated ventx1.1 repression by foxd4l1.1. Our results indicated that foxd4l1.1 not only reduced C-terminal phosphorylation of endogenous Smad1  phosphorylation in our system  expression with and without foxd4l1.1 in gastrula embryos and animal cap explants. Foxd4l1.1 strongly inhibited xbra expression in whole embryos  to induce linker region phosphorylation (pSer-206) of Smad1, which leads to cytoplasmic retention of Smad1 and contributes to transcription inhibition of neural repressor, ventx1.1. (2) Foxd4l1.1/FGF/MAPK axis mediated Smad1 inactivation may lead to inhibition of zygotic expression of bmp4, resulting in reduction of BMP4 levels, leading to reduced C-terminal phosphorylation of Smad1 and its downstream target genes including ventx1.1. Taken together, Foxd4l1.1-mediated inhibition of BMP4/Smad1/Ventx1.1 axis may contribute to preventing neuroectoderm to ectoderm transition in dorsal ectoderm (neuroectoderm) region of Xenopus embryos.ventx1.1. Foxd4l1.1 physically interacts with Xbra  promoter construct which contains all three response elements including BRE, XbRE and FRE1 (Foxd4l1.1 binding site). Results showed that ectopic expression of EnRfoxd4l1.1 significantly decreased synergistic activation of ventx1.1 (− 233) promoter activity which was increased by smad1 and xbra in absence of EnRfoxd4l1.1  promoter activity. Foxd4l1.1 may be inhibiting the physical interaction of Xbra and Smad1. Foxd4l1.1 actually reduced the physical interaction of Xbra and Smad1  construct up to 0.5-fold  promoter region may contain more than one consensus cis-acting FRE, jointly involved in negative regulation of ventx1.1 transcription. Thus, we further mapped the ventx1.1 (− 2481) promoter region and found 6 more putative cis-acting FRE (RTAAAYA) sites in − 2481 promoter region of ventx1.1  promoter construct  constructs were reduced by co-injection with EnRfoxd4l1.1 mRNA albeit the reduction was less than those of wild type ventx1.1  may also contribute to EnRFoxd4l1.1-mediated reduction of ventx1.1 (− 2481) mFRE1 reporter activity. The EnRFoxd4l1.1-mediated reduction was recovered by reducing the number of putative FREs in the ventx1.1 promoter  mFRE nor BRE-mutated ventx1.1 (− 233) mBRE constructs completely abolished EnRfoxd4l1.1-mediated reduction of the relative promoter activity  m(BRE + FRE1) promoter construct  m(BRE + FRE) construct  is essential for epidermis specification or the details of the relevant GRNs among various non-neural TFs still remain to be addressed.In the present study, we wanted to uncover a potential mechanism for non-neural ectoderm exclusion seen in neural ectoderm areas as part of an established transcriptional gene regulatory network (GRN). Here, we focused on elaborating the repressive activity of the neural specific transcription factor (TF), ventx1.1 as a target TF to examine Foxd4l1.1 function in preventing cells from reverting to a non-neural fate. The reasons on why we focused on ventx1.1 are the following: First, ventx1.1 is expressed throughout the embryonic ectoderm in blastula to gastrula embryos such as with msx1 and ventx2. Second, ventx1.1 induces the epidermis and inhibits the formation of the dorsal mesoderm and neural tissue17. Third, ventx1.1 is a neural repressor, being a direct target TF of BMP418. Fourth, inhibition of ventx1.1 induces neural ectoderm together with FGF in animal cap explants, similar to DNBR treated animal caps24. Fifth, the reporter activity of ventx1.1 promoter construct is reduced by foxd4l1.115. Ventx1.1 transcript is found in the ventral ectoderm and mesoderm, but is completely absent in the organizer and the neuroectoderm region in early gastrula embryos43. Although Ventx1.1 has yet to be proven as an essential master TF among the direct target BMP TFs in epidermal specification, our previous study that ventx1.1 directly represses foxd4l1.1 led to examine the reciprocal repression between them in specifying the non-neural versus neural ectoderm fates at the transcription level. For a given ectodermal region, among exclusively expressed neuroectoderm or ectoderm genes, certain TFs may function as repressors in excluding expression of genes of alternate fates. Such a proposal has been put forward by Sasai25. Once, the neural ectoderm is formed and neural transcription factors (nTFs) are expressed, some of the earliest expressed nTFs are needed to prevent cells from reverting to a non-neural fate26. The primary neuroectoderm expresses various transcription factors that may block it from becoming an epidermis28.In this study, we selected Foxd4l1.1  transcript is exclusively expressed in the superficial layer, namely neuroectoderm, of cells above the dorsal lip of the Spemann organizer territory at the early gastrula43. Both Foxd4l1.1 and Ventx1.1 contain a strong repressive domain44 with mutually exclusive expression in the ectoderm/ventral mesoderm and the neuroectoderm/organizer, respectively, at the time of the ectoderm/neuroectoderm commitment for early gastrula. We hypothesized that BMP signal modulation leads to expression of non-neural versus neural TFs, with at least one being essential in preventing cells from reverting to alternate fates. We thus examined foxd4l1.1 as one of the earliest expressed nTFs, which block neuroectoderm from becoming epidermis. Exclusive presence of a repressive gene in a given territory would at least be a strategy to exclude essential gene expression involved in alternate germ layer commitment. For example, ectopic overexpression of organizer genes including gsc, chordin and noggin in ventral region of 4-cell stage embryos leads to two-axis formation in whole embryos. On the other hand, overexpression of ventral specific genes including vent1.1, vent1.2 and bmp4 in dorsal region of 4-cell stage embryos leads to headless embryos. As such, overexpression studies in developing embryos suggest that ectopic signaling or presence of certain TF(s) in competent cells convert their GRNs.gsc and ventx1/2 has also been proposed to in part mediate dorsoventral patterning to ensure robust and reproducible embryonic development through triple depletion of gsc, ventx1, and ventx245. Various TFs have been proposed and examined for being reciprocal means of repression in early vertebrate embryogenesis47. Involvement of foxd4l1.1 and ventx1.1 in neuroectoderm versus ectoderm specification cannot be ruled out in the present study and require further study. However, in this work, we mainly focused on the maintenance/protective role of foxd4l1.1 in the expressed cells of neuroectoderm for fate reversion.Reciprocal transcriptional repression of ventral and dorsal opposing homeobox genes foxd4l1.1 expression, which is directly repressed by Ventx1.118. In the present study, foxd4l1.1 induced neuroectoderm formation which was also inhibited by co-injection of ventx1.1 . It was shown that Foxd4l1.1 directly binds to one of the cis-acting FREs of ventx1.1  to inhibit ventx1.1 transcription  of ventx1.1 promoter . However, the exact mechanism remains to be demonstrated.From our previous work, inhibition of BMP signaling induces ter Fig. b–f. In afoxd4l1.1 mediated Smad1 inhibition, it has been documented that ventrally injected foxd4l1.1 reduces the number of phosphorylated Smad1 (activated Smad1) positive cells and inhibits BMP4/Smad1 downstream targets epi-keratin and ap2 in Xenopus embryos34. Furthermore, foxd4l1.1 increases expression of szl, suggesting that foxd4l1.1 reduces nuclear localization of Smad1 either by inhibiting bmp4 expression or by Sizzled-mediated inhibition of BMP4 in a Chordin-dependent manner34. In the present study, we found that foxd4l1.1 reduced bmp4 expression    and xbra with both foxd4l1.1 and EnRfoxd4l1.1 injected embryos. Neither injection induced xbra or fgf4 in the animal cap explants, suggesting that FGF4/MAP-kinase/xbra loop is not involved in foxd4l1.1 mediated Smad1 linker phosphorylation. Furthermore, whether fgf8a/b induction occurs through Foxd4l1.1 activator function or indirectly through Foxd4l1.1 repressor function would be interesting to explore. Both foxd4l1.1 mRNAs induced fgf8a/b, implying that fgf8a/b induction may occur indirectly through Foxd4l1.1 repressor function and Xbra/FGF positive feedback loop was not the reason for Smad1 inactivation. Similar to Foxd4l1.1, Zbtb14 reduces the levels of phosphorylated Smad1/5/8 in Xenopus51. Overexpression of zbtb14 promotes neural induction similar to that of foxd4l1.1. While BMP inhibition and overexpression of foxd4l1.1 induces anterior neural tissue, zbtb14 promotes posterior neural tissue and suppresses anterior neural tissue. Presently, we cannot delineate the connection between Foxd4l1.1 and Zbtb14 in neural induction and Smad1 inactivation, and details of foxd4l1.1 mediated BMP/Smad1 signal attenuation and fgf8 involvement during neuroectoderm formation await more elaboration.For ventx1.1 transcription in Xenopus embryos15. Foxd4l1.1 reduced Smad1 and Xbra physical interaction possibly, first, due to reduced C-terminal phosphorylation of Smad1 via reduced BMP expression, and second, via competition for Xbra since both Foxd4l1.1 and Smad1 bind to Xbra protein  promoter construct by up to eightfold while it reduced that of ventx1.1 (− 157) construct by up to 0.5-fold. This may be due to ventx1.1 (− 2481) promoter containing more than one functional FRE for Foxd4l1.1. The ventx1.1 (− 2481) promoter region contains an additional six putative cis-acting FREs for Foxd4l1.1  promoter constructs indicated that the 5′-flanking region of ventx1.1 contains more than one consensus FRE, actively participating in Foxd4l1.1-mediated negative regulation of ventx1.1 transcription  mBRE promoter constructs  m(BRE + FRE) promoter construct  expression by EnRFoxd4l1.1. Our study demonstrates that gsc inhibited ventx1.1 expression as well as the relative activity of ventx1.1 promoter construct16. Ventx1.1 (− 233) promoter contained the direct binding response element for Gsc  cycle and at 18℃ according to the guidelines of the Institute of Laboratory Animal Resources.This animal study was conducted in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Hallym University . All the research members attended both the educational and training courses for the appropriate care and use of experimental animals at our institution in order to receive an animal use permit. Adult EnRfoxd4l1.1: T7, BamHI, Myc-xbra: Sp6, Asp718, HA-foxd4l1.1: Sp6, Asp718, and Flag-smad1: Sp6, SalI. In vitro transcription was performed by using a MEGAscript kit accordingly to the manufacturer’s instructions . All synthesized mRNAs were quantified by a spectrophotometer at 260/280 nm .All mRNA used for this study were synthesized from linearized vectors. All vectors were linearized with the appropriate restriction enzymes, including ventx1.1 gDNA of Chr-3S was performed into the pBluescript SK(-) plasmid  as described by Lee et al.16.The cloning of ventx1.1 promoter mutants and triple-repeated BMP4-response element (BRE) were generated from − 2481 bps construct and subcloned into a pGL-2 basic plasmid by PCR amplification16. Numbering of the reporter construct was for the distance 5′-upstream of the translation start site (ATG).The 2.5 kbs of 5′-flanking region of positive clone was subcloned into the pGL-2 basic plasmid  and was designated as the − 2481 bps construct. Serially-deleted Xenopus laevis adults were obtained from the Korean Xenopus Resource Center for Research . Xenopus embryos were obtained by in vitro fertilization after induction of female frogs with 500 units of human chorionic gonadotropin . RNAs were injected into the animal pole of 1–2 cell stage embryos; animal caps explants were dissected from injected embryos at stage 7–8 and incubated to stage 11 and 24 in L-15 medium for RT-PCR experiments.EnRfoxd4l1.1 (280 pg/embryos) and other mRNA (Myc-xbra (1 ng/embryo), Flag-smad1 (1 ng/embryo) and HA-foxd4l1.1 (3 ng/embryo)) was injected into the animal pole of one-cell stage embryos that were then cultured in 30% MMR solution until stage 8 and also for control non-injected embryos. Animal caps were then dissected from the injected and non-injected embryos and incubated until stage 11 and 24 in L-15 medium. Total RNA was isolated from whole embryos or animal caps using RNA-bee reagent following the manufacturer’s instructions  as described by Kumar et al. (2018)15. PCR was performed by using oligonucleotides according to the following conditions as described in Table 57 was not repeated. All qPCR reactions were repeated three time using independent samples to present data with standard deviations and statistical significance.The qPCR reactions were performed by using an Applied Biosystems StepOnePlus Real-Time PCR System with KAPA SYBER FAST qPCR Master Mix. All the real-time values were averaged and compared using the threshold cycle (CT) method, in which the amount of target RNA (2 − ΔΔCT) was normalized against the endogenous expression of ODC (ornithine decarboxylase) (ΔCT). The qPCR reactions were performed when RT-PCR reaction results need to be quantified Fig. s. The con15.Levels of relative luciferase activity were measured as described by Kumar et al. (2018)15.The binding sites of transcription factors including Foxd4l1.1 (FRE1) and Smad1 (BRE) were identified using serially-deleted reporter gene constructs Table  and siteThe mRNAs were injected at the one-cell stage of embryos and collected at stage 11 for western blots. Non-injected embryos served as the negative control. Collected embryos were lysed in lysis buffer with phosphatase and protease inhibitors, in preparation for resolving the proteins with 10% SDS-PAGE and transfer to a PVDF membrane, with the membrane blocked and incubated with either pSmad1 pSer-463/465 (CS-9511S) or pSmad1 pSer-206 (CS-9553P) antibodies. Following washes of the PVDF membrane, it was incubated with the enzyme-labeled secondary antibody . The protein signals were visualized by an ECL detection kit .smad1, HA-foxd4l1.1 and Myc-xbra mRNA constructs in four different groups. The injected embryos were collected at stage 11.5. They were then homogenized in lysis IP buffer. The composition of the IP buffer is described in Kumar et al. (2018)15. Cell lysates were cleared by centrifugation and were then incubated with Flag-Smad1  monoclonal antibody and α-HA  polyclonal antibody overnight at 4 °C with the immunocomplexes precipitated by protein A/G beads . Proper amounts of precipitated beads-protein complex were boiled in the sample buffer, and resolved by electrophoresis in 10% SDS–polyacrylamide gels. Western blotting of Myc-Xbra  was performed by using an anti-Myc and secondary antibody anti-mouse . Immune complexes were visualized by using an ECL detection kit .Embryos were co-injected at one-cell stage with Flag-58. Dissected animal caps were fixed in 4% paraformaldehyde for 2 h and incubated in PBSTB  for 1 h. The animal caps were incubated with primary antibody for overnight at 4 °C and washed with PBS. The animal caps then were incubated with fluorescent secondary antibodies for 2 h at room temperature. Mounted animal cap images were then obtained and analyzed by confocal microscopy   at a magnification of 63X.Embryos were injected at one-cell stage into animal pole with mRNAs. Injected and un-injected embryos were dissected at stage 9.5 to perform immunofluorescence as described in Lee et al. (2018)59 Embryos were injected at one-cell stage with mRNA encoding HA-foxd4l1.1, Myc-xbra and Flag-smad1 (1 ng/embryo) either separately or in combination. The antibodies used to immunoprecipitate chromatin were anti-HA , anti-Myc  polyclonal antibody and anti-Flag  monoclonal antibody. Normal rabbit IgG  and normal mouse IgG  used as a negative control. PCR were performed with immunoprecipitated fragmented chromatin using ventx1.1 (− 233 and − 157) promoter region primers. The Fold Enrichment Method was used to analyze ChIP-qPCR values60. “Fold enrichment” is by calculating the ΔCT for the difference between CT values for the ChIP samples using the antibody of interest and the negative control antibody. The primer sequences are shown in Tables Chromatin immunoprecipitation assay was performed as described in Blythe et al. 2009)00959 Embventx1.1  cDNA sequence has been submitted to GenBank16.The p < 0.05 was considered to be significant for difference. Notations: **p ≤ 0.01; ***p ≤ 0.001; n.s., not significant.Data were analyzed by GraphPad Prism5 . Statistical analysis was established using one-way ANOVA. Supplementary information