AHR −129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that −129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with −129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than −129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo.Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of  Vitiligo is a chronic depigmentation disorder resulting from melanocyte destruction. The incidence of vitiligo is approximately 0.5 ∼ 8% worldwide, and over 50% of the patients develop the disease between the ages of 10 and 30 years124678Ahr-deficient mice showed decreased melanocyte number and hypopigmentation in the tail skin caused by reduced levels of SCF and C-KitThe aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and belongs to the basic-helix-loop-helix family12AHR polymorphisms and vitiligo susceptibility. Our data demonstrated that the T allele of −129C > T variant (rs10249788) in the AHR promoter region is associated with a protective effect on vitiligo in Han Chinese populationsAHR transcription process. The promoter of human AHR gene lacks TATA and CCAAT boxes but possesses several putative SP1 transcription factor (SP1) binding sites within a highly GC-rich region2/His2-type zinc-finger transcription factor that binds to GC box elements (5′-GGGCGG-3′) within promoter regionAHR gene via binding to these GC-rich motifsAHR promoter is responsible for AHR down-regulation in human diseases22AHR gene and is juxtaposed 5′ to the SP1 recognition sequence, we therefore hypothesized that the AHR −129C > T polymorphism could influence its transcription and downstream effectors in melanocyte biology or immune system, which could further affect the development of vitiligo. In the present study, we performed functional research to explore the molecular mechanisms underlying such genetic marker for vitiligo.In consideration of the critical role of AHR in both melanocyte and cellular immunity and aberrant AHR pathway in vitiligo, we previously evaluated the potential association between AHR promoter activity related to −129C > T polymorphism, C or T promoter constructs were transiently transfected in human normal melanocyte PIG1 cells, human malignant melanoma LiBr cells and human embryonic kidney 293T cells. As shown in P = 0.018 for PIG1 cells; P = 0.010 for LiBr cells; P = 0.023 for 293T cells). These results suggest that the AHR −129T allele possesses an increased AHR transcriptional activity.To assess the AHR −129C > T variant on DNA local structure, bioinformatic analysis was performed using the OH Radical Cleavage Intensity Database (ORChID) and showed different hydroxyl radical cleavage patterns among variants of the AHR −129C > T polymorphism. As shown in Both nucleotide sequences and DNA shape influence specific binding of proteins to DNA24262728AHR promoter and transcriptional regulators, we carried out electrophoretic mobility shift assays (EMSA) and found the DNA-protein complex with stronger intensity for the −129T allele than −129C allele  from 23 vitiligo patients and 23 age- and sex-matched controls harboring different AHR −129 genotypes . We observed positive correlation between AHR and SP1 expressions . We detected significantly increased TNF-α concentration and decreased levels of IL-10 and TGF-β1 in the serum of vitiligo patients compared with controls  underlying its association with vitiligo. Our data demonstrated that the protective T allele of this polymorphism dramatically enhanced AHR promoter activity through promoting the binding activity of SP1. We also found decreased AHR transcript expression in the PBMCs of vitiligo patients, which was negatively correlated with disease duration. Further genetic analysis showed that −129T carriers had higher AHR expression than −129C carriers. We further detected AHR related cytokines in vitiligo patients and observed declined serum IL-10 level and its association with the AHR −129C > T variant. Thus, our scientific evidence strengthens our previous findingAHR −129C > T polymorphism is indeed a functional genetic marker for vitiligo.In the current study, we investigated the molecular mechanisms of AHR promoter function, which might influence AHR transcription by altering its interaction with transcriptional regulators. Human AHR gene contains features of a housekeeping gene as possessing GC-rich regionsAHR gene to 20%AHR promoter caused by SP1 inhibitor or AHR hypermethylation led to AHR downregulation2223AHR genes. Given the pivotal role of SP1 in AHR expression and AHR −129C > T variation located nearby the core binding site for SP1, we then performed EMSA and ChIP assays and observed the −129T allele with higher SP1 binding affinity than the −129C allele. Li D. et al. previously reported that the −129C > T variant interfered the binding activity of nuclear factor 1-C, which might down-regulate AHR transcription via antagonizing SP1 occupancyAHR transcription remains unclear, it is conceivable that the AHR −129C > T variant may affect the binding of SP1 to the AHR promoter in a direct or indirect manner.In this report, we first proved the contribution of −129C > T variation to AHR expression and vitiligo remains to be elucidated. Gene expression analysis of PBMCs from independent cases and controls showed apparently decreased AHR mRNA level in vitiligo, which was clearly negatively correlated with disease duration of vitiligo patients, suggesting that down-regulation of AHR transcription is relative to vitiligo. We also found that declined SP1 mRNA level in vitiligo patients and a strongly positive correlation between SP1 and AHR expressions. It has been reported that the expression levels of SP1 and AHR vary greatly among different tissuesSP1 and AHR were observed in the same tissues, such as thymus, lung, and spleen3522AHR promoter activity could eventually influence AHR transcript expression, we performed genetic analysis showing that the protective −129T allele is associated with elevated expression of AHR mRNA. Both in-vitro and in-vivo transcriptional analyses provided consistent results, indicating the potential effect of −129C > T mutation on AHR expression, which might in turn influence its function in regulating melanogenic factors including TYR, TYRP, SCF and C-Kit143841424345AHR −129C > T polymorphism might be associated with vitiligo through influencing AHR expression and its downstream melanogenic factors.The association between AHR −129C > T polymorphism, we assessed the impact of this functional variant on the immune cytokines in vitiligo. Consistent with previous findings47AHR −129T allele than −129C allele. Recent studies have demonstrated that AHR could affect IL-10 expression via mediating the differentiation of Treg cells49Ahr null mice inefficiently generated Tregs with decreased IL-10 levels53485758AHR −129C > T mutation might be related to vitiligo partially by affecting IL-10 production, which further supports previous findings about AHR function in immune regulation. Nevertheless, immune system contains many biological structures and complex processes, which could be modulated by mutual effort of multiple factors including AHR. Thus, functional studies may be needed to explore the regulatory mechanisms of AHR pathway in the immune pathology of vitiligo further.To further understand another significance of There are several limitations within our study. First, owing to absence of melanocytes in vitligo epidermis and Koebner phenomenon exiting in vitiligo, it is hard to acquire primary melanocytes or tissue samples from vitiligo patients and we therefore used PBMCs instead of melanocytes to confirm these findings. Second, multiple genes and various environmental factors make collaborative contribution to vitiligo. These highlight the need to explore the gene-gene or gene-environment interaction in vitiligo in our future genetic studies.AHR –129C > T variant could lead to allele-specific binding of SP1 to AHR promoter and further modify its transcription and downstream effectors in vitiligo. Therefore, our findings have not only advanced our understanding on AHR physiological function but also provided significant insight into the pathogenesis of degenerative disorders or autoimmune diseases including vitiligo.In summary, we provide evidence that AHR −129C > T polymorphism.ORChID (dna.bu.edu/orchid) was used to predict the hydroxyl radical cleavage pattern of local variation in solvent-accessible surface area of duplex DNA, and provided information on the local structural DNA profiles from nucleotides sequence containing the Human normal melanocyte PIG1 cell line  was cultured in Medium 254  supplemented with Human Melanocyte Growth Supplement  and 5% Fetal Bovine Serum . Human embryonic kidney 293T cell line and human malignant melanoma LiBr cell line were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12  supplemented with 10% FBS. For SP1 inhibition, PIG1 and LiBr cells were treated with 100 nM chemical inhibitor mithramycin A  or control vehicle DMSO  for 48 h.AHR −129C > T variant have been described previouslyFor assays of gene expression, 23 patients with vitiligo and 23 sex- and age-matched control subjects were enrolled in the present study. We obtained 10 ml of peripheral blood from each participant for PBMCs isolation as describedAHR promoter region was directly synthesized with added KpnI and MluI sites at 5′ ends in forward and reverse sequences, respectively . The DNA sequence was inserted between KpnI and MluI sites in the firefly luciferase reporter vector pGL3-Basic . The −129C > T polymorphism was introduced into the recombinant pGL3 reporter vector by performing site-directed mutagenesis. The sequence of constructs was confirmed by direct DNA sequencing.A 534-bp DNA fragment (−534 to −1) of human For transfections, PIG1, LiBr and 293T cells were plated onto 24-well plates, respectively. The cells were transfected with 0.8 μg of the recombinant pGL3 reporter vector with either −129C or −129T allele using Lipofectamine 2000 Transfection Reagent . The pGL3-Basic vector without an insert and pGL3-Control vector  were used as a negative control and a positive control, respectively. All plasmids were cotransfected with 0.02 μg of pRL-SV40 vector  as an internal standard. At 48 h after transfection, cells were collected and luciferase activity was measured with Dual-Luciferase Reporter Assay System  according to the manufacturer’s protocols. Luciferase activity was normalized against the activity of pRL-SV40 with the Renilla luciferase gene. Independent triplicate experiments were done for each plasmid construct.GGGCGGGGG-3′ (SP1 binding site underlined) were synthesized by Viagene Biotech Inc . Nuclear extracts (10 μg) were isolated from PIG1 cells using Viagene Subcellular Protein Fraction Kit  and protein concentration was determined by BCA Protein Assay Kit . For each gel shift reaction (15 μl), a total of 300 fmol labeled probes were combined with 10 μg of nuclear extract, 1.5 μg of poly (deoxyinosinic-deoxycytidylic acid) and 10 × binding buffer. For competition assays, a 100-fold molar excess of unlabeled either −129C or −129T probes was preincubated for 20 min at room temperature with nuclear extracts before addition of the labeled probes. For each supershift reaction (15 μl), 3.5 μl of anti-SP1 antibody  was incubated with nuclear extracts at 4 °C for 15 min, followed by an additional incubation for 20 min at room temperature with the labeled probes. After incubation, samples were separated on a native non-denaturing 6.5% polyacrylamide gel and then transferred to a nylon membrane. The positions of biotin-labeled probes in the membrane were detected by a chemiluminescent reaction with the stabilized streptavidin– horseradish peroxidase conjugate according to the manufacturer’s instructions and visualized by autoradiography.The DNA-protein interactions were detected using LightShift Chemiluminescent EMSA Kit . The biotin labeled and unlabeled double-stranded oligonucleotides corresponding to a 30-bp sequence 5′-BIO-AAGAC/TGGAATGGAATCCAGAT6 of PIG1 cells and LiBr cells were treated with 2.7% formaldehyde for 10 min, followed by 2.5 M glycine stop solution. Cells were then collected and centrifuged at 4 °C and resuspended in lysis buffer for 30 min. The extracts were sonicated, and the DNA was sheared using enzymatic shearing cocktail until the DNA fragments were 200 ∼ 500-bp in size. Sheared chromatin (10 μl) was incubated with protein G magnetic beads and 3 μg of rabbit polyclonal anti-SP1 antibody , rabbit control IgG (Santa Cruz Biotechnology) or rabbit polyclonal anti-RNA polymerase II antibody  overnight at 4 °C. After extensive washing of the beads, the samples were reverse cross-linked and treated with ribonuclease A and proteinase K. After DNA purification, the immunoprecipitated chromatin fraction was amplified by PCR with primers specific for the AHR promoter . The mRNA levels of AHR, SP1 and ACTB were measured by real-time quantitative PCR using specific primers  and then converted to cDNA using PrimeScriptmers see  online. SP1 and AHR protein expressions in PIG1 and LiBr cells were examined by Western blots analysis. Western blots (50 μg protein/lane) were performed as described previouslyThe amounts of TNF-α, IFN-γ, IL-10, TGF-β1, IL-17A and IL-22 in the serum from each participant were determined using a corresponding ELISA kit  according to the manufacturer’s instructions.t test was performed to evaluate the difference in luciferase activities between different constructs. Unpaired t test with Welch’s correction was used to assess the different DNA-protein binding affinities between −129T and −129C probes. Non-parametric Mann–Whitney U-test was applied to analyze the results of peripheral AHR and SP1 mRNA expressions and serum cytokine concentrations between vitiligo patients and controls with different AHR −129 genotypes. Correlation studies were performed using Pearson’s correlation test. All tests were two-sided by using SPSS software . Statistical significance was defined as P < 0.05.Student’s How to cite this article: Wang, X. et al. AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo. Sci. Rep.5, 13542; doi: 10.1038/srep13542 (2015).


We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.DOI:http://dx.doi.org/10.7554/eLife.06942.001 Many genes working within large gene networks influence the development of heart muscle cells in humans and other animals. The activity of these genes is controlled in part by proteins called transcription factors, which bind to DNA and act as molecular switches. One transcription factor that is particularly important for the development of heart muscle cells is called NKX2-5.Mice lacking NKX2-5 have abnormal hearts and many humans who are born with congenital heart disease carry mutations in the gene that encodes this protein. Many of these mutations alter a section of the protein called the homeodomain, and therefore interfere with the ability of NKX2-5 to bind to DNA or associate with other important cardiac proteins called cofactors. However, it is not clear how such mutations alter the behaviour of NKX2-5 across all of its targets.Bouveret et al. have now used a technique called ‘DNA adenine methyltransferase identification’ to study how NKX2-5 interacts with other proteins and DNA. The experiments found that, as expected, the mutant NKX2-5 proteins were unable to associate with many of the usual gene and protein targets of normal NKX2-5. However, the mutant proteins were still able to bind to some of their usual targets, plus many other targets that the normal NKX2-5 protein was not able to bind to.A particular NKX2-5 mutant protein that the experiments analysed was missing the entire homeodomain, yet it was still able to associate with the normal NKX2-5 protein and bind to cofactors that help NKX2-5 to find its usual targets. This finding led Bouveret et al. to discover the role of a section of the NKX2-5 protein called the tyrosine-rich domain, which in the absence of the homeodomain can direct interactions of NKX2-5 with itself and its cofactors.Bouveret et al.'s findings suggest that protein cofactors of NKX2-5 help mutant NKX2-5 proteins retain some of their normal activities, but also direct the mutant proteins to abnormal gene targets, which could contribute to congenital heart disease. The next steps are to carry out experiments in animals to confirm these findings, and to understand the activities of mutant NKX2-5 and other mutant transcription factors across the whole genome. This could lead to new therapeutic approaches to treat congenital heart disease and other conditions.DOI:http://dx.doi.org/10.7554/eLife.06942.002 Nkx2-5 in mice leads to arrested heart morphogenesis due to blocked progenitor growth, defective chamber and conduction system development, and a deranged GRN  . The carnged GRN . In humanged GRN . Many CHnged GRN , which snged GRN . It is wnged GRN , often iHere, we take a functional genomics approach to understanding the mechanism of NKX2-5 CHD at the chromatin level. We applied the technique of DNA adenine methyltransferase identification (DamID)  to identEscherichia coli DNA adenine methyltransferase (Dam). When expressed, Dam fusions bind to DNA, whereby Dam locally methylates adenine within its target sites (5′GATC3′). Target DNA can then be released using DpnI, a nuclease specific for the methylated Dam site, and analysed on arrays , which was sufficient for Dam enzymatic activity on chromatin (DpnI sites (5′GATC3′), which occur on average every 260 bp in the mouse genome, as in the fly genome , suggesting that DamID captures the vast majority of NKX2-5 direct targets.Here, we used DamID in HL-1 cells, which resemble atrial cardiomyocytes, to profile NKX2-5 WT and mutants that mimic those found in CHD . Mouse Nromoters . Only N-romoters . Transduromoters . DamID fhromatin  but avoiy genome . After D-2000 bp . We deteCisGenome after subtraction of signal obtained from cells expressing Dam alone .Weeder or Trawler    in vitroursor A) .10.7554/Surprisingly, in HL-1 cells, both NKX2-5Y191C and NKX2-5∆HD associated with a large number of loci  than that mutants , which diffused more slowly than paGFP alone (The ability of NKX2-5Y191C and NKX2-5∆HD to bind a large number of targets was unexpected. Hence we sought to confirm using an independent approach that these mutants could interact with chromatin in vivo. We employed photoactivatable fluorescence correlation spectroscopy (paFCS) , measuriFP alone . This coFP alone  and suggNppa. Therefore, the HD mutations result in a severe loss of function. However, a high proportion of mutant peaks overlapped with NKX2-5 WT peaks .We compared NKX2-5 WT and mutant peaks and adopted a simple nomenclature  for peak subsets, where A is bound by NKX2-5 WT only, B is bound by both WT and a mutant, and C is bound only by a mutant . In bothClover (TRANSFAC (BIOBASE) and JASPAR databases  as a B set gene bound by NKX2-5 WT, Y191C, and ΔHD for detailed analysis  cells in which expression of exogenous NKX2-5 WT or mutants could be conditionally induced by doxycycline (dox)-dependent activation of a ubiquitous promoter , 2011. FActc1, Nkx2-5, Hand2, and Gata4 were repressed by both NKX2-5 WT and NKX2-5Y191C, while Nppa and Tbx3 were activated by both. These data show that while NKX2-5Y191C loses its ability to bind and regulate most normal NKX2-5 targets, it nonetheless retains some of the regulatory capabilities of NKX2-5 WT.To gain deeper insights into the underlying effects, we therefore measured alterations in gene expression in the ES cell lines at day 5–6 in three independent experiments, testing a selection of A and B-set genes derived from the NKX2-5WT:Y191C comparison. The majority of both A (8/12) and B (12/13) set genes tested were modulated >1.5 fold by NKX2-5 WT , providing strong evidence that DamID selects NKX2-5 WT developmental targets. Within B-set genes, most were repressed by NKX2-5 WT, consistent with the repression of cardiogenesis by NKX2-5 WT at early stages of cardiac lineage specification . A subseOur results showing that the NKE is enriched in both B-sets suggest that NKX2-5 mutants can bind targets by dimerising with NKX2-5 WT. While dimerisation to NKX2-5 WT has been demonstrated for NKX2-5Y191C , it was Renilla luciferase protein fragment complementation assay (Rluc-PCA)   with a wluc-PCA) . It alsoluc-PCA) . We furtluc-PCA) , detectiluc-PCA) , the latY−A mutation in which all 9 tyrosines of the YRD are mutated to alanine. We have previously shown that NKX2-5YRDY−A does not affect DNA binding, although it has a strong dominant-negative activity leading to lethal CHD-like phenotypes in high-level heterozygous ES cell chimaeras, and when placed over the null allele creates a phenocopy of NKX2-5 loss-of-function  for the NKE is reduced by 10-fold, while its affinity for the TAAT core was unchanged . We focused on the enrichment of TF-binding sites in the C-set of NKX2-5∆HD, based on the hypothesis that DNA-binding cofactors of NKX2-5 guide NKX2-5∆HD to its off-targets. The motifs for E−26 transformation-specific (ETS) TFs occurred most frequently in NKX2-5∆HD C-set peaks (42%) and were significantly enriched compared to random peaks, suggesting that ETS factors could play a role in guiding NKX2-5∆HD to its off-targets. ETS factors are broadly expressed TFs that are activated by phosphorylation downstream of tyrosine kinase receptors. They are important for extracellular signal-gating in specification of cardiac progenitors in Ciona , eve-pososophila , and for mammals . HoweverMany ETS factors are expressed in embryonic hearts , and we Antibodies specific for total ELK1 or its phosphorylated form (pELK1) showed that in HL-1 cells ELK1 was mostly cytoplasmic and unphosphorylated, although in a minority of cells ELK1 was phosphorylated and nuclear, where it co-localised with endogenous NKX2-5 . In CV-1Rluc-PCA in HEK 293 cells, we confirmed that both ELK1 and ELK4 interacted with NKX2-5 WT and NKX2-5∆HD, as well as SRF and TBX5  were bound by ELK1/4, representing more than half of the 26% of this C-set predicted by Y−A and NKX2-5 WT targets revealed robust B and C-set  substitutions in the YRD because this mutant does not interact with ELK1/4. Comparisons between NKX2-5YRD2-5Y191C , support2-5Y191C .Rad50 and Snai2, which bind to NKX2-5∆HD, NKX2-5Y191C, ELK1, and ELK4, but not to NKX2-5 WT, into a luciferase reporter. In HEK 293 cells, both promoters were stimulated modestly by NKX2-5 WT but repressed by ELK1 or ELK4  could be accounted for by NKX2-5 WT binding to a distinct DamID peak, and consistently, NKX2-5 WT and Y191C had opposite effects on these two genes. Thus, NKX2-5 WT influenced a minority of NKX2-5Y191C off-targets. NKX2-5Y191C repressed Dock4 and activated 7/22 other C-set genes with 6 of these not regulated by NKX2-5 WT at the >1.5 fold significance threshold. Other genes showed a similar trend. Thus, NKX2-5Y191C could regulate ≥36% of its off-targets in this system.We next tested whether NKX2-5 mutants could alter off-target gene expression in the inducible ES cell lines described above. For off-targets from the NKX2-5WT:Y191C comparison, NKX2-5 WT modulated the expression of 6/22 C-set genes tested by > 1.5-fold , althougNkx2-5, Actc1, Id2, and Id3), the NKX2-5 and ELK1/ELK4 peaks overlapped. Overlaps between SRF and ELK1 targets, and between SRF and NKX2-5 targets have been documented previously, although not in the same cell type  and generic  terms  terms . SRF targ) terms , consistg) terms .Fos gene—the defining target of ternary SRF-ELK complexes  or of NKX2-5 and ELK1/4 were most over-represented in GO terms heart development, blood vessel morphogenesis, and cytoskeleton organisation. These data show that, overall, ELK1/4, SRF, and NKX2-5 regulate many generic cellular functions, while NKX2-5 and SRF also have more executive roles in regulating heart development and contraction. Furthermore, ELK1/4 appear to control very specific subsets of the cardiac GRN. By integrating the targets of all WT TFs profiled with those of other cardiac TFs previously published, we found that Elk1/4 were embedded in the developmental cardiac GRN with many cross and feedback connections  fail to bind the majority of WT targets (A sets) even in the presence of normal levels of NKX2-5 WT. Loss-of-binding of mutant proteins will be associated with alterations in the level of expression of many genes in the cardiac network through haploinsufficiency, which will be a major contributor to the structural and functional heart defects found in human CHD patients.Our study using DamID confirms for the first time at a genome-wide level in vivo, the prevailing view that severe mutations in cardiac kernel TF . B-sets included direct and indirect NKX2-5 WT target genes and were enriched in heart- and muscle-related GO terms. In the ES cell system, NKX2-5 mutant proteins retained WT-like functionality on some B-set genes. We propose that B-sets are recognised by mutants via dimerisation with cofactors or with NKX2-5 WT. The NKX2-5 HD is a recognised interface for NKX2-5 homo-dimerisation and interactions with cofactors, such as GATA4, HAND1, TBX5/20, and MEF2. NKX2-5∆HD lacks the HD and embedded nuclear localisation domain and represents a severe mutation that has lost the ability to bind to the majority (81%) of normal NKX2-5 WT targets (A-set). However, even this severe mutant retained binding to a subset of direct and indirect NKX2-5 WT targets (B-set). NKX2-5∆HD binding to B-set peaks could ogenesis . In humaogenesis .Y−A), and thus B-set and C-set size and content may prove to be valuable signatures for understanding the relationship between genotype, phenotype, and clinical outcomes in CHD. NKX2-5∆HD off-targets were associated with GO terms chromatin organisation and cell cycle, and peaks were enriched in the binding sites for ETS family and many other broadly expressed TFs, attesting to an underlying logic in their selection. A high percentage of NKX2-5 mutant off-targets (14.2% for NKX2-5∆HD and 6.5% for NKX2-5Y191C) were occupied by ELK1/4 in HL-1 cells, compared to <1% when randomly generated peaks were overlapped. Furthermore, only 3% of C-set peaks of the NKX2-5YRDY−A mutant were bound by ELK1/4. Although ETS factors have previously been predicted to be involved in heart development based on motif enrichment in predicted cardiac enhancers in fly and humans , which we term off-targets. The relative size of the C-set within the total mutant target set (C-set/B-set ratio) correlates with the location and predicted severity of NKX2-5 mutations  was constructed by replacing the HD with 8 glycine residues. Vectors used for lentivirus production were obtained from Prof D Trono/Addgene: pMLDg/pRRE , pRSV-Rev , and pMD2.G   .The HL-1 cell line was donated by Prof W C Claycomb  . Human eNkx2-5 WT, Nkx2-5∆HD, and Nkx2-5Y191, open reading frames were amplified by PCR from the plasmids described in section 'Plasmids/cloning', sequence-verified and cloned in place of Mesp13XFlag in the p2LoxMesp1-3XFlag-IRES-EGFP vector (For doxycycline (dox)-inducible mouse ES cell lines, P vector . These cP vector .ES cells were cultured on irradiated mouse embryonic fibroblasts , 2011. BImmunofluorescence on fixed cells was performed as described by . Antibodcis-element PCR-amplification from mouse HL-1 cell DNA: ACGCTCCTGACTTGACTGTTTT and GCAGCGGCCGACTCTTATAG for the Id3 promoter; CCCGAGTCCCTTGGCTAACT and AACCCCAGCCCTTCCTACTAAC for the Id3 downstream region (DR); ACCCTGCAGGACATGAACTCA and TGGGCTAGGAGGCTCTGTAATA for Rad50; and GCGCTACAAAGGGAGGAAGTC and CATGCAGGAGACTCCATAGGAG for Snai2. All cis-elements were cloned into the pGL4.24 Luciferase reporter vector .Transactivation experiments were performed in HEK 293 FT cells . We usedNkx2-5+/+, Nkx2-5LacZ/+ heterozygous, and Nkx2-5LacZ/LacZ  using the Stratagene Absolutely Nanoprep Kit . Six hearts for each genotype wild type acZ/LacZ  were pooTbp and Actb.For gene expression analysis in mouse ES cells, RNA extraction, DNase treatment, and RT-PCR were performed as previously described , 2011. qAAGTGGGGGCCTC for the Nppa-NKE; GGGCTGCTTCTGGCAGAATGGAG for NF-1 #I; and GGGCCTCTTCTGGCAGGAGGGAG for NF-1 #II.Electrophoretic mobility shift assays (EMSAs) were performed according to . ProteinAnimal experimentation was performed with approval of the Garvan Institute/St Vincent's Hospital Animal Ethics Committee (Project numbers 10/19 and 10/01).ChIP experiments were performed in HL-1 cells following the Abcam Cross-link Chromatin (X-ChIP) protocol. Proteins and DNA were cross-linked with 0.75% formaldehyde at room temperature for 10 min. Immunoprecipitation of chromatin complexes was done using Santa Cruz antibodies cat no sc365207 for NKX2-5 and sc2025 for IgG control. Target gene levels were quantitatively measured by PCR. Primers were designed to validate DamID peaks and are provided in http://genome.ucsc.edu/cgi-bin/hgTracks?db=mm9&type=bed&hgt.customText=ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE44nnn/GSE44902/suppl/GSE44902_DamID.bed.gz. Microarray data were deposited in NCBI's Gene Expression Omnibus (GSE44902).DamID experiments were performed after modification of published protocols . In brieQuality control was first performed using the ‘affy’ and ‘affyPLM’ R packages to assess the distribution of probe-levels effects via the RLE and PLM methods . RB_NS12Microarrays (.CEL files) and statistical output from CisGenome (.COD files) were deposited in NCBI's Gene Expression Omnibus, accession number GSE44902 . A bed fhttp://www.r-project.org/). Peak overlaps were calculated using the BEDTools package using a default minimum of 1 bp overlap  was assessed using GREAT . SignifiWeeder 1.4.2   .Clover (Clover  was usedA pre-processed series matrix file containing the expression profiles of 91 mouse cell and tissue types was downloaded from the NCBI Gene Expression Omnibus (GSE10246). Replicates were transformed to log2 normalised mean expression (NME), and gene symbols were assigned using the mouse4302.db library in R. Using the background gene assignments from section ‘GO Analysis’, 1000 genes were drawn at random, and the median of each tissue/cell expression profile for these genes was calculated from the NME of all samples. This was repeated 100 times and the average taken to form a static random background expression profile. For any given set of input genes, a tissue/cell type expression profile was formed by the ratio of the randomly generated median expression profiles to the median tissue/cell type expression profile of the input group of genes.BioTapestry  with 5 mg/ml fibronectin and 20 mg/ml gelatine and transfected with indicated paGFP fusion proteins and histone 2B (H2B)-RFP (to identify nuclei) using Lipofectamine 2000 (Invitrogen), as per manufacturer's instructions. Fluorescence correlation spectroscopy (FCS) measurements were performed 48 hr post-transfection, following photoactivation of the paGFP fusion proteins with 405 nm light, as previously described  using thRenilla luciferase protein fragment complementation assay (Rluc-PCA) was performed based on a modified version of the technique described by Renilla luciferase fragment 1  or Renilla luciferase fragment 2 (Rluc8.2 amino acids 111–312), each followed by a 2×(GGGS) linker, upstream of a Gateway cloning cassette allowing insertion of the gene of interest. The resulting fusion is under the control of a truncated form of the CMV promoter (CMVd1), allowing for reduced levels of protein expression (Promega). Transfection efficiency was normalised by a third vector encoding LacZ, downstream of a CMV promoter (CMV-LacZ). The day before transfection, 20,000 HEK 293T cells were plated in clear-bottomed, white-walled 96-well plates in 100 ml DMEM/10% FCS growth media. A master mix for each quadruplicate was prepared, containing 16 µl Optimem (Life Technologies), 40 ng CMV-LacZ, 180 ng R.luc8.1The Renilla luciferase readings were then normalised to levels of ß-galactosidase and the four replicates averaged. As Glutathione transferase dimerises, fusions to Rluc8.1 and Rluc8.2 were used as a positive control and were used separately with other proteins of interest as negative controls.Fusion, 180 ng R.luc8.2-Fusion, and 0.8 µl FuGeneHD (Roche). The mix was incubated for 10 min, and equal amounts of mix added to each well of the quadruplicate. After 24 hr, cells were washed in PBS and 100 µl of PBS added to each well. A 10 mM solution of Benzyl Coelenterazine in PBS  was prepared from a 10 mM stock solution in methanol, and 100 µl was injected into each well by luminometer . Readings were taken for 5 s, 2 s after injection, and again for 5 s 15 min after initial injection of substrate. Following readings, cells were washed with PBS and ß-galactosidase detected using the Galacto-Star System (Life Technologies). CV-1 cells were seeded onto coverslips, and upon reaching ∼70% confluency were transfected with the appropriate plasmids encoding Cerulean- and Venus-TF fusions, following the protocol supplied with Lipofectamine LTX reagent (Life Technologies). Briefly, for each well of a 6-well plate containing 2 ml of DMEM (minus FBS), 500 µl of Optimem was added to each plasmid mixture and 2.5 µl of Plus reagent added. This mixture was left to incubate at room temperature for 5 min, before 6.25 µl Lipofectamine LTX was added to each DNA/Optimem/Plus reagent mixture.Following 30 min incubation at room temperature, 500 µl of the resulting DNA-lipid complexes were added drop wise to the appropriate well. Complexes were left on cells for 4–5 hr, before the serum-free media was replaced with DMEM containing 10% FBS and incubated 16–24 hr. Prior to live imaging, media were changed to phenol-red free DMEM, and the coverslip was mounted in a POCmini-2 cultivation system (Zeiss) in DMEMgfp medium  containing 10% FCS on a LSM 7Duo confocal microscope equipped with a 32-channel spectral detector. 12-bit spectral images were acquired using a 63× Plan-Apochromat 1.40 oil objective at a spectral resolution of 10 nm, first with 405 nm laser excitation and then with a 514 nm laser. Donor, raw FRET, and acceptor images were obtained by linear-unmixing spectral data using newly acquired reference spectra in the Zen 2009 software (Zeiss). Images with average pixel values above 300 and below 2900 were processed through the psFRET algorithm to remove donor spectral bleed-through from the raw FRET signal .S. cerevisiae strain AH109 (Clontech). Double transformants were selected for growth on -Leu/-Trp selection plates, before being selected for interaction on -Ade/-His/-Leu/-Trp selection plates.Protein-coding sequences were cloned into pGADT7 AD or pGBKT7 DBD expression vector backbones , which were modified to contain a Gateway cloning cassette. pGADT7-AD and pGBKT7-DBD fusions were co-transformed into chemical competent  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and four reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets” for consideration at This paper makes an important contribution to the field of cardiogenesis and to understanding NKX2-5 driven mechanisms underlying congenital heart disease. It leads to a shift in the conceptual framework, from the conventional view that mutations in NKX2-5 result in a failure of the transcription factor to correctly activate its downstream targets to the idea that the mutant factor participates in promiscuous interactions that lead to inappropriate activation of genes that are not normally NKX2-5 targets. This is a very data rich study that represents a technical tour de force. The results are complex and the manuscript densely written. In order to make it more accessible, some simplifications and clarifications are required in the text. Further experimentation is not required. However the authors should strengthen the validation of their results by more comparative analyses of data and tone down some of their conclusions. Specific points to be taken into account when modifying the text are as follows:1) The results are based on the Dam-ID approach to discover transcription factor binding sites. It is essential to compare the findings with available published ChIP-Seq data. The authors compared their DamID peaks with NKX2-5 ChIP-Seq from HL-1 cells  mentioniDpnI sites in the enriched DamID peaks and should discuss whether the requirement for Dpn sites introduces bias.2) The enzymatic function of Dam is supposed to be local. The authors should indicate the average distance from NKE to the nearest (predicted) methylated 3) The experiments were performed using overexpression in cell culture and the data should also be compared with results obtained by others on NKX2-5 chromatin interactions using endogenous NKX2-5 ex. .4) The authors understandably restrict their analysis of target sites to promoter regions and do not attempt to analyse more distant regulatory regions. It would be useful to discuss what fraction of DamID selected genomic fragments are estimated to fall within these promoter regions. This could be illustrated by comparison of promoter versus known enhancer sequences for one or two genes in their data set. Mention of comparison with the data of He et al. (in the subsection “DamID identification of NKX2-5 WT targets”) is unclear.5) Conclusions should be toned down, with inclusion of reference to a number of caveats – overexpression of proteins in cultured cell lines, limited in vivo validation, no evidence provided that expression of off target genes is increased in mouse models or patients with NKX2-5 mutations.A specific caveat concerns the presence of wild type NKX2-5 protein in HL-1 cells. The authors should justify their assumption that they are not pulling down mutant protein interacting with endogenous wild type protein which might mean that the binding effects are secondary to the binding of wild type NKX2-5. In the context of the ES cell experiments, it is not clear why complex formation with the endogenous NKX2-5 protein does not permit more nuclear translocation (in the first paragraph of the subsection headed “DamID identification of NKX2-5 WT targets”).Drosophila. Immunofluorescence shown for NKX2-5  require more explanation. The authors should discuss their thoughts about the potential mechanism in repressive functions of NKX2-5.6) What about the activity of NKX2-5 as a repressor, previously demonstrated by the Harvey lab and also for Tinman in r NKX2-5  only co-7) Results are mentioned for NF1-B1/3 (in the fourth paragraph of the subsection headed “DamID identification of NKX2-5 WT targets”) but the in vivo significance of this is not clear and the factors are not integrated into the gene regulatory network . Is it n8) It is not clear that the ELK complex is playing a highly important role. It only came up towards the bottom of the motif enrichment shown in Ciona model should be discussed. In addition to showing the importance of ETS factors in the Ciona cardiac gene regulatory network, this study found an adjacent ATTA motif similar to the over-represented motif identified by Bouveret et al.Concerning the role of ETS factors in cardiogenesis, the highly relevant paper of Woznica et al. (Dev Biol 2012) in the  1) The results are based on the Dam-ID approach to discover transcription factor binding sites. It is essential to compare the findings with available published ChIP-Seq data. The authors compared their DamID peaks with NKX2-5 ChIP-Seq from HL-1 cells  mentioning 77.8% overlap but commenting that genes associated with ChIP-Seq peaks overwhelmed DamID targets, making comparison difficult. However, since a high quality ChIP-Seq dataset provides genome-wide TF occupancy information with higher resolution, the authors should show the percentage of ChIP-Seq peaks that are overlaid for the DamID data. This will serve as a validation.We agree that this is an important issue. In response to the reviewer’s request, we calculated the overlap of NKX2-5 DamID peaks (this study) and ChIP-seq peaks , both daThe overlap between our DamID data and the ChIP-seq data of He et al. data, both generated in HL-1 cells, was relatively low (18% of DamID peaks and 6.8% of ChIP-seq peaks). Importantly, the overlap between the He et al. peaks and those of van den Boogaard peaks, both established using ChIP-seq, was similarly low .We note the following observations that relate to these specific comparisons of NKX2-5 targets:Drosophila reported “a high degree of overlap” (A) Previous studies comparing DamID and ChIP-chip experiments performed in overlap” . Althoughsp68). The He et al. data was generated from stable HL-1 cell lines over-expressing bio-tagged NKX2-5 protein (4-5x based on RNA detection). As highlighted by the editor, overexpression may lead to a larger number of apparent target peaks and we note that in the He et al. study, >20,000 peaks were detected.B) Contrary to the understanding of one of the reviewers (point 3 and response below), in DamID experiments extremely low, undetectable, levels of NKX2-5 were expressed from the uninduced heat shock protein68 promoter (C) We performed DamID using 3-4 biological replicates for each experiment. It is known that ChIP-seq experiments can be noisy ; therefoD) Within our total DamID peaks, de novo motif discovery identified the perfect known NKE (T/CAAGTG) as the top enriched motif, with at least one perfect NKE detected in 79% of peaks and 2 or more perfect NKEs detected in 40% of peaks. Up to 12 such NKEs were detected in NKX2-5 peaks. NKX2-5 ChIP-seq peaks determined by He et al. and van den Boogaard et al. were most enriched in a variant form of that NKE (GAGT/AG) which was determined from a limited selection of either the top 500 peaks or 600 randomly-selected peaks, respectively. This variant has not been documented previously using techniques such as SELEX or protein-binding microarrays. We note from our calculations that the high affinity NKE (AAGTG) was present in 59% and 43%, respectively, of their ChIPseq peaks included in bio-tagged NKX2-5 fusions both before and after enrichment by immunoprecipitation were highly degraded. ChIP-seq data presented in this paper therefore likely represents targets of heterodisperse NKX2-5 processed products.E) Western blots presented in Figure S1D  show thaF) NKX2-5 target genes identified by DamID were most enriched in cardiac GO terms with very low p-values. When restricted to promoter regions, NKX2-5 ChIP-seq peaks were not enriched in any GO terms containing “cardiac”, “muscle” or “heart”  or were We strongly believe that our DamID data should be evaluated on its own merits. There are many findings in our DamID data and in previously published DamID data that suggest that this technique is highly specific. As we have highlighted above, there are potentially simple and well-documented experimental design explanations (e.g. inclusion of replicates) for the differences we have observed. Although we agree that the issues facing the field in this regard are important and warrant further investigation, we prefer to deal with them briefly in our paper and to follow up with a subsequent meta-analysis of comparable experimental designs.For the present paper, we present the overlap of DamID peak with those of He et al. and van den Boogaard et al., as requested, and provide a brief commentary in the second paragraph of the Discussion.2) The enzymatic function of Dam is supposed to be local. The authors should indicate the average distance from NKE to the nearest (predicted) methylated DpnI sites in the enriched DamID peaks and should discuss whether the requirement for Dpn sites introduces bias.DpnI sites in proximity of binding sites, which occur on average every 260bp in the mouse genome. In our experiments on HL-1 cells, DpnI digestion and amplification lead to fragments of ∼200-2000bp . We have also added a new track in 3) The experiments were performed using overexpression in cell culture and the data should also be compared with results obtained by others on NKX2-5 chromatin interactions using endogenous NKX2-5 (ex.).hsp68 (heat shock) promoter to avoid skewing of the network. Such low levels of expression are nonetheless sufficient for specific methylation of chromatin. Please note that overexpression shown in The statement above about overexpression is incorrect. We expressed DamID fusions in HL-1 cells at extremely low levels (undetectable by western blotting) from an uninduced We have clarified this point in the text (in the subsection headed “DamID identification of NKX2-5 WT targets”).In response to the second part of Question 2, we have compared NKX2-5 DamID peaks with peaks determined using ChIP-seq in the adult mouse heart  Figure . See our4) The authors understandably restrict their analysis of target sites to promoter regions and do not attempt to analyse more distant regulatory regions. It would be useful to discuss what fraction of DamID selected genomic fragments are estimated to fall within these promoter regions. This could be illustrated by comparison of promoter versus known enhancer sequences for one or two genes in their data set. Mention of comparison with the data of He et al. (in the subsection “DamID identification of NKX2-5 WT targets”) is unclear.The Affymetrix promoter microarray covers an estimated 7.3% of the mouse genome. This microarray would capture 20% to 30% of NKX2-5 peaks previously determined using ChIP-seq by He et al. and van den Boogaard et al., respectively.This was noted in the third paragraph of the subsection headed “DamID identification of NKX2-5 WT targets”. We have also clarified the text in the second paragraph of the Discussion with respect to the comparison of DamID peaks and ChIPseq peaks of He et al.5) Conclusions should be toned down, with inclusion of reference to a number of caveats – overexpression of proteins in cultured cell lines, limited in vivo validation, no evidence provided that expression of off target genes is increased in mouse models or patients with NKX2-5 mutations.We confirm again for clarity that DamID fusions were expressed in HL-1 cells at extremely low levels (undetectable by western blotting). See our response to Question 3 above.We have been generally more cautious about our claims and have included the caveats as suggested above at the end of the Discussion.A specific caveat concerns the presence of wild type NKX2-5 protein in HL-1 cells. The authors should justify their assumption that they are not pulling down mutant protein interacting with endogenous wild type protein which might mean that the binding effects are secondary to the binding of wild type NKX2-5.must occur indirectly. We provide evidence and a mechanism for how this occurs.Response: DamID experiments do not involve “pulling down” mutant protein. NKX2-5 WT and mutant Dam fusions are expressed individually at very low levels in HL-1 cells, which express endogenous NKX2-5 protein. We make very clear in the text that expressed NKX2-5 WT and mutant fusions bind to chromatin via protein:protein interaction with endogenous NKX2-5 or with other cofactors. Indeed, the binding of NKX2-5 mutants that have a crippled or missing homeodomain to genuine NKX2-5 targets In the context of the ES cell experiments, it is not clear why complex formation with the endogenous NKX2-5 protein does not permit more nuclear translocation (in the first paragraph of the subsection headed “DamID identification of NKX2-5 WT targets”).In the ES cell differentiation experiments, we used the embryoid body (EB) method . Only a fraction of cells differentiate into cardiomyocytes and express endogenous NKX2-5 at day 6 of differentiation. The vector used for exogenous expression utilised a tet-responsive promoter which is likely ubiquitous in tissue competence. Thus, after application of doxycycline, exogenous NKX2-5 is induced in all cells, the majority of which do not express NKX2-5 or cardiac cofactors and therefore do not localize NKX2-5ΔHD to the nucleus. It was therefore not possible to compare results with NKX2-5ΔHD directly to those using nuclear NKX2-5 proteins (WT or Y191C).We have modified the text in the second paragraph of the subsection headed “Functionality of NKX2-5 mutants” to make clear that endogenous NKX2-5 is only activated in a minority of cells at day 6 of differentiation, while exogenous NKX2-5 is activated in most or all cells.6) What about the activity of NKX2-5 as a repressor, previously demonstrated by the Harvey lab and also for Tinman in Drosophila. Immunofluorescence shown for NKX2-5  only co-localises with active histone marks in HL-1 cells. What about its effect on Id3 transcription? The comments on activation/repression in the context of the ES cell experiments  require more explanation. The authors should discuss their thoughts about the potential mechanism in repressive functions of NKX2-5.NKX2-5 mutant embryos, the majority of differentially expressed genes were down-regulated (suggesting that these were positively-regulated targets of NKX2-5) although a limited number of progenitor genes were up-regulated in the transition from progenitor state to differentiated cardiomyocytes, suggesting repression of some targets. In Watanabe et al., we show that repression of the Fgf10 genes is direct. Consistent with an early repressive role for NKX2-5, cardiac reprogramming of fibroblasts is stimulated by expression of MEF2C, GATA4 and TBX5 but inhibited by NKX2-5 expression  NKX2-5 protein with active histone marks. However, our immunofluorescence results do not exclude a repressive role for NKX2-5 and indeed show that some NKX2-5 protein does not localise with active histone marks. Overall, it is clear that there is a repressive role for NKX2-5 on some genes, specifically progenitor genes, including We have modified the text in the subsection headed “Probing the mechanisms of CHD using DamID” to make these issues clearer.7) Results are mentioned for NF1-B1/3 (in the fourth paragraph of the subsection headed “DamID identification of NKX2-5 WT targets”) but the in vivo significance of this is not clear and the factors are not integrated into the gene regulatory network . Is it necessary to include this?The co-enrichment of NKX2-5 and NF1 motifs and the physical interaction between NKX2-5 and NF1-B1/3 suggests that these factors co-operate to regulate NKX2-5 targets. However, we have not yet investigated this relationship further. This will be the subject of further studies. We include these data here because the NF1 motif is the third most overrepresented DNA motif in NKX2-5 peaks, after the known NKX2-5 motif (NKE) and the GATA4 motifs.8) It is not clear that the ELK complex is playing a highly important role. It only came up towards the bottom of the motif enrichment shown in. The authors should explain why they chose to concentrate on the association of this complex with NKX2-5. The authors claim that the GO analysis shows that SRF and ELK1/4 regulate many cellular functions. This may be the case, although target genes appear to be expressed at a low level in the heart, suggesting that the major impact of these factors may be in the hijacking phenomenon? In the absence of complete transcriptome studies and functional promoter analysis statements about the importance of the role of the ELK complex should be qualified .We chose to focus on ETS transcription factors because the known ETS motif was significantly enriched in NKX2-5∆HD C-set peaks compared to random peaks and occurred most frequently (in 42% of NKX2-5∆HD C-set peaks) . We specWe clarify the occurrence and enrichment (relative to random peaks) of the ETS motifs in the second paragraph of the subsection headed “ETS factor binding sites are enriched in NKX2-5∆HD off-targets”.Concerning the role of ETS factors in cardiogenesis, the highly relevant paper of Woznica et al. (Dev Biol 2012) in the Ciona model should be discussed. In addition to showing the importance of ETS factors in the Ciona cardiac gene regulatory network, this study found an adjacent ATTA motif similar to the over-represented motif identified by Bouveret et al.Ciona heart development focusing on the role of Ets/2 are relevant.We agree that studies in We include this in the fifth paragraph of our Discussion. We have also added the relevant references  here and


FIT1/FITM1) gene is a member of the conserved gene family important for triglyceride-rich lipid droplet accumulation. FIT1 gene displays a similar muscle-specific expression across pigs, mice, and humans. Thus pigs can act as a useful model of many human diseases resulting from misexpression of FIT1 gene. Triglyceride content in skeletal muscle plays a key role in pork meat quality and flavors. An insertion/deletion mutation in porcine FIT1 coding region shows a high correlation with a series of fat traits. To gain better knowledge of the potential role of FIT1 gene in human diseases and the correlations with pork meat quality, our attention is given to the region upstream of the porcine FIT1 coding sequence. We cloned ~1 kb of the 5′-flanking region of porcine FIT1 gene to define the role of this sequence in modulating the myogenic expression. A canonical E-box element that activated porcine FIT1 promoter activity during myogenesis was identified. Further analysis demonstrated that promoter activity was induced by overexpression of MyoD1, which bound to this canonical E-box during C2C12 differentiation. This is the first evidence that FIT1 as the direct novel target of MyoD is involved in muscle development.Fat-induced transcript 1 ( FIT1/FITM1), also named fat-inducing transcript 1, is a member of the conserved gene family (including FIT1 and FIT2), important for triglyceride-rich lipid droplet (LD) accumulation [et al. [FIT1 gene coding region was highly associated with a series of fat traits that significantly affect pork meat quality. As shown previously in our lab, porcine FIT1 exhibited a particular and high expression in skeletal muscle tissues with extremely low expression in fat tissue [FIT1 in regulating myogenic expression among these mammals. In addition, very limited reports [FIT1 gene was found differentially expressed in muscles from normal individuals and facioscapulohumeral muscular dystrophy (FSHD) patients. Collectively, study on the transcriptional regulatory mechanism of porcine FIT1 gene is of high agricultural and medical value.Fat-induced transcript 1  with the lowest level at the embryonic stage while the level dropped dramatically  at 6 months old. This indicates that FIT1 expression is regulated with development of skeletal muscles in pigs.Previous reports showed that the transcript of porcine ectively . ResultsFIT1 gene in C2C12 myoblasts was regulated in a similar way, FIT1 mRNA was quantitated using myoblast cultures and myotubes differentiated for 2, 4, and 6 days, respectively. As shown in p < 0.001) whereas the expression level was reduced markedly at day 4 . Following differentiation for another two days, though the decrease continued, the expression level of FIT1 remained higher than that of C2C12 myoblasts . The data above demonstrates that the expression of FIT1 in C2C12 cells is induced with differentiation. In summary, our results illustrated that FIT1 was temporally expressed in the similar pattern in C2C12 cells as that in skeletal muscle.To further determine whether the expression of FIT1 5′-flanking region  was obtained using porcine genomic DNA as the template. Using TESS software, several putative regulatory factor-binding sites related to muscle development were predicted, including CEBPβ, NFκB, MyoD, and MEF2C. A graphical summary of transcription factor binding sites of FIT1 5′-flanking region was shown  and the adjacent genes of FIT1 were identified, including PSME1, PSME2 and PCK2  whereas the region from −1037 to +3 bp had slightly lower luciferase activity. Results from multiple comparisons showed that the luciferase activity variation was not significant between these two fragments (p > 0.05). Moreover, the shorter fragment from −397 to +3 bp displayed higher activity  excluding the potential E-boxes (p < 0.001) as well as fragments (−397/+3) and (−384/−245) (p < 0.01) in C2C12 myotubes, respectively  of  E-boxes C accordi E-boxes C. Constr E-boxes D. By comectively D. This sFIT1 promoter, we introduced site-directed mutagenesis to E-box1 and E-box2 of the fragments −1037 to +3 bp and −397 to +3 bp respectively  in muscle-specific gene promoter or enhancer regions . In combFIT1 gene displayed a typical characterization of high and primary muscle expression patterns among mammals. In addition, it was shown that there was an enhancer site present in the porcine FIT1 promoter containing the conserved E-box motifs that were required for myogenic regulation and the promoter activity was induced by MyoD1 overexpression. It was previously reported that MyoD transcription factor bound to the cis-element that contained a core consensus sequence specified by CANNTG [FIT1 interacted with MyoD1, we carried out ChIP experiments in C2C12 myotubes using the MyoD1 monoclonal antibody. PCR amplification was performed with immunoprecipitated chromatin samples and qRT-PCR was carried out to determine the enrichment of this region relative to the amount of total input. A schematic of the primers used in ChIP-qPCR was shown in FIT1 promoter from −123 to +3 bp containing simply E-box1 site , type IIA (fast oxido-glycolytic), and type IIX and IIB (fast glycolytic) [FIT1 gene may participate in the type I to type II fiber change during postnatal muscle development. Besides the skeletal muscle fiber transitions, other factors may influence meat quality as well, such as body weight, lipid content, PH, and tenderness [FIT1 gene coding region was highly associated with a series of fat traits [FIT1 gene in skeletal muscle is very important in agriculture.and mice . We isoln factor , was invo humans . It was patients , thus oucolytic) . In addicolytic) . In contcolytic) ,14,15. Tnderness ,17. Intrt traits . TherefoFIT1 promoter activity during C2C12 myotube formation. On the other hand, the promoter activity of porcine FIT1 gene was detected exclusively in C2C12 myotubes rather than in porcine PK15 cells, which may suggest the lack of myotube environment required for transcription initiation in this cell type. Alternatively, it may be as a consequence of absence of the mouse-specific cofactor in PK15 cells. Despite the fact that additional experiments are required to confirm this hypothesis in porcine myoblast cells, it is likely that muscle-specific regulatory elements reside in this region and are thus implicated in next step searching for myogenic transcription factors binding to this region. In addition, as the RNA samples used for expression pattern analysis are from large White pigs and the templates to isolate the proximal promoter from Meishan pigs, we sequenced ~1 kb of the promoter sequences from two breeds to confirm whether there is any difference in promoter sequences between large White pig and Meishan pig. Our results showed that only one SNP was detected in this region, which was located at −880 bp upstream the coding region. However, our work demonstrated that this region from −1037 to −875 bp did not contain elements required for transcription activity. Therefore, we can conclude that the difference is not the result of the pig breed difference.The progressive deletions showed that the fragment from −397 to +3 bp maintained almost the full activity exclusively in C2C12 myotubes while lost the activity in C2C12 myoblast cells, C3H10T 1/2 fibroblast cells as well as PK15 cells. This finding demonstrates the crucial role of this promoter sequence in activating porcine FIT1 transcription during C2C12 differentiation. Interestingly, two E-box elements were present in fragment from −397 to +3 bp and by aligning the proximal promoter sequence among pig, mouse, rat, and human were found conserved across these species. E-box is typically bound by MyoD and is critical to the induction of plenty of myogenic genes such as MCK [FIT1 promoter, we report an essential role of the region from −245 to +3 bp in modulating the strong transcriptional induction of the porcine FIT1 gene during C2C12 myotube formation. This finding suggests that there may exist a myogenic enhancer. Furthermore, by mutating the specific nucleotides in the E-box1 and E-box2 sequence, we first report the base alterations in either E-box are the key to inducing the strong FIT1 promoter activity during the myotube formation. We concluded that the proximal consensus E-box1 sequence (CACCTG) plays the dominant role in activating FIT1 transcription in C2C12 myotubes. Interestingly, our results also indicate that the non-canonical E-box2 sequence (CAGCTG) in this region remained functional in part, as the luciferase activity was adequate to be modestly reduced by this site mutation.We next sought the specific sequence that could be accountable for the myogenic activation of h as MCK . By remoFIT1. Results show that over-expression of MyoD1 dramatically enhanced the promoter activity of FIT1 while its inhibition eliminated most of the activity, indicating the essential role of MyoD1 in FIT1 transcription induction. These findings suggest MyoD is a transcription activator of FIT1 gene, consistent with the previous report [FIT1 gene through specific binding to the consensus E-box 1 (CACCTG) support this. It might to some extent demonstrate the previous finding that MyoD-E2A heterodimers form the complex with the canonical E-box sequence more tightly than MyoD homodimers with tetraplex/E-box complexes [FIT1 promoter activity. Moreover, our result that mutation of non-canonical E-box2 simply partly eliminated the promoter activity was in agreement with the finding of Shklover et al. [FIT1 promoter may offer indirect evidence to the report of Winokur et al. [Given the previous finding that E-box can be bound by MyoD homodimers or MyoD-E2A heterodimers , as wells report . Intrigus report  neighbors report , howevers report . Our finomplexes . This mar et al.  that a fr et al.  that manet al. showed that overexpression of PGC-1α activated the expression of FIT1 and protected the muscle from atrophying as well as increasing the triglyceride level and glucose transport in cultured human skeletal muscle cells [FIT1 gene could be activated by myogenic transcription factor MyoD1, it is interesting to explore the relations between PGC-1α and MyoD. It is previously known that PGC-1α contains a key functional domain used for interacting directly with MEF2 [FIT1 gene may require the additional cooperative involvement of MEF2C and MyoD. It is not yet clear whether FIT1, as a target of PGC-1α, is activated by the direct interaction of PGC-1α and MyoD. However, at least it is an alternative to mediate this activation of FIT1 by PGC-1α through cooperating with MyoD, with MEF2C as a partner. The possible model is shown in FIT1 promoter activity, the proposed pathway involved in the activation of FIT1 via the interaction between PGC-1α and MyoD could therefore be reasonable. Although more evidence is still needed to prove whether PGC-1α is a coactivator of MyoD, this hypothesis could be helpful to further study the interactions of transcription factors with FIT1 promoter during muscle development.In addition, Mormeneo le cells . Combineith MEF2 , and MEFith MEF2 . We therFIT1 promoter activity during C2C12 differentiation, and propose this to some extent explains the myogenic expression of FIT1 gene across different mammals. We also demonstrate that porcine FIT1 promoter is transactivated by MyoD1 that binds to the canonical E-box1 site in C2C12 myotubes. This is the first demonstration of the interaction between MyoD1 and FIT1 promoter during myogenesis and MyoD1 is the first transcription factor recognized to function in FIT1 promoter region. We therefore extend the previous work on mechanisms of FIT1 gene in skeletal muscle regulation except for its role in TG accumulation [FIT1 gene in muscular dystrophy through targeting the E-box sequence.Taken together, for the first time we identify an enhancer site encompassing two E-box motifs  that govern FIT1 5′-flanking region was obtained by alignment with its cDNA sequence (Accession number FJ393218) to the genomic DNA sequence. Primers used to amplify the fragment including the promoter and 5′ untranslated region were listed in http://www.cbil.upenn.edu/cgi-bin/tess/tess).Genomic DNA of Chinese Meishan pig was acquired from the skeletal muscle following the instructions of the genomic DNA isolation kit . The porcine FIT1 expression with 1 µL cDNA, 10 µL SYBR Green I Super-mix , and 10 nM specific primers in a total volume of 20 µL. HPRT (hypoxanthine guanine phosphoribosyl transferase) gene was also amplified for internal standard and primers were shown in FIT1 gene was calculated by 2Ct−∆∆ [Total RNAs were extracted from skeletal muscle of Large White pigs at embryonic, 2-, 4-, 6-month of age respectively, using the RNA extraction kit . cDNAs were synthesized according to the instructions of the kit . Real time RT-PCR was performed on the CFX96 Real-time System  to analyze y 2−∆∆Ct .2. For induction of differentiation, DMEM medium with 2% horse serum was added to C2C12 myoblast cells for 72 h. A series of progressive 5′-deletion fragments were amplified by PCR, then ligated into pGL3-basic vectors  and confirmed by sequencing . The MyoD1 eukaryotic expression vector was a gift from Wangjun Wu . Cells were plated in 24-well plates and transiently transfected with plasmids when they reached ~80% confluence according to the protocol of Lipofectamine 2000 (Invitrogen).Porcine PK15 cells, C3H10T 1/2 fibroblast cells, and C2C12 myoblast (All from China Center for Type Culture Collection (CCTCC), Wuhan, China) cultures were fed in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  at 37 °C with 5% COThe transfected cells were lysed and the supernatants were obtained by centrifugation after transfection for 24 h. The transcriptional activity was then measured with the dual-luciferase reporter assay kit (Promega) in triplicates for each treatment in luminometer . A pRL-TK (50 ng) plasmid was co-transfected with deletion constructs to correct for the transfection efficiency.The siRNA against MyoD1 was designed as described previously  and synt3) was employed to elute the beads, and 5 M NaCl was to reverse the cross-linked DNA-protein complex at 65 °C overnight. Post-treatment by proteinase K at 45 °C for 1 h, the expected DNA targets were obtained by PCR using the following protocol: 3 min at 94 °C; 26 cycles of 94 °C for 20 s, 66 °C for 30 s, and 72 °C for 8 s. ChIP-qPCR was performed to quantitate relative enrichments of the regions containing E-box in the FIT1 promoter. Primers used are listed in ChIP assay was conducted using chromatin immunoprecipitation kit . Specifically, C2C12 myoblasts were differentiated for 3 days and fixed with 1% formaldehyde for 10 min at 25 °C followed by glycine neutralization for 5 min. Prior to sonication, cell lysates were rinsed by ice-cold PBS, and then fragmented to the size between 200 and 750 bp. The ChIP Dilution Buffer containing Protease Inhibitor Cocktail II was mixed with the sonicated chromatin above, followed by overnight incubation with anti-MyoD1 antibody  at 4 °C with rotation. Precipitated by Protein G Agarose, the resulting complex was washed in the cold buffers as indicated by the instructions (Millipore). Lastly, Elution Buffer  was used to indicate the results. Comparisons between two groups were made by student’s


We used mouse ENCODE data along with complementary data from other laboratories to study the dynamics of occupancy and the role in gene regulation of the transcription factor TAL1, a critical regulator of hematopoiesis, at multiple stages of hematopoietic differentiation. We combined ChIP-seq and RNA-seq data in six mouse cell types representing a progression from multilineage precursors to differentiated erythroblasts and megakaryocytes. We found that sites of occupancy shift dramatically during commitment to the erythroid lineage, vary further during terminal maturation, and are strongly associated with changes in gene expression. In multilineage progenitors, the likely target genes are enriched for hematopoietic growth and functions associated with the mature cells of specific daughter lineages (such as megakaryocytes). In contrast, target genes in erythroblasts are specifically enriched for red cell functions. Furthermore, shifts in TAL1 occupancy during erythroid differentiation are associated with gene repression (dissociation) and induction (co-occupancy with GATA1). Based on both enrichment for transcription factor binding site motifs and co-occupancy determined by ChIP-seq, recruitment by GATA transcription factors appears to be a stronger determinant of TAL1 binding to chromatin than the canonical E-box binding site motif. Studies of additional proteins lead to the model that TAL1 regulates expression after being directed to a distinct subset of genomic binding sites in each cell type via its association with different complexes containing master regulators such as GATA2, ERG, and RUNX1 in multilineage cells and the lineage-specific master regulator GATA1 in erythroblasts. Dynamic changes in the locations and actions of transcription factors (TFs) are thought to drive much of the differential gene expression that determines cell fate, morphology, and function . Recent Tal1-null murine embryos die of anemia with failed yolk sac hematopoiesis , a TF that is indispensable at multiple stages of hematopoiesis. This basic helix-loop-helix (bHLH) protein is required to establish hematopoietic stem cells during embryogenesis and also to differentiate along the erythroid and multiple myeloid cell lineages, including those leading to megakaryocytes, mast cells, and eosinophils. The requirement for TAL1 in these processes has been demonstrated by multiple in vivo and in vitro genetic experiments. Homozygous opoiesis . Furtherric mice . Conditiic cells . TAL1 isic cells . In contic cells . The pleCAGATGGT, which contains a subset of E-box motifs (CANNTG)  still support primitive erythropoiesis  and found that one-fifth of the wild-type TAL1 binding sites were also occupied in the mutant mice  , binds t(CANNTG) . Other s(CANNTG)  and CAGC(CANNTG) , implyinopoiesis , and moumice die . These rmice die . Anotherant mice . This abcis-regulatory elements in erythroid cells (Some of the TAL1 in the nucleus is in a multiprotein complex with the TFs GATA1 (or GATA2), LMO2, and LDB1; this complex binds to specific id cells . In the id cells , additioid cells . Cobindiid cells . Furtherid cells  and is did cells . In contid cells . These sThe widely differing phenotypes of cells expressing active TAL1 predict that its regulated gene targets differ significantly. Consequently, the DNA segments occupied by this protein should differ between cell types. This prediction can now be evaluated comprehensively and quantitatively in mouse cell models. Recent studies from our laboratory, as part of the Mouse ENCODE Project , and oth− fetal liver cells, which contain erythroid progenitors (Epro) (+ erythroblasts (Ebl) from fetal liver  . TAL1 Chal liver , and culal liver . G1E celor cells . A subliroblasts . Hormonaroblasts . Cell liGata1-null G1E progenitor cell model or in the GATA1-restored and activated ER4 cells. Of these, ∼50% were bound by TAL1 only in the absence of GATA1, ∼30% were bound before and after GATA1 was restored, and the remaining 20% were bound only after GATA1 is restored  across myeloid hematopoiesis was constructed by taking the union of all the peak calls for these six cell types and merging overlapping segments. These were used to generate a data matrix with each TAL1 OS on a row and the value for the TAL1 ChIP-seq read count for a given cell type in each column . The sites of occupancy differ substantially, with the vast majority occurring in only one of the cell types . To ensurestored . A comparestored . The occk-means (k = 16) of the ChIP-seq signal strength at each TAL1 OS in the six cell types revealed the dynamics of TAL1 occupancy during differentiation (−) erythroid progenitor cells (cluster 8) but lost TAL1 in the more differentiated erythroid cells. Conversely, most of the erythroid TAL1 OSs were not bound in HPC-7 cells (clusters 2–6 and 11–14). One group of TAL1 OSs was bound predominantly in megakaryocytes (cluster 7). Three clusters showed binding in both HPC-7 cells and megakaryocytes , perhaps related to the “priming” of these genes in multipotential progenitors for subsequent expression in megakaryocytes .Unsupervised clustering by ntiation . Very fentiation  or as 15ntiation . Most ofntiation . Anotherryocytes . The preEven in a single committed lineage, TAL1 occupied DNA segments dynamically, according to maturational stage. Specifically, most DNA segments were bound by TAL1 predominantly in only one erythroid cell type .The presumptive gene targets of HPC-7-specific TAL1 occupancy were highly enriched for functions associated with hematopoiesis, proliferation, and apoptosis. Examples of hematopoietic genes  are Kit,− progenitors, were highly enriched for functions associated with the differentiated myeloid cells, perhaps reflecting the maintenance of multiple lineage potentials. The HPC-7 cells can be induced to differentiate into several myeloid cell types such as granulocytes and monocytes , for which HPC-7 cells serve as a proxy, and from primary erythroblasts and cultured megakaryocytes (Pf4 (Ppbp (data not shown) were expressed in multilineage precursor cells and were further induced in megakaryocytes, but expression levels were lowered in erythroblasts. Similarly, TAL1 was bound near the TSS of Pf4 in HPC-7 cells and megakaryocytes. A weak signal for TAL1 was seen in erythroid progenitors, and this declined further as erythroid maturation progressed .The case for lineage priming by TAL1 is quite strong for megakaryocytic genes. Several pathways, phenotypes, and Gene Ontology (GO) terms associated with megakaryocytes were enriched for the sets of genes that are presumptive targets of TAL1 OSs observed in both HPC-7 cells and in megakaryocytes . Thus, tryocytes , we fountes , a co-repressor , GATA2, and RUNX1 . The binding of TAL1 to distinctive sites in different cells for a given gene means that, in our analysis, the gene was placed in multiple categories of presumptive targets for TAL1, which contributes to the appearance of functional enrichment terms in unexpected cell types, such as enrichment for megakaryocytic functions and innate immunity in G1E cells  derived from the MEME analysis matched this consensus, and we used it to find instances of this particular E-box in the TAL1 OSs. This motif was enriched in binding sites from all six cell types , showingSurprisingly, the level of enrichment for the TAL1 E-box motif was only about half that observed for the motif corresponding to a GATA factor binding site, which was the most strongly enriched of any motif . This moPf4, Cpox, and Cbfa2t3 , was enriched only in TAL1 OSs from HPC-7 cells and megakaryocytes, and it was depleted in TAL1 OSs from erythroblasts , but we The KLF binding site motif was also enriched in TAL1 OSs in HPC-7 cells, but it tended to localize away from the center of the TAL1-bound DNA segment . The meaGata1 gene complementation system (G1E and ER4 cell lines) to examine directly the effects of GATA1 restoration on TAL1 binding. Most TAL1 OSs in either G1E or ER4 cell lines  overlapped with the DNA segments bound by GATA1 in ER4 cells . Motifs that contribute strongly to occupancy are expected to generate dense concentrations of motif instances close to peak centers and to do so throughout most of a data set . ConsistThe presence of GATA binding site motifs in the centers of TAL1 OSs that were not bound by GATA1 suggests that GATA2 could also direct TAL1 binding in G1E cells. GATA2 bound at specific sites in erythroid progenitor cells can be replaced by GATA1 in maturing erythroblasts; these GATA switch sites have been implicated in repression during maturation . To examcis-regulatory modules  contributes to the specific binding by TAL1 (as a heterodimer with TCF3), we find that the GATA binding site motif is a much stronger determinant of specific occupancy. Previous studies revealed a role for the GATA motif and GATA factors in binding of TAL1 to some sites , and ourThe cobinding of TAL1:TCF3 and GATA factors can occur by the binding of large multiprotein complexes. A pentameric complex composed of the TAL1:TCF3 heterodimer and GATA1 (or GATA2) connected by the bridge proteins LMO2 and LDB1  is preseThe differential occupancy at progressive stages of differentiation and in distinct lineages is further directed by the binding of additional, lineage-specific TFs. We confirmed that TAL1-bound sites in HPC-7 cells were frequently co-occupied by the ETS proteins ERG and FLI1 along with RUNX1 and GATA2 . Our curThe major role played by GATA factors and ETS proteins in directing binding of TAL1:TCF3 to specific sites is reminiscent of observations on the binding of the TFs SMAD and TCF, which are the targets of the BMP and Wnt signaling pathways, respectively. These two TFs co-occupy DNA segments bound by lineage master regulators such as GATA1 and CEBPA, thereby directing SMAD and TCF to target genes . In a siThe redistribution of TAL1 binding profoundly affects gene expression during hematopoietic differentiation, leading to dramatic changes in cell morphology and function. The new genomic sites to which TAL1 binds with each wave of redistribution place TAL1 in positions to regulate new sets of target genes. The cobinding master regulatory TFs not only direct TAL1 binding to the appropriate locations, but they also help to determine the impact of TAL1 on the target genes. In the cases illustrated in Our current study examined more closely particular aspects of the changes in regulation associated with the GATA switch, i.e., the replacement of GATA2 by GATA1 during maturation after erythroid commitment . We confAs is the case for almost all genome-wide mappings of TF occupancy, we found TAL1 occupying a very large number of genomic DNA segments. The 18,595 OSs in the combined data set were largely differentially bound in specific cell types, but each cell type still had thousands of bound segments. Consequently, each predicted target gene was associated with multiple TAL1 OSs, each of which was placed into categories based on binding across cell types or association with other TFs. These several multiplicities added complexity to the functional analysis of the predicted gene targets for the various categories of TAL1 OSs. We adopted two different methods for assigning gene targets to focus on the more robust results. It is important to realize that virtually every gene associated with TAL1 occupancy actually has several TAL1 OSs in its vicinity. Some of this multiplicity of binding likely reflects complex regulatory interactions that ensure correct timing and amount of expression, and some may reflect redundancy to achieve more robust regulation. Future work uncovering consistent trends in these networks of interactions should help illuminate the associated regulatory mechanisms. On the other hand, we cannot rule out the possibility that some of the binding sites are nonfunctional, e.g., they could be DNA segments with favorable motifs located in accessible chromatin, and TAL1 (or other TFs) not actively engaged in regulation could be bound there. Such “opportunistic” binding  may expl+ erythroid precursor cells led to a model of GATA1 binding before TAL1  under accession number GSE30142. The peak sets and signal files of ChIP-seq data in HPC-7 and Ter119− cells were downloaded from publications (TF occupancy was measured by chromatin immunoprecipitation (ChIP) followed by sequencing of the ChIP DNA on the Illumina Genome Analyzer IIx or HiSeq 2000 for a minimum of two biological replicates. ChIP was done for TAL1 in G1E cells, G1E-ER4 + E2 cells, Ter119chnology . The reachnology . Followichnology , qualitychnology  or measuchnology , with thications  or from ications . Certainications ; peaks f2-transformed FPKMs (fragments per kilobase of exon model per million mapped fragments). Genes whose expression level exceeded our threshold for active expression (log2 FPKM > 3) in both cell types and whose differential expression passed a threshold of FDR = 0.05 were declared as DE.Total RNA was extracted from 5 million to 10 million G1E and G1E-ER4 (treated with estradiol for 30 h) cells using Invitrogen’s TRIzol reagent. Subsequent steps—including polyA selection, fragmentation, and cDNA synthesis—were performed as previously described , with twk-means, hierarchical, and model-based clustering on the quantile-normalized values. Heatmaps were generated with segments sorted by the clustering order. For better visualization, the signals that exceeded the 0.5% or 99.5% quantile in each cell type were forced to be the same color as the 0.5% or 99.5% quantile, respectively . These selected motifs were also shown to be overrepresented in a comprehensive TF OS set from previous literature /(average of b/average of d)].To quantitatively measure the motif enrichment in each panel, we first counted the number of motif occurrences within 50 bp on each side of the center of each OS (a) and in the remainder of the OS (b). Then we counted motif occurrences in comparably sized random DNA segments, again within 50 bp on each side of the center (c) and in the remainder of the OS (d). Enrichment (or depletion) was computed as log2 (true percentage/ percentage after shuffling) gives an empirical estimate of the enrichment or depletion for induction or repression of the presumptive gene targets in each TAL1 OS category. Since the permutations were done 1000 times, we could evaluate a distribution of these estimates for each TAL1 OS category.Co-location within an EPU was used to assign potential gene targets for each TAL1 OS. Each gene with a TSS in the same EPU as the TAL1 OS was assigned as a potential target. The expression response for each gene during maturation induced by restoration and activation of GATA1 in ER4 cells was determined from the RNA-seq data. Thus each TAL1 OS was associated with a specific number of genes that are induced, repressed, or not responding, and the percentage of DE genes that were induced or repressed was computed. Data for all the TAL1 OSs in each of the groups defined by overlaps in binding patterns  were comhttp://mouseencode.org/) and have been submitted to the NCBI Gene Expression Omnibus  under accession numbers GSE36029, GSE36028, and GSE51338. Data are also available from the UCSC Genome Browser (tracks ENCODE/PSU on the mouse mm9 assembly) and a customized browser maintained at Penn State (http://main.genome-browser.bx.psu.edu).Data from this study were generated as part of the Mouse ENCODE Project (


CRYAA promoter region for association with ARC and elucidates the mechanisms of this association. Three SNPs nominally associated with ARC were identified in the promoter region of CRYAA: rs3761382  = 1.5), rs13053109 , rs7278468 . The C-G-T haplotype increased the risk for ARC overall , and both alleles and haplotypes show a stronger association with cortical cataract . The C-G-T risk haplotype decreased transcriptional activity through rs7278468, which lies in a consensus binding site for the transcription repressor KLF10. KLF10 binding inhibited CRYAA transcription, and both binding and inhibition were greater with the T rs7278468 allele. Knockdown of KLF10 in HLE cells partially rescued the transcriptional activity of CRYAA with rs7278468 T allele, but did not affect activity with the G allele. Thus, our data suggest that the T allele of rs7278468 in the CRYAA promoter is associated with ARC through increasing binding of KLF-10 and thus decreasing CRYAA transcription.CRYAA plays critical functional roles in lens transparency and opacity, and polymorphisms near CRYAA have been associated with age-related cataract (ARC). This study examines polymorphisms in the  Age-related cataract (ARC), is defined as a lens opacity that occurs after age 45 or 5026CRYAA have been shown to cause congenital cataracts (see Cat-Map)CRYAA gene has also been reported to be associated with ARCCryaa knockout mice display a small lens, lens epithelial cell death, reduced proliferation, cataract, and inhibition of pathological neovascularization1213CRYAA was reported to be decreased in ARC patients’ lenses8CRYAA transcriptional regulation in ARC lens have not been fully elucidated. In addition, a previous paper has suggested association of a synonymous G > A transition c.6G > A with ARCIn addition to being a major structural protein component of the lens, CRYAA  can protect other crystallins against thermally induced inactivation or aggregation and is thus critical for the maintenance of lens transparency over time9CRYAA promoter region was sequenced in Northern Italian cataract patients and compared with ethnically and age-matched unaffected control individuals. We identified three SNPs that show association with ARC in this population. These SNPs are in strong linkage disequilibrium, and the haplotypes of these SNPs are also associated with ARC. To analyze the possible functional roles of these SNPs in CRYAA transcription further, we examined the effects of the individual SNPs, and the results show that rs7278468 has the greatest effect on the transcriptional activity of CRYAA. This SNP is located in the binding motif of the transcriptional repressor KLF10 , and a single-base change from the G to T allele of rs7278468 enhanced the binding affinity of KLF10 and thus inhibited the transcription of CRYAA. Knockdown of KLF10 in HLE cells can partially rescue the transcriptional activity of CRYAA with rs7278468 T allele but has no effect on the G allele. Our results thus suggested that the rs7278468 T allele in the CRYAA promoter decreases its transcriptional activity through enhanced binding of KLF10 which would be expected to increase susceptibility to ARC.The DNA sequence of the promoter region is a major determinant of gene expression. Functional noncoding SNPs in a promoter or enhancer region have been shown to influence transcriptional activity1617CRYAA promoter region that might be associated with age-related cataract (ARC), the CRYAA promoter regions (−1 to −1000) of 215 ARC patients and 106 normal control individuals were sequenced. Three polymorphisms were identified in the CRYAA promoter region of both patients and control individuals: rs3761382, rs13053109 and rs7278468  or protective haplotype (T-C-G). Transcriptional activity of the CRYAA promoter was estimated using a dual-luciferase reporter assay 72 hours after transfection of HLE cells. The CRYAA promoter has transcriptional activity in HLE cells: compared with the vector, the luciferase activity was more than 10 times higher in cells transfected with the vector containing the CRYAA promoter  was most responsible for regulating the transcriptional activity of CRYAA. In order to address this question, we carried out site–directed mutagenesis to alter each individual SNP from the allele present in the protective haplotype to that present in the risk haplotype and analyzed the resulting transcriptional activity of the CRYAA promoter. As can be seen in CRYAA promoter with the T-C-T haplotype decreased about 12%, significant with a P < 0.01. This suggests that the sequence change seen in rs7278468 might directly affect CRYAA expression, while rs13053109 and rs3761382 show association with ARC through linkage disequilibrium with rs3761382.While the previous data implicated the SNP haplotypes in CRYAA transcriptional activity, the mechanism through which this might occur remained unclear. Since rs7278468 lies 361 bp upstream of the transcription start site and does not reside within the actual consensus promoter itself, it seemed possible that it might affect binding of one or more transcription factor(s) and affect the transcriptional activity of CRYAA in that fashion. We analyzed transcription factor binding sites in the CRYAA promoter region using the Genomatix system (https://www.genomatix.de). Based on these results, rs7278468 lies in the binding motifs of several transcription factors including KLF10 (Kruppel-like factor 10)  . KLF10 iCRYAA transcription, even though the precise mechanisms have not been fully elucidated20CRYAA in the lens, we initially examined Klf10 expression in the C57BL/6 mouse lens. Total RNA was isolated from E16.5, P2 and P7 mice, and Klf10 mRNA expression was examined by RT-PCR. Klf10 was seen to be expressed in mouse lens at all time points, perhaps somewhat higher in the embryonic period , knocking down KLF10 increased in luciferase activity of the CRYAA promoter with the rs7278468 T allele by about 35%  was mixed with 4 ul PepMute reagent into 100 ul transfection buffer. After mixing, the mixture was maintained at room temperature for about 15 min, then the transfection mixture was added to the cells, which were then cultured for 5 hours, after which the culture medium was changed with fresh complete culture medium. Forty eight hours later, the knock down efficiency or further tests were carried out. The sequence of the KLF-10 siRNA (5′ to 3′) was UUAUCCUUGAUGAAUCAAUCUGAGG (Invitrogen).All experiments in lens cells utilized the HLE cell line FHL124, which has 95% similarity in transcriptional profile to human lens epitheliaPlasmid transfections were carried out using a PolyJet Transfection Kit (SignaGen) according to the manufacturer’s protocol, in 6-well plates, 1 ug plasmid was used for transfection and 72 hours later the cells were collected for luciferase activity testing, or 48 hours after transfection the cells were used for ChIP analysis.CRYAA proximal promoter was amplified using CRYAA-Promoter-F and CRYAA-Promoter-R as described above. The PCR product was cloned into the PCR 2.1-TOPO (Invitrogen) vector for sequencing. After sequencing, the CRYAA promoter region was cloned into the PGL4.17 vector (Promega) using the restriction enzymes Hind III and EcoR V (NEB). The CRYAA promoter containing each SNP mutation was constructed using site–directed mutagenesis with the primersf: rs3761382-mut F: TACATCGAGGGGACGATGGCCAT, R: ATGGCCATCGTCCCCTCGATGTA; rs13053109-mut F: GGTGAGACTCTGAGGACGATGTGT, R: ACACATCGTCCTCAGAGTCTCACC; rs7278468-mut F: GGGTGTGTGCTCTCCCTCCTCT, R: AGAGGAGGGAGAGCACACACCC and confirmed by sequencing once more.A 1111 bp region of the human HLE cells were cultured in 6-well plates to approximately 80% confluency, 1ug of either PGL4-CRYAA Promoter or PGL4 plasmid and 30ng pGL4.75 [hRluc/CMV] were transfected into cells using a LipoJet Transfection Kit (SignaGen). Seventy two hours after transfection luciferase activities were checked using a dual-luciferase reporter assay system (Promega) according to the supplier’s protocol.GAPDH. The fold change relative to the control was calculated using the ΔΔCt method. All procedures with mice in this study were performed in compliance with the tenets of the National Institutes of Health Guidelines on the Care and Use Animals in Research and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Primers used for real-time PCR were: Human GAPDH F: AGGGCTGCTTTTAACTCTGGT, R: GACAAGCTTCCCGTTCTCAG. Human KLF10 F: CCACACGGGTGAGAAGAAAT, R: CCCGCTGAGACCAAAGTTAG. And mouse Klf10 RT-PCR primer F: GAAGTTTGCCTGTCCCATGT, R: ACTTCCATTTGCCAGTTTGG. Mouse Cryaa RT-PCR primer F: GAGATTCACGGCAAACACAA, R: ACATTGGAAGGCAGACGGTA. Mouse Gapdh F: CGTCCCGTAGACAAAATGGT, R: TCAATGAAGGGGTCGTTGAT. All PCR reactions used for RT-PCR had an efficiency greater than 95%, and single products were confirmed by melting profiles.Mouse lens or HLE cell total RNA was isolated using Trizol reagent (Life Technologies) and was reverse transcribed into cDNA using a reverse transcriptase kit (Invitrogen) with random primers. The cDNA was processed for real-time PCR using SYBR Green (Life technologies). All reactions were run in triplicate and data were normalized to CRYAA promoter 48 hours after transfection using a ChIP-IT express Enzymatic Magnetic Chromatin Immunoprecipitation kit as per the supplier’s suggested protocol (Active Motif). Antibodies used for ChIP included: Anti-Human IgG (Abcam), and Anti-KLF10 (Proteintech). Primers used for ChIP PCR were: CRYAA ChIP F: TGGTGAGTGTAACGGAGGTTC, R: CCAGGGACCATGCTAGTTCT. CRYAA ChIP NC: F2: GAGAGCGATGGACTCTGGTC, R2: GCTGATGGAGGAAAGCAAAG.ChIP analysis was carried in HLE cells transfected with a plasmid containing the SNP genotype frequencies, Chi square p values, odds ratios with 95% confidence intervals, and HWE (Hardy–Weinberg equilibrium) were analyzed using the SVS software package . Since the SNP haplotype extended over only 334 bp recombination was assumed to be 0 for these markers. The Chi-square P value was corrected using the Bonferroni Correction and a corrected P < 0.05 was considered to be statistically significant. The experiments of mRNA, protein and luciferase activity test were repeated three time and results presented as mean ± standard deviation (SD). The statistical significance between experimental and control groups was assessed with a Student’s t-test and a P < 0.05 was considered significant.How to cite this article: Ma, X. et al. Polymorphism rs7278468 is associated with Age-related cataract through decreasing transcriptional activity of the CRYAA promoter. Sci. Rep. 6, 23206; doi: 10.1038/srep23206 (2016).