A preliminary speculative framework is outlined, based on the evidence of what appears to be a cohesive set of interactions at molecular, biophysical and electrostatic level between the various components contributing to chromatin conformation and dynamics. It proposes that within plant nuclei, general and localized ionic homeostasis plays an important role in maintaining chromatin conformation, whilst maintaining complex genomic regulation that involves specific patterns of epigenetic marks. More generally, reversible changes in DNA methylation appear to be consistent with the ability of nuclear chromatin to manage variation in external ionic and temperature environment. Whilst tentative, this framework provides scope to develop experimental approaches to understand in greater detail the internal environment of plant nuclei. It is hoped that this will generate a deeper understanding of the molecular mechanisms underlying genotype × environment interactions that may be beneficial for long-term improvement of crop performance in less predictable climates.Crop plants encounter thermal environments which fluctuate on a diurnal and seasonal basis. Future climate resilient cultivars will need to respond to thermal profiles reflecting more variable conditions, and harness plasticity that involves regulation of epigenetic processes and complex genomic regulatory networks. Compartmentalization within plant cells insulates the genomic central processing unit within the interphase nucleus. This review addresses the properties of the chromatin hardware in which the genome is embedded, focusing on the biophysical and thermodynamic properties of DNA, histones and nucleosomes. It explores the consequences of thermal and ionic variation on the biophysical behavior of epigenetic marks such as DNA cytosine methylation (5mC), and histone variants such as H2A.Z, and how these contribute to maintenance of chromatin integrity in the nucleus, while enabling specific subsets of genes to be regulated. Information is drawn from theoretical molecular  Crop plants are sessile autotrophs, represented by relatively few monocotyledon and dicotyledon angiosperm species which lack the internal thermoregulation of hot blooded animals. Modern breeding programs have contributed to increases in yield, with major advances made during a period of relative climate stability. However, the planet has entered a period of climate variability, in which higher global temperatures also increase amplitude and temporal variance of climate parameters, and temperature accounts for over 30% of global crop yield variability . These eSuch issues require a deeper understanding of the molecular mechanisms underlying plant responses to the environment . Crop peThe detection of temperature by plants is required for appropriate responses on multiplexed timescales covering periods from seconds to years , with suAlthough many crop traits and developmental phase changes are dependent upon thermal and ionic signals, conventional genetic models have not provided a complete understanding of the relevant signal transduction pathways and behavior . More reEnvironment is detected in a number of ways, including via effector proteins, small RNAs  and direPlant growth and development progress within biophysical and thermodynamic constraints imposed by the molecular composition of cells . In eukaThe performance potential of crop plants relies on maximizing harvest index, the ratio of harvestable to total biomass . This isin vitro and molecular modeling studies is placed where possible in the context of in vivo observations for model species and crop plants. The contribution of ions to mediating electrostatic interactions of chromatin and epigenetic marks is placed in the context of ionic variation at whole plant level. Recent advances in understanding how specific epigenetic marks mediate plant thermosensory signaling and other responses to abiotic environment are placed in the context of chromatin dynamics and biophysics.This review describes the macromolecular components of the chromatin hardware in which plant genomes are embedded. It explores their biophysical behavior in the context of the ionic and thermal environment of the nucleus, and how this is affected by the local distribution of DNA and histone epigenetic marks. Evidence from A preliminary speculative framework is outlined, based on the evidence of what appears a cohesive set of interactions at molecular, biophysical and electrostatic level between the various components contributing to chromatin conformation and dynamics Figure . It propArabidopsis (125 Mbp), crop genome sizes vary over 60-fold, from around 265 Mbp  to the larger cereal genomes  and other interacting proteins, as well as having greater sensitivity to DNase I , which provide an accurate experimental indication of where the DNA is exposed and accessible . For exaith DHSs , indicatbidopsis . In ricebidopsis . This isbidopsis . Rice DHbidopsis . For probidopsis , while fbidopsis .cis-regulatory and gene body sequences  occurring in all context in plants compared with solely CG in animals, the presence of hydroxymethylated cytosine (5 hmC) in animals, the distinct plant methyltransferases CMT3, DRM1/2, and MET  and defiequences . In addiequences , and tigequences  comparedequences .Various experimental approaches have shown that DNA cytosine methylation contributes to regulating higher order chromatin structure in plants , primariin vitro by 3 bp  in regulation of gene expression makes a significant contribution to definition of cellular identity and coordination of ontogeny . The speleosomes , and a sThe processes underlying hypomethylation that are associated with reprogramming, particularly in establishing pluripotency and imprinting effects in plant and animal systems, are now starting to be uncovered . DynamicThe RNA-directed DNA methylation (RdDM) epigenetic pathway is the primary mechanism by which plants mediate responses involving small RNAs, and is dependent upon the RNA polymerases Pol IV and Pol V, which are specific to plants, along with various accessory proteins currently being characterized . There ide novo methylation of cytosines in each of the CG, CHG, CHH contexts, and although Pol V-mediated RdDM operates over many genomic regions, there appears to be a preference toward euchromatin, more recently acquired intergenic TEs, and genes containing TEs or other repeat sequences in their promoters and introns  affects a wide range of DNA biophysical properties, with variation in the localized patterns of steric and conformational energy, as well as hydrophobic modifications to polarity . TogetheIn addition to DNA flexibility, stacking energies also contribute to the cooperative melting associated with the DNA helical-coil transition that is observed both in naked form  as well These contributions of 5mC to increased DNA duplex stability and reduced flexibility also appear to affect some aspects of nucleosome positioning, as well as the ability of nucleotide sequences to wrap around the histone complex . A pictuArabidopsis, H3.1 is enriched in silent areas of the genome, including those with the H3K27me3 and H3K9me3 modifications that contribute to transcriptional repression, as well as with DNA methylation tetramer . These dtetramer , and cantetramer .Sequence analysis of human H2A.Z and H2A-containing nucleosomes has also indicated a prominent association with DNA flexibility at nucleosome boundaries , with H2By adding a negative charge, phosphorylation of H1 generally has the effect of weakening the electrostatic interaction between H1 and DNA, thus increasing H1 mobility . Althoug+ to sustain nuclear function  had the effect of increasing meiotic chiasmata frequency at 30°C, although with little effect at 20°C  is the most abundant inorganic cation in plants, representing up to 10% of dry weight  analysis suggesting a significant role for variation in K+ transporters  present in soil solution, allowing about 2% to be transported in the xylem to shoots . Na+ sev+ ratios . Thus, w+ ratios . Na+ toxribution . Rice apribution , and so nd 80 mM .Medicago callus cultures indicated that while short term exposure to NaCl in had no effect on histone variant composition, it did lead to major increases in acetylation of H3.1, H3.3, and H4  hypermethylated in shoots  provides a useful demonstration of the consequences of temperature variation at different phases of development, with each phase transition typically sensitive to a genotype-specific optimal temperature range , the mobile integrator gene of the floral pathway.The autonomous flowering pathway was conventionally regarded as being independent of environmental signals such as photoperiod. However, mutant analysis has demonstrated that genes of this pathway are also directly involved in mediating the effects of ambient temperature , with coFT locus mediates the thermosensory flowering pathway , where candidate genes at such loci appear to account for differences between over-wintering and summer crop types  has been repressed by transposon insertion, with high levels of 5mC in both B. napus and the ancestral B. oleracea. Meanwhile the FT-A7/C6 homologs are specifically silenced in winter type B. napus, but abundantly expressed in spring type cultivars under vernalization-free conditions.Detection of accumulated thermal units or growing day degrees above or below a threshold has been resolved to major QTLs , even inop types . NaturalFT locus . In B. nbidopsis  with probidopsis  indicatiArabidopsis, dissection of the molecular mechanisms underlying vernalization has uncovered the role of epigenetic marks, particularly the polycomb-mediated additive effect of histone modifications, which under cold conditions regulate silencing of the flowering repressor FLOWERING LOCUS C  albeit transitory effect of hypomethylation in mature leaves compared with untreated controls at the same stage of development. These phenomena appeared to be reversible, as after 7 days growth at 22°C DNA methylation in seedlings developed from the vernalized controls was comparable to those of control seedlings. Cold stress has since been found to lead to genome-wide demethylation in maize seedlings (Antirrhinum majus in low-temperature conditions results in hypomethylation of the transposon Tam3 (An early study by eedlings , while gson Tam3 . A relatson Tam3 .Arabidopsis cold-responsive genes COR15A and ATGOLS3 (Histone methylation at H3K27me3 has also been shown to decrease gradually during cold exposure in two  ATGOLS3 . It appeOur current understanding of the direct involvement of ionic and temperature variation on chromatin structure and transcription is fragmentary. However, it is clear that some key biophysical properties of chromatin components and epigenetic marks are affected by electrostatic and thermal interactions, and these more fundamental observations are starting to align with observations at cellular and whole organism level. In order to unravel these relationships, experimental approaches need to distinguish between direct effects and those mediated by signal transduction pathways that sense variation in external ionic or temperature environment and are then apparent at the level of epigenetic modifications.Few studies have systematically evaluated the ensemble of interactions that place the ionic and thermal environment, the biophysical attributes of 5mC, H2A.Z and other epigenetic marks in the context of chromatin dynamics and genomic regulation . Thus, aTaken together, the various lines of evidence outlined here appear to be internally consistent in describing contributions to accessible versus inaccessible chromatin. The framework that emerges is based on what appears to be a cohesive set of interactions at molecular, biophysical and electrostatic level between the various components that affect chromatin conformation and dynamics. This is represented in a simple schematic Figure  that outThe contributions of 5mC to local DNA stability and reduced flexibility appear to be consistent with the association of 5mC with stable, more ordered nucleosomes and localized transcription, with denser methylation leading to tighter chromatin and gene repression. The complementary contributions of 5mC and H2A.Z to less accessible chromatin is consistent with their observed relative mutual exclusivity in chromatin , and H2A+ and K+ in chromatin condensation (+ and K+ gradients are observed within some eukaryote nuclei (Complex electrostatic interactions within the nucleus contribute to the condensation state of chromatin, with the localized net charge state of the interaction between DNA and histone affecting position and stability of nucleosomes. Post-translational histone modifications play a major role, although it is currently unclear how these may be affected by the proposed differential roles played by Naensation . The late nuclei . Given te nuclei  and concWhilst tentative, this framework provides scope to develop experimental approaches to understanding in greater detail the internal environment of plant nuclei. It is hoped that this will generate a deeper understanding of the molecular mechanisms underlying genotype × environment interactions that may be beneficial for long-term improvement of crop performance in less predictable climates.Arabidopsis (Arabidopsis, spontaneous transgenerational epiallelic variation can occur at a rate 103 times higher than the genetic mutation rate (A high proportion of crop traits exhibit quantitative inheritance, many with relatively low penetrance. For example, in rapeseed hundreds of significant environment-specific QTL have been identified for yield and other traits in a “fixed” segregating population of homozygous lines grown in 10 environments, with relatively few coinciding in multiple environments . In manybidopsis . It is wion rate , with hyion rate . This stion rate . For croAt present we have only a partial understanding of how the various epigenetic components confer dynamic functional information content in the context of the ionic and thermal environment of the nucleus. It is becoming clear that most regions of complex eukaryotic genomes play some role in gene regulation . HoweverThe ability of plants to accommodate fluctuations in thermal and ionic environment is an essential fitness attribute and a key determinant for crop performance, and requires a deeper understanding of the interactions at intra-cellular and intra-nuclear level, including those with epigenetic marks and processes. The availability of comprehensive tissue-specific epi-genome, nucleosome and snRNA datasets will contribute to more comprehensive models of interactions between genome organization, chromatin dynamics, and epigenetic signaling systems. These can help provide new tools and approaches for breeding selection and agronomic management of crops able to perform in changeable environments.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin  and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation.Virulent  Agrobacterium tumefaciens and include the oncogenes IaaH, IaaM and Ipt, which, according to the tumor-inducing principle, are essential for crown gall development. The oncogenes are involved in auxin and cytokinin production. This results, when at appropriate hormone ratios, in enhanced cell proliferation. The T-DNA transformation process and the encoded oncogene enzymes have been intensively studied, but knowledge of oncogene expression in plant cells and the regulatory host factors is missing. We set out to fill this gap, providing evidence that expression of the Ipt gene is host-cell controlled, whereas the IaaH and IaaM genes are ubiquitously expressed at low levels in T-DNA transformed tissue. This is achieved by A. tumefaciens, which first hijacks transcription factors of the plant pathogen response pathway to activate Ipt oncogene expression and initiates cell proliferation. With increasing auxin levels during the infection process, a transcription factor of the auxin-signaling pathway is recruited, potentiating Ipt gene expression. Thus, for crown gall development, two host-signaling pathways are combined through the interaction of transcription factors that adjust the ratio of cytokinin to auxin.Crown gall development requires the expression of agrobacterial genes in the plant host. These genes are transferred by the T-DNA of the plant pathogen  Agrobacterium tumefaciens is a pathogenic bacterium that infects several plant species. A region in the tumor inducing (Ti) plasmid, the transfer DNA (T-DNA), is integrated into the plant genome causing crown gall disease  without the addition of phytohormones, but with 100 mg/L ticarcillin disodium/clavulanate potassium . The GFP expressing crown gall cell cultures were generated in the same manner except the A. tumefaciens strain C58 was used. This harbored, in addition to its pTiC58 plasmid, the binary vector pMDC206  at a 1:2 dilution (v/v) twice a week. mutants . Plants  pMDC206  with theA. tumefaciens strain C58 nocc  and the disarmed derivative of C58, strain GV3101 (pMP90) were used for plant inoculations. The strains were cultivated on YEB-agar plates  at 28°C for 2 days. GV3101 was cultivated in the presence of rifampicin (10 mg/L) and gentamicin (25 mg/L). Before plant inoculation, the A. tumefaciens strains were transferred into King’s liquid medium  and grown overnight at 28°C and 140 rpm. King’s medium was removed by pelleting the bacteria three times at 8000 rpm for 1 min and resuspension in Agromix buffer . For recovery, the resuspended cells were cultured at 28°C and 140 rpm for 2 to 3 hours. The optical density (OD600) was measured at 600 nm  and adjusted to OD600 1.0 for leaf infiltrations and OD600 0.5 for inflorescence stem inoculations. A. tumefaciens suspensions were infiltrated into the abaxial side of 5-week-old Arabidopsis (Col-0) leaves by tightly pressing the orifice of a 1 mL syringe onto the leaf surface. For induction of crown gall growth, young inflorescence stems (3 to 10 cm) of A. thaliana plants were inoculated by injecting A. tumefaciens suspensions four times with a 5 mL syringe and a needle attached to it. Crown galls were separated from the inflorescence stems 25 days after inoculation with a scalpel using a dissecting microscope (Leica MZ6) and their weight was immediately determined. Leaves infiltrated or stems inoculated with A. tumefaciens strain GV3101 served as reference.The virulent IGR1a::GFP, IGR1b::GFP and IGR2::GFP), the vector pMDC206 was used, which contains the coding sequence (CDS) of GFP including an intron  adenosine 5’-triphosphate . About 5 ng labeled probe and 150−300 ng 6×His-WRKY40 protein were mixed in DNA-protein binding buffer , 5% (v/v) glycerol]. The binding reaction mixture was incubated on ice for 30 min and separated in a 6% (w/v) native polyacrylamide gel  at 4°C for 3 h at 200 V in 0.5 × TBE buffer . The gel was fixed in 5% acetic acid for 10 min and dried for approximately 1 h , exposed at −70°C to an x-ray film  overnight and then developed.Protein synthesis was induced in the bacterial suspension of the transgenic A. tumefaciens strain C58. The analysis was performed as described in [Crown gall materials used for cytokinin analysis were obtained from Wassilewskija (WS-2) stems inoculated with ribed in .For the BiFC assay, 20 μg of each cYFP and nYFP protein fusion constructs  were transfected into mesophyll protoplasts using the PEG-calcium transfection method . After iFluorescing plant cells and tissues were inspected and documented using an epifluorescence microscope  and the software program (BZ observation application). For the inspection of intact plants, a dissecting microscope  was used and pictures of crown galls were taken using a Leica DFC500 camera .Arabidopsis gene indexes (AGI) of genes mentioned in the text are AT2G33860 (ARF3), AT1G19850 (ARF5), AT3G16857 (ARR1), AT1G10470 (ARR4), AT1G04550 (IAA12), AT5G63790 (NAC102), AT4G31800 (WRKY18), AT1G80840 (WRKY40), AT4G23810 (WRKY53) and AT2G25000 (WRKY60). AGI codes are from The Arabidopsis Information Resource database .The S1 FigNucleotide sequences of indole-3-acetamide hydrolase (IaaH) promoters including the 5’ untranslated regions of T-DNAs from the nopaline-type , octopine-type  and agropine-type (pTiBo542) Ti plasmids. The arrow indicates the position of the transcription start sites (TSS) +1. Negative numbers indicate the nucleotide positions upstream and positive numbers downstream of the TSSs. TATA box and CAAT box sequences are written above the aligned sequences.(PDF)Click here for additional data file.S2 FigNucleotide sequences of tryptophan monooxygenase (IaaM) promoters including the 5’ untranslated regions of T-DNAs from the T-DNAs of the nopaline-type , octopine-type  and agropine-type (pTiBo542) Ti plasmids. An arrow indicates the position of the transcription start sites (TSS) +1. Negative numbers indicate the nucleotide positions upstream and positive numbers downstream of the TSSs. TATA box and CAAT box sequences are written above the aligned sequences.(PDF)Click here for additional data file.S3 FigIpt element.Nucleotide sequences of isopentenyl transferase (Ipt) promoters including the 5’ untranslated regions of T-DNAs from the nopaline-type , octopine-type  and agropine-type (pTiBo542) Ti plasmids. An arrow indicates the position of the transcription start sites (TSS). Negative numbers indicate the nucleotide positions upstream and positive numbers downstream of the TSSs. TATA box and CAAT box sequences are written above the aligned sequences. The framed “TATAAA” sequence indicates the TATA box, only conserved in the octopine type Ti plasmids. The sequence from −150 to −91 shows the conserved region, which is named as (PDF)Click here for additional data file.S4 FigIpt promoter-driven luminescence in the presence of WRKY18, WRKY40 and WRKY60 transcription factor expression plasmids in the protoplast transactivation system. The relative luminescence induced by the Ipt promoter in protoplasts without transfection of any of the transcription factor expression plasmids was set to 1. Bars show mean values (±SD) of three independent experiments.Fold induction of (PDF)Click here for additional data file.S5 FigPositions of TATA boxes , auxin responsive elements , W-boxes  and transcript start site (TSS arrow) in the sense (above the line) and anti-sense strand (below the line) of the intergenic region (IGR1) and IGR2.(PDF)Click here for additional data file.S6 Figp-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; NS: not significant (Student’s t-test).(A) Relative transcript numbers of the Ipt oncogene in 25-day-old crown galls of wild-type plants and wrky18, wrky40 and wrky60 single mutants, (B) in stems of wild-type plants 2 days and 6 days post-inoculation (2 dpi and 6 dpi) of A. tumefaciens strain C58 and (C) of ARF5 and IAA12 in crown gall tumors of the wild-type Col-0 and wrky single mutants. Relative transcript numbers were quantified by qRT-PCR and normalized to 10,000 molecules of ACTIN2/8. Bars show mean values (±SD) of three independent samples. NS: not significant. * (PDF)Click here for additional data file.S7 FigARR1 and ARR4 genes in crown galls 25 days after inoculation with the virulent A. tumefaciens strain C58  and in stems inoculated with the disarmed strain GV3101 (GV3101 Stems). Relative transcript numbers were quantified by qRT-PCR and normalized to 10,000 molecules of ACTIN2/8. Bars show mean values (±SD) of three independent samples. (B) Fold induction of IaaH, IaaM, Ipt promoter-driven luminescence in Arabidopsis mesophyll protoplasts transfected with ARR1 and ARR4 transcription factor expression plasmids and in the presence or absence of trans-zeatin. The relative luminescence in the absence of ARR1, ARR4 expression plasmids and trans-zeatin was set to 1. Bars show mean values (±SD) of three independent experiments.(A) Relative transcript numbers of the (PDF)Click here for additional data file.S1 Tablehttp://planttfdb.cbi.pku.edu.cn/index.php?sp=Ath) for annotation. Genes are listed according to the following criteria: Fold change (FCh) ≥ 2 or ≤ 0.5, p value < 0.01. hpi: hours post inoculation; dpi: days post inoculation.Analyses are based on existing microarray data ,46 using(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Biochemical and genomic studies have shown that transcription factors with the highest reprogramming activity often have the special ability to engage their target sites on nucleosomal DNA, thus behaving as “pioneer factors” to initiate events in closed chromatin. This review by Iwafuchi-Doi and Zaret focuses on the most recent studies of pioneer factors in cell programming and reprogramming, how pioneer factors have special chromatin-binding properties, and facilitators and impediments to chromatin binding. A subset of eukaryotic transcription factors possesses the remarkable ability to reprogram one type of cell into another. The transcription factors that reprogram cell fate are invariably those that are crucial for the initial cell programming in embryonic development. To elicit cell programming or reprogramming, transcription factors must be able to engage genes that are developmentally silenced and inappropriate for expression in the original cell. Developmentally silenced genes are typically embedded in “closed” chromatin that is covered by nucleosomes and not hypersensitive to nuclease probes such as DNase I. Biochemical and genomic studies have shown that transcription factors with the highest reprogramming activity often have the special ability to engage their target sites on nucleosomal DNA, thus behaving as “pioneer factors” to initiate events in closed chromatin. Other reprogramming factors appear dependent on pioneer factors for engaging nucleosomes and closed chromatin. However, certain genomic domains in which nucleosomes are occluded by higher-order chromatin structures, such as in heterochromatin, are resistant to pioneer factor binding. Understanding the means by which pioneer factors can engage closed chromatin and how heterochromatin can prevent such binding promises to advance our ability to reprogram cell fates at will and is the topic of this review. Cell fate control represents the most extreme form of gene regulation. Genes specific to the function of a particular type of cell may be antagonistic to the function of another type of cell, so nature has evolved diverse regulatory mechanisms to ensure stable patterns of gene activation and repression. In prokaryotes, self-sustaining regulatory networks of transcription factors bound to DNA can be sufficient to regulate gene activation and repression . In eukaWhich transcription factors are the first to access a developmentally silent gene and initiate its expression to promote a cell fate change? Genetic studies alone cannot answer this, as a transcription factor may appear necessary or sufficient to elicit a regulatory change but dependent on the prior binding of other factors in the cell. Why does knowing “who’s on first” matter? Because the hierarchical mechanisms by which transcription factors engage target sites in chromatin provide insights into ways to modulate the process and hence cell fate control.In diverse contexts where groups of transcription factors have been tested for their ability to convert cell fate, a subset of factors consistently has the greatest effect in cell conversion. Biochemical and genomic studies indicate that such factors can be considered “pioneers” by virtue of their ability to engage target DNA sites in closed chromatin prior to the apparent engagement, opening, or modification of the site by other factors . NucleosEngage their target sites in closed (nuclease-resistant), silent chromatin prior to gene activity.Increase accessibility of a target site that makes other proteins  accessible to the site.Play a primary role in cell programming and reprogramming and establish the competence for cell fate changes.Observe the chromatin state of target sites for a transcription factor before and after the factor is expressed in a cell. Pioneer factors can target sites that are closed  or shown to be nucleosomal (MNase-Seq) prior to binding and often result in chromatin opening upon binding. Merely correlating open chromatin at sites where a transcription factor is already bound does not predict pioneer factors.Analyze direct binding between a transcription factor and a reconstituted mononucleosome or a nucleosomal array in vitro .Analyze the effect of a transcription factor binding on DNA accessibility at the target sites in vitro  or in vivo (ectopic expression or deletion of a transcription factor).We start by reviewing the latest genome-mapping studies of chromatin states before and after transcription factor engagement in the contexts of development and cell reprogramming, which provide evidence that pioneer factors have special chromatin-binding properties suitable for their pioneering function. We then review the molecular mechanisms that underlie chromatin binding, the limits of such binding, and the importance of overcoming chromatin impediments in order to control cell fate.Drosophila activator) plays a primary role for the onset of zygotic genome activation in Drosophila embryos , and the functionally redundant SoxB1 group of transcription factors  are highly enriched and bound to their target sites prior to zygotic genome activation. They also play primary roles in the onset of zygotic genome activation . In miceReprogramming of terminally differentiated cells was first shown by somatic cell nuclear transfer into enucleated oocytes , indicatOf these factors, Oct3/4, Sox2, and Klf4 act as pioneer transcription factors in that they can access closed chromatin whether they bind together or alone . “ClosedSingle-molecule tracking studies in live cells suggested that during iPSC reprogramming, Sox2 is bound to the target DNA first, followed by binding of Oct3/4 to stabilize the ternary complex . These lmyogenin through cooperation with the Pbx factor, which is constitutively bound to MyoD target sites in fibroblasts prior to MyoD expression (Direct cell conversion (transdifferentiation) by individual transcription factors was shown for the first time when MyoD was found to induce muscle-specific properties when ectopically expressed in fibroblasts . Howeverpression . There apression  and GATApression .The first evidence of efficient transdifferentiation across different germ layers was the generation of functional glutaminergic neurons (inducible neurons [iNs]) from fibroblasts by the three transcription factors Ascl1, Brn2, and Myt1l . Of thesFurthermore, Ascl1 was characterized as an “on-target” pioneer factor in that most of its initially bound sites, when ectopically expressed in fibroblasts, correspond to sites that remain bound when the cells finally differentiate into neurons . This inAlternatively, there could be an inherent difference in the overall chromatin structure of pluripotent cells that takes multiple phases to recapitulate by reprogramming to iPSCs, whereas direct conversion among differentiated cells may not involve as dramatic an overall chromatin change. For example, fluorescence recovery after photobleaching experiments have suggested that pluripotent cell chromatin is more “hyperdynamic” than somatic cell chromatin, allowing for a more rapid exchange of chromatin components than in differentiated cells . Thus, gArf (Another example of transdifferentiation across germ layers is when hepatocyte-like cells are induced from fibroblasts (iHep) by exogenous expression of either forkhead box protein A1 (FoxA1), FoxA2, or FoxA3 with HNF4a  or exogeArf . These fArf . CellNetArf . They alArf . It appeArf , and addArf .CellNet analyses further revealed that most transdifferentiation events fail to extinguish the expression programs of the starting cell type and thus impair the fidelity of cell type conversion . Therefoalbumin (Alb1) gene contains six transcription factor-binding sites  can bind to nucleosomal DNA and recruit other factors to bind . Along tGiven that pioneer factors can bind their targets on nucleosomal DNA, where other factors cannot, we suggest that, at enhancers involved with cell and tissue identity, nucleosomes act as a filter to prevent binding of nonpioneer factors that prefer free DNA and would otherwise perturb a cell identity network. By this logic, if the target sequence became stably nucleosome-free, the nucleosome could no longer serve as such a filter. We need further studies of nucleosome stability at higher eukaryotic enhancers to assess these issues.Genome-wide mapping studies show that all transcription factors, including pioneer factors, bind to only a subset of their putative binding motifs in the genome and show distinct binding patterns in different cell types. As an example of the requisite analysis, see the application of the computational tool MultiGPS to compare ChIP-seq data sets . The obsThe binding of multiple transcription factors that function combinatorially is inherent to eukaryotic gene regulation, particularly for cell-specific control . As descThe most commonly known cooperative binding is based on protein–protein interaction between transcription factors. However, the nucleosome structure of DNA enables cooperative binding without such protein–protein interactions; that is, nucleosome-mediated cooperativity between transcription factors can be achieved by their simultaneous competition with histones for the underlying DNA . Such coDroshophila genomes lack distinctive histone modifications and are considered to be in a “low signal” state  follow or are concomitant with FoxA binding but are not prior to FoxA binding during differentiation induced by retinoic acid in pluripotent ES or P19 embryonal carcinoma cells . HoweverAre there chromatin features that prevent a pioneer factor’s binding to nonfunctional or alternative lineage sites? Comparing between MCF7 breast cancer and LNCaP prostate cancer cell lines, FoxA1 binds differentially to more than half of its target sites . This ceHeterochromatic blocks to pioneer factor binding may provide a means for cells to stably retain their fate. We suggest that a reason why transdifferentiation typically fails to shut off an initial genetic program may relate to the failure of resetting heterochromatic domains for new cell type . UnderstPioneer transcription factors play an important role in cellular memory during mitotic growth. During mitosis, the chromosomes markedly condense, RNA polymerase is excluded from the chromatin, and transcription ceases. Recent chromosome conformation capture studies show that the three-dimensional partitioning of genomic domains in interphase is almost completely lost during mitosis . During Notably, the genes associated with GATA1 and FoxA1 binding in mitosis were among the first to be reactivated during mitotic exit . A carefSox2 is not expressed in the normal epidermis but is up-regulated in mouse and human cancer stem cells in skin squamous cell carcinomas  impede pioneer factor binding?How are heterochromatic domains established or broken down in order to impede or enhance pioneer factor binding?Despite their special nucleosome-binding ability, how is it that pioneer factors recognize only a subset of their target sites in native chromatin? As we discussed here, perhaps the subset of sites that are targeted by pioneer factors involve ensembles of positioned nucleosomes in a particular cell type that favor pioneer binding. More detailed mapping of nucleosome positions in cells, along with how they may or may not be targeted by transcription factors, should provide insight.How do different DNA-binding domain structures enable or inhibit pioneer activity? This question seems most efficiently addressed by detailed biochemical studies, comparing how different pioneer and nonpioneer factors bind (or not) to purified, defined nucleosomes. Are there preferred motif variants for targeting a closed chromatin site in the genome versus open sites, and how does the ability of a DNA-binding domain to adapt to nucleosomal DNA relate to nucleosome-binding capacity? This could be modeled on nucleosome substrates in vitro and the information used to modify a DNA-binding domain in order to enhance its initial targeting to nucleosomal DNA, thereby enhancing cellular reprogramming. This strategy is feasible because the transcription factors that are used to elicit cellular reprogramming appear to be needed only for the initial activation of the appropriate cellular networks. After initiation, the exogenous reprogramming factors can be extinguished, while the new networks self-sustain and complete the fate conversion in the absence of the initial reprogramming factors .How might pioneer protein domains outside of the DNA-binding domain affect nucleosome architecture and stability? Other protein domains could interact with the core histones to stabilize nucleosome binding. Furthermore, it remains to be determined whether pioneer factors “tickle” the nucleosome in a way that destabilizes the core histones or changes nucleosome conformation in a way that modulates how other factors can bind. In addition, it will be important to perform detailed biochemical studies on polynucleosomal templates so that protein domains that affect higher-order folding of the local chromatin can be assessed . ConceivExactly how does highly compacted heterochromatin exclude pioneer factor access? Understanding how tissue-specific heterochromatic domains are made and can be broken down will provide much insight into ways to enhance cellular reprogramming. Assuming that the terminal establishment of a new cell fate after reprogramming will involve the resetting of heterochromatic domains, it seems likely that transient rather than permanent methods for heterochromatin diminution at the onset of cellular reprogramming will be the most useful.It is presently possible to use small molecules to target chromatin modifications that globally affect most cell networks at once. We anticipate that understanding the mechanisms by which pioneer factors target specific sites in the genome, initiate regulatory events, and maintain them through mitosis and other contexts will provide more specific means for modulating cell networks that control cell fate and function in health and disease.


Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5. The majority of myocardial infarctions and ischemic strokes are caused by intravascular clotting that, when unopposed, rapidly can progress into an occluding arterial thrombus. To protect against this, the vascular endothelium of healthy individuals has the ability to acutely release the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) , causingConsequently, there is a clinical need to find a pharmacological tool for prevention of atherothrombotic events that targets the endogenous fibrinolytic system. We recently showed that valproic acid , a clinically available antiepileptic and mood-stabilizing drug, powerfully upregulated t-PA in cultured endothelial cells and enhanced t-PA release in atherosclerotic man as well as in a porcine coronary ischemia model –9. This However, the mechanism by which VPA induces t-PA expression is currently unknown. Based on our previous findings, we hypothesize that VPA mediates its effect by inhibiting HDACs. This causes a more acetylated histone status around the t-PA promoter, resulting in a permissive and transcriptionally active chromatin state. When the t-PA promoter is accessible, the binding or posttranslational modifications of transcription factors can mediate induction via cis-acting elements. If this hypothesis is correct, dysfunctionality of critical cis-element(s) or unavailability of key transcription factors would have great impact on the responsiveness to VPA.The t-PA gene is transcribed primarily from a TATA-independent transcription initiation site (TIS) , 19. ThrThe investigation conformed to the principles of the Declaration of Helsinki. Use of human umbilical cords cells was approved by the Regional Ethics Review Board of Gothenburg (number 449-93). The personnel at the maternity ward of the Sahlgrenska University Hospital were given verbal informed consent from the donors regarding the use of umbilical cord cells for research purposes. Given that the study material was nonidentifiable, written consent was uncalled for and the use of oral consent was approved by the ethical review board.Na valproate was purchased from Sigma-Aldrich  and stock solutions (0.3 M) were prepared in complete endothelial cell culture medium  and stored at −80°C. The compound was diluted in cell culture medium immediately before use.Due to their suitability as model system for transfection of large reporter constructs, HT-1080 fibrosarcoma cells were utilized in reporter studies. HT-1080 cells  were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin. The validity of using HT-1080 was ensured by verifying a consistent and significant induction of t-PA mRNA upon stimulation with 3 mM VPA.Human umbilical vein endothelial cells (HUVECs) were used as a physiologic relevant cell type in both siRNA studies and confirming reporter studies. HUVECs were extracted by collagenase (Sigma-Aldrich) treatment  of freshGGCTATCA, to create t-PA-318-GCIImut-pGL4, the GC box II site CCCGCCC was mutated to CACGCAC, and to create the t-PA-318-GCIIImut-pGL4, the GC box III site CCCACCC was mutated to ACTAGTC. These particular base pair substitutions have previously been reported to interrupt transcription factor binding to these sites [318 bp of the proximal t-PA promoter (−246 to +72) was PCR amplified from the insert of the previously described t-PA9578-CAT vector  and clonse sites , 23. AllOn the day prior to transfection, cells were seeded at a density of 100 000 cells/well in 12-well plates (HT-1080) or 50 000 cells/well in 24-well plates (HUVECs). Cells were transfected using the FuGENE 6 (HT-1080) or FuGENE HD (HUVECs) reagent according to manufacturer's protocol (Promega) using a mixture of 500 ng of t-PA promoter firefly luciferase construct and 10 ng of the SV40 driven pGL4.73 renilla luciferase control vector. On the following day, cells were treated with 3 mM of VPA for 24 h after which cells were harvested in passive lysis buffer (Promega) and analyzed on a GloMax® Luminometer using Dual Luciferase Assay reagents (Promega). Firefly luciferase signals were normalized to renilla luciferase signals and data are presented relative to unstimulated controls.p < 0.001) by VPA in a microarray experiment [http://dx.doi.org/10.1155/2016/7928681. A detailed siRNA protocol has been presented elsewhere [Short interfering RNAs (siRNAs) specific for selected Sp/KLF transcription factors, p300, and PCAF were obtained from Dharmacon . The selection criteria for Sp/KLF factors were either a significant regulation  and genomic DNA was removed using RNase-free DNase I set (Qiagen). Levels of t-PA, Sp1, Sp2, Sp3, Sp4, KLF2, KLF4, KLF5, KLF6, KLF9, KLF10, KLF13, KLF15, p300, and PCAF mRNA were analyzed with real-time PCR, performed on an Applied Biosystems 7500 Fast Real-Time PCR System using cDNA and Taqman reagents obtained from Applied Biosystems. t-PA mRNA was analyzed with a Custom Taqman Expression Assay (Applied Biosystems) using the following sequences: t-PA forward primer: 5′-GGC CTT GTC TCC TTT CTA TTC G-3′, t-PA reverse primer: 5′-AGC GGC TGG ATG GGT ACA G-3′, and t-PA probe 5′-TGA CAT GAG CCT TCA GCC GCT-3′. Confirmation of knock-down of respective Sp/KLF factor and PCAF was performed using Taqman Gene Expression Assays (Applied Biosystems) and of p300 using a QuantiTect Primer Assay from Qiagen . HPRT was used as endogenous internal standard in all experiments except in KLF9 studies where GUSB was used.t-test and a p value of less than 0.05 was considered statistically significant. All comparisons are specified in the figure legends and were performed between samples from the same experiment and timepoint. The primary focus of this study was to examine if the VPA-response could be modulated by an approach involving site-directed mutagenesis of regulatory t-PA promoter elements or by siRNA mediated knockdown of candidate transcription factors interacting with the same. The secondary focus of the study was to evaluate if siRNA knock-down of the transcription factors affects basal t-PA gene expression. For the latter, Bonferroni correction for multiple analysis was performed.Data are presented as mean and standard error of the mean (SEM). The statistical evaluation was performed using paired Student's p < 0.05) with 3 mM VPA . Moreover, VPA-mediated induction of either construct was greatly attenuated by each mutation. Mutation of GC boxes II and III lowered the induction from being 40-fold in unmutated constructs to being 17- (p = 0.06) and 8-fold (p < 0.05), respectively. The basal signal level in empty pGL4.10 vector (no t-PA insert) was 3% of the level in constructs with intact insert.To evaluate the role of the t-PA CRE and GC box II and III elements in basal and VPA-induced t-PA promoter activity, HT-1080 cells were transfected with nonmutated and mutated variants of a 318 bp insert of the t-PA proximal promoter Figures . Nonmuta3 mM VPA . Mutatio3 mM VPA  nor VPA 3 mM VPA . Howeverp < 0.001) (p < 0.01)  . Similar < 0.01) .To identify candidate transcription factors binding the two promoter GC boxes and to evaluate their involvement in the VPA-response, a set of Sp and KLF factors were individually knocked down with siRNA. Each tested siRNA resulted in a target reduction of 75% or below at 96 h, except KLF10 which was only depleted by 68%. Since we failed to deplete KLF10 mRNA to cut-off levels, t-PA mRNA was not analyzed in these samples.p < 0.05), from 9.2- to 5.7-fold (p < 0.01), and from 9.4- to 7.6-fold (p < 0.05), respectively.Confirming our recent data , an apprTo verify the specificity of these positive hits on the VPA-response, each of the three siRNAs was tested for potential off-target effects on two other related mRNAs . All siRp < 0.01) and 3.8- (p < 0.05) fold relative to nontransfected cells. Moreover, a significant fall in t-PA expression was observed with knock-down of KLF2 .Evaluation of basal t-PA mRNA expression after siRNA knock-down of each factor revealed enhanced expression with depletion of Sp4 and KLF9 Figures . RespectThe induction of basal t-PA expression in response to knock-down of either Sp4 or KLF9 was reflected by an elevated magnitude of the t-PA response after VPA stimulation. Relative to nontransfected control cells, Sp4 and KLF9 knocked cells showed VPA-inductions in the order of 13- and 22-fold, respectively.p < 0.01) (p = NS). Treatment of cells with PCAF siRNA had no effect on either basal or VPA-induced t-PA mRNA expression.A role of the histone acetyl transferases (HATs) p300 and PCAF has recently been proposed in t-PA induction by the HDACi MS-275 . Therefo < 0.01) . VPA-medToday there is no clinically available pharmacological tool to enhance or restore an impaired endogenous fibrinolysis. Therefore, the potent induction of t-PA by the clinically used antiepileptic drug valproic acid (VPA) opens up an opportunity for pharmacological modulation of the system. Thus, it is important to clarify the exact mechanism by which VPA induces t-PA.In our recent paper on VPA, we showed that the induction of t-PA is most likely due to the HDAC inhibitory effect of the drug, as we observed significant increases of acetylated histones H3 and H4 associated with the t-PA promoter after VPA-treatment . In suppTranscription of the human t-PA gene is predominantly initiated from a TATA-less transcription initiation site , 19 and  in vitro approaches; no one has actually confirmed a correlation between Sp1 and altered t-PA expression in an intact chromatin context.With respect to the common view of dependence on Sp1 for t-PA expression, it was somewhat surprising that the Sp1 knock-down experiments showed unaltered t-PA expression during both control and VPA-treated conditions. One explanation could be an insufficient depletion of Sp1 protein. To dismiss this doubt, we confirmed knock-down of Sp1 protein using Western blotting and validated mRNA suppression of the positive control eNOS  in Sp1 dIn contrast to Sp1, our data indicate that Sp2 and Sp4 are essential for regulation of t-PA expression. The t-PA gene was induced upon knock-down of Sp4, thus suggesting that this factor acts as a repressor of the t-PA gene under constitutive conditions. Moreover, VPA-induction of t-PA mRNA was attenuated in Sp2 and Sp4 depleted samples. If the VPA-response was independent of Sp4, one would expect a relatively stronger t-PA induction after depletion of this repressive factor. Instead, it is possible that VPA mediates acetylation of the Sp4 protein itself, since HDAC inhibitors are known to acetylate many nonhistone proteins including transcription factors . This maAlthough an intact VPA-response was observed after depletion of each of the other tested KLF factors, KLF2 and above all KLF9 showed involvement in basal t-PA gene expression. Our data indicate that KLF2 acts as an activator of t-PA expression. Interestingly, this feature of KLF2 is in line with extensive documentation on antithrombotic and anti-inflammatory properties of this factor (reviewed in ). RegardDunoyer-Geindre and Kruithof recently evaluated the involvement of the HATs p300/CBP and PCAF in t-PA induction after using the HDACi MS-275. Based on the documented selectivity of the pharmacologic inhibitors used in the study, they proposed a role for PCAF rather than p300/CBP . In ordeWe examined the mechanism of how the histone deacetylase (HDAC) inhibitor valproic acid (VPA) induces the endothelial fibrinolytic enzyme t-PA. In summary, we show that VPA-mediated induction of t-PA is dependent on the proximal GC boxes in the t-PA promoter and possibly involves interaction with (acetylated) transcription factors Sp2, Sp4, and KLF5.The supplementary information includes three tables (A–C) showing: (a) Gene regulation data of transcription factors from the Sp/KLF family in response to VPA. (b) The Sp/KLF transcription factors that were selected for siRNA knock down experiments and the re-agents that were used. (c) The reagents that were used for siRNA knock down experiments of p300 and PCAF.


Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT). In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs) at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism’s ability to respond on a phenotypic level to various stimuli within an inconstant environment.A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and subsequent binding the activated AHR to xenobiotic response elements (XREs) on DNA. The model was based on effects observed in cells lines commonly used as  As science begins to comprehend the workings of transcription through new techniques such as microarrays, chromatin immuno-precipitation (ChIP), and fluorescence visualization techniques, the overall and inescapable conclusion is that the inside of the nucleus is a very busy place. Transcription factors appear to interact with DNA via a number of interdependent mechanisms that occur on multiple time scales, and these complex dynamics may be important for producing appropriate and coordinated gene expression programs; these meThe basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) family of transcription factors occurs ubiquitously in eukaryotes. –9. CellsThe molecular machinery of transcription also includes a number of cofactors., 15 The Despite these mechanisms for resource allocation, competition for transcriptional resources among independent pathways of gene expression has been observed for a number of years.–28 In thThe aryl hydrocarbon receptor (AHR) is one of the most intensively studied transcription factors.  After biRecently, there has been much discussion about nonmonotonic dose-response curves (NMDRCs), especially with regard to endocrine disrupting chemicals (EDCs).–45 A higCYP1A1) gene was developed. The model uses the Gillespie stochastic simulation algorithm (SSA) , 8887, 8This model was developed to explore the role of competition in ligand-activated gene transcription. Hankinson (2005) provides a list of possible candidates for the cofactor including histone acetylases, histone methyl transferases or receptor-interacting protein 140 (RIP140). RecentlyNuclear factor erythroid 2-realted factor 2 (Nrf2) interacts with the AHR in its role in the expression of both CYP1A1 and NQO1.–105 Nrf2Yet another candidate for the identity of the cofactor in the model is nuclear factor-κB (NF-κB). Inhibition of NF-κB reduced the expression of mRNA for both AHR and CYP1A1 in U-937 derived dendritic cells.2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase  co-localizes with AHR in the nucleus. TiPARP enhances the production of CYP1A1 mRNA but also increases the proteolytic destruction of ligand-bound AHR.  This negAt present, data on binding of transcription factors to DNA is obtained by two primary methods—chromatin immuno-precipitation and fluorescence techniques—with the former conducted on populations of cells and the latter on single cells. These two methods give very different kinetic results. Hager (2009) has proposed that several explanations for the oscillations of transcription factor binding observed in ChIP experiments. ChIP expCYP1A1 were not observed in any of the model runs.Oscillations in the binding of either AHR, ARNT, cofactor or RNA polymerase to CYP1A1 was represented by four different promoter states, each with a slightly different rate constant. This simplified model was an attempt to introduce variable transcription rates into the model. For the sake of parsimony, more complex models of transcription were not used.[Current knowledge of the process of transcription suggests it is complex and not yet well understood, 111 In not used., 111 Ratnot used. Distribunot used.–115Whether ARNT is considered a transcription factor or a cofactor is somewhat moot. The basic helix-loop-helix-PAS proteins appear to have been evolutionarily conserved and serve multiple roles. ARNT dimerizes with the bHLH-PAS protein hypoxia inducible factor 1 (HIF-1) that acts to produce cellular responses to hypoxia., 116 ARNNonmonotonic U-shaped dose response curves have been shown to occur for steroid hormone-mediated gene expression. Such shapes in the dose-response occur when there exist receptors unoccupied by endogenous hormones and recruitment of cofactors by receptors bound to xenobiotic ligands is weaker than to hormone-bound receptor. For the ARNT serves as a coactivator for ERα and ERβ, similar to the generic cofactor modeled here. Hence, oTransforming growth factor-β1 (TGF-β1) appears to act as an inhibitor or repressor of AHR-mediated effects in a cell line derived from human prostate cells. The cros2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase  co-localizes with AHR in the nucleus. TiPARP enhances the production of CYP1A1 mRNA but also increases the proteolytic destruction of ligand-bound AHR. This negOther processes that could be included in the model are:Induction of AHR by including AHR synthesis dependent on AHR activation level;Repression of AHR by TIPARP or otherTranscription of other genes in the AHR core battery at different transcription rates.While the model is somewhat complex, the actual inter-relationships between receptors involved in gene induction are even more extensive and inter-related. Extensive crosstalk and overlap exists between the AHR, ERα and Nrf2. A specifStochastic effects resulting from transcription factor binding likely produce heterogeneity in gene expression across cells within organs or tissues in metazoans. This heterogeneity could result from stochasticity in gene activation and inactivation processes and could be an overall compensatory mechanism for the limitations on resources within a single cell. Hence, at the organ tissue levels, the ability to respond to stimuli or environmental changes by gene expression would be maintained., 122–124Within a single cell, a small number of mRNA molecules can potentially be amplified into a sufficient number of protein molecules to produce a phenotypic change in that cell. Cell-to-cell signaling may occur either by extracellular chemical signals such as cytokines or through gap junctions between adjacent cells. One possible function of such signaling could be to regulate gene expression on a tissue-wide basis. The fact that gene expression in metazoans occurs within nuclear structures that maintain locally high concentrations of transcription components is testament to these resource limitations., 20, 125else is going that leads to an observed phenotypic change?If the oscillations observed in ChIP data represent different levels of nuclear receptor binding in cell populations within tissues, this could be another aspect of this mechanism of tissue-wide coordination. What theS1 FileThis file contains: Code A, MATLAB Script for Model Runs. Code B, SBML Code for the Model. Equation A, Calculation of Percent Recruitment from the model results. Equation B, Calculation of Time-Averaged Numbers of ligand-bound AHR. Equation C, Calculation of Time-Averaged Numbers of cofactor bound to AHR-ARNT. Equation D, Equations used for Baseline Projection to determine Transitional Dose Values from Simon et al., (2014). Fig A, Dose-response to the number of ligand-bound AHR molecules as a function of TCDD concentration and fitted Hill functions. Fig B, Histograms of CYP1A1 mRNA copy number induced by TCDD. Table A, Compartments and Species. Table B, Reactions and Rate Constants. Table C, Comparison of classical rate constants, molecular rate constants and propensities. Table D, Hill Model fits of the Percentage of Cells with one or more CYP XRE bound. Table E, Goodness of fit of Poisson distribution for modeled CYP1A1 mRNA copy number.(DOCX)Click here for additional data file.


TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome). TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5′-AGGTGTGAAGGTGTGA-3′. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations  do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) to further elucidate the molecular pathogenesis of VCFS/DGS.Haploinsufficiency or mutation of  Brachyury, the founding member of this family was first identified due to the presence of a short tail phenotype found in heterozygous mice and lack of axial development with early lethality in null mutant embryos Brachyury, nineteen different T-box genes have been identified and are evolutionarily conserved from flies to humans TBX3 lead to Ulnar Mammary Syndrome, and mutations in TBX5 cause Holt-Oram Syndrome, both of these presenting disease specific limb and heart defects T-box genes encode a large family of transcription factors that are required during embryonic development. All T-box family members share an evolutionarily conserved, DNA binding domain comprising approximately 180 amino acids. The Brachyury protein binds as a homodimer to a palindrome of two AGGTGTGA “half-sites” TBX1 gene encodes a T-box transcription factor that maps to the 22q11.2 region, which is hemizygously deleted in individuals with velo-cardio-facial syndrome and DiGeorge syndrome . Since most have a typical 3 million base pair deletion, it is also referred to as 22q11.2 deletion syndrome (22q11DS). Historically, TBX1 was found to bind to the palindromic T-site, but unlike for other transcription factors, it did not significantly activate nor repress transcription of a reporter construct TBX1 have been reported in rare non-deleted patients with related physical defects to that of VCFS/DGS. It is believed that these are loss of function mutations resulting in haploinsufficiency The Tbx1 is required in a dose dependent manner for normal mouse embryogenesis Tbx1 results in neonatal lethality and embryos have a cleft palate, abnormal inner ears, absent thymus, parathyroid glands and persistent truncus arteriosus Tbx1Fgf8, Fgf10, Pitx2, Chd7, Vefr3, Eya1 and Wnt5aAs for other T-box genes, in silico genome wide search for these motifs, we tested 30 and validated 11 putative direct binding sites, including sites in the Fgf8, Bmper and Otog-MyoD genomic loci. These and others are strong candidates to be pursued as direct downstream targets in future by in vivo functional experiments.To expand the repertoire of direct transcriptional downstream target genes, we performed Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to identify the mouse TBX1 consensus site 2.Animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Albert Einstein College of Medicine Animal Institute Committee . All embryo dissections were conducted after euthanizing mice by direct inhalation with COTbx1 was PCR amplified from cDNA with the flanking restriction enzyme sites of EcoRI and XhoI. These sites were used to subclone the DNA fragment into the bacterial expression vector, pGEX4t3 , to generate a GST-TBX1 fusion protein. The vector was transformed into BL21(DE3)LysS competent cells (Stratagene) and grown on LB ampicillin agar plates. Colonies were picked and grown in 2XYT media, 10 mg/ml Amp, 1 M MgCl2 and 20% glucose. Cultures were grown at 29°C and protein expression was induced by the addition of 100 mM IPTG . After induction with IPTG, protein was detected via Coomassie blue staining and the fusion protein was subsequently purified with glutathione Sepharose 4B beads  and detected via western blot. The same protocol was followed when inducing the F148Y, H194Q and G310S mutated TBX1 proteins.The T-box region (amino acids 90–303) of mouse 18CTCGAGGATCCGTGCTCAGTCCCTATCG-3′5′-GTAACGTCGAGACGGAATTCGCGGCCGCN, where a random 18-mer sequence flanked by two 28-mer flanking fixed sequences used for sequencing, was synthesized by Fisher Scientific (HPLC purification) as previously described 5′-CGATAGGGACTGAGCACGGATCCCT-3′. The dsDNA samples were separated on a 4.5% UltraPure Agarose 1000 gel (Invitrogen) and purified by Qiaquick Gel Extraction Kit (Qiagen). PCR was performed to amplify the dsDNA products. After six rounds of selection with recombinant GST-TBX1 protein, the PCR products from round 0 , two, four and six were labeled with [α-32P] dCTP  by Taq DNA polymerase. Oligonucleotides from each round were captured using glutathione Sepharose beads . Each labeled round of oligonucleotides was tested via EMSA to determine the round with the highest enrichment. Amino acids 90-303 were digested from a plasmid containing full length Tbx1 cDNA (Tbx1-pCDNA3.1) and subcloned into the pGEX 4t3 vector for bacterial induction (http://weblogo.berkeley.edu) and two motifs were generated.A 76-mer single-stranded library of oligonucleotides nduction . ProteinProteins that were induced by IPTG and visualized with Coomassie staining were also tested via western blot with specific antibodies. Proteins were denatured with 6x Laemmli loading buffer to 95°C for 5 minutes. Samples were then loaded onto a 10% acrylamide/bisacrylamide gel for 1 hour at 120 volts and subsequently transferred onto a PVDF membrane (BioRad). Antibodies used were: 1°- rabbit polyclonal α mouse Tbx1 1∶500 (Zymed); rabbit polyclonal α GST 1∶500 (Abcam); 2°- ECL donkey anti-rabbit IgG, horseradish peroxidase linked whole antibody 1∶10,000 (Amersham Biosciences).32P] dATP and T4 Polynucleotide Kinase (NEB), and purified with G-50 Sephadex columns (Roche). EMSA reactions were carried out in 12.5 ul total volumes. GST-TBX1 (5–25 µg) was pre-incubated with 1 µg/ul poly dI-dC, 100x unlabeled self-competitor in A100 buffer  for ten minutes at room temperature as previously described 32P] dATP labeled oligonucleotide was added together with 20% Ficoll, and 10X EMSA buffer  and incubated for an additional ten minutes at room temperature. Reactions were then loaded onto a 5% acrylamide gel and the protein-DNA complexes were electrophoresed at 200 volts for 80 minutes.Oligonucleotides were end-labeled with [γ-AGGTGTGAAGGTGTGA (6x TR) and half site partial site – AGGTGTGATCGCGTCAT (6x ½ SPS) in tandem respectively, were generated synthetically from Genscript. The 6x tandem oligonucleotides were digested with XhoI and NheI and subcloned into a pGL3-promoter vector (Promega) Tbx1-pCDNA3.1, we determined that 100 ng of this plasmid led to the highest fold change (Fc-27) when compared to the control experiment where the empty activating vector (Empty pCDNA3.1) was co-transfected with the reporter. Mutated binding sites were generated synthetically from Genscript. The AGGTGTGA sequence was mutated to AATTTTGA (Mutated 6xTR-pGL3p and Mutated 6x½SPS-pGL3p) Two oligonucleotides containing six copies of the tandem repeat-Tbx1-pcDNA3.1, F148Y Tbx1-pCDNA3.1, H194Q Tbx1-pcDNA3.1, G310S Tbx1-pcDNA3.1) and 5 ng of the internal control pRL TK vector (Promega). Cells were grown in 10% FBS in DMEM (Invitrogen), trypsinized and plated onto 48 well plates. The following day, cells were transected with the above constructs together with Lipofectamine LTX (Invitrogen), in MEM (Invitrogen). Four hours later, the media was changed to 10% FBS in DMEM. Luciferase assay readings were carried out 48 hours later using the Dual Luciferase Reporter Assay System (Promega). All data are presented as means ±SD; n≥3. P-values were determined using the Student's t-test.Jeg3 cells (ATCC) were co-transfected with 25 ng of one reporter construct , 25-200 ng of activating construct . Cells were fixed with 4% paraformaldehyde and 4% sucrose 48 hours later, for 15 minutes at room temperature. Cells were permeabilized with 0.3% Triton X-100 and blocked with 10% BSA/PBS for 30 minutes at 37°C. Cells were then incubated with rabbit polyclonal α mouse TBX1 1∶500 (Zymed) for two hours at 37°C and then with Alexa Fluor 488 goat α mouse IgG (Invitrogen) and DAPI stain (1∶500) for one hour at 37°C.Jeg3 cells were grown in 10% FBS in DMEM, trypsinized and plated onto 6-well plates. Cells were transfected 24 hours later with 500 ng of Tbx1-pCDNA3.1 was used as a template for the PCR reaction. Primers were designed with the nucleotide changes: F148Y Sense- 5′-CCCCACGTTCCAAGTGAAGCTTATGGAATGGATCC-3′; H194Q Sense- 5′-CTGGCCGAGTACAGTACCACCCGGACT-3′; G310 Sense- 5′-AACCACCGGCCCAGTGCGCTGCCGCTC-3′. After DpnI digestion, plasmids were transformed into XL10-Cold Ultracompetent cells and plated overnight on LB-ampicillin plates, per instructions supplied with kit. Colonies were picked and grown in liquid culture overnight. Plasmid DNA was isolated using the Qiagen mini-prep kit and subjected to Sanger sequencing.The Quick Change Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) was used to generate mutations in TBX1. Full length http://genome.ucsc.edu). Motifs were then assigned to the nearest RefSeq genes and were then grouped based on conservation and distance to transcriptional start sites (TSS) defined as −100 kb to +1 kb and −1 kb to +100 kb. Gene ontology software tools, GREAT (Genomic Regions Enrichment of Annotations Tool- http://bejerano.stanford.edu/great/public/html/index.php) and DAVID (http://david.abcc.ncifcrf.gov), were used to generate functional groups of genes harboring the motifs, by inputting the chromosomal positions of the putative TBX1 motifs.An in-house bioinformatic program was created to search the mouse genome for the consensus T-box motif, AGGTG(T/C)(G/T)A, identified by the SELEX experiment. These sites were then compared to a list of the most conserved elements produced by the phastCons database based on whole-genome alignment of placental mammals in situ hybridization assay was carried out as previously described . Anti-sense digoxigenin labeled RNA probes to Tbx1 and Fgf8Bmper probe was generated from templates amplified from E10.5 mouse cDNA using the following primers: Fwd . Each RNA template was created by PCR with a T3 RNA polymerase binding site at the 5′ end and a T7 RNA polymerase binding site at the 3′ end. Embryos were dissected at E9.5 and E10.5. Mice were maintained in a 12 hour dark/12 hour light cycle in compliance with the Albert Einstein College of Medicine of Yeshiva University Institutional Animal Care and Use Committee (IACUC).Embryos were fixed in 4% paraformaldehyde overnight at 4°C. The embryos were then serially dehydrated to 100% methanol and stored at −20°C. On day 1 of the protocol, the embryos were rehydrated to 1xPBS/.01%Tween-20 and the in vitro selection method termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. We created a GST-TBX1 fusion protein containing the T-box DNA binding domain in vitro SELEX selection method and after six rounds of selection, we identified a clearly distinguishable protein-DNA complex . To further test the specificity of the newly derived motifs, gradients of both poly(dI-dC) and specific cold-competitor were generated and tested by EMSA  Tbx1-pCDNA3.1 transfection was compared to the simultaneous transfection of empty pCDNA3.1 vector. The mutated 6xTR-pGL3p showed a dramatic decrease in transcriptional activation when compared to the wild-type (WT) reporter construct , 11 (P11) and 13 (P13) as these nucleotides seemed to vary the most when comparing complex intensities on gel shift assays (data not shown) . When thTBX1 have been previously identified in a subset of patients with VCFS/DGS but with no deletion. These mutations, including F148Y, H194Q and G310S, were previously tested in transcription reporter assays in cell culture using the Brachyury palindrome sequence to determine whether they altered transcription Tbx1 would suppress the effect of the mutated allele. Jeg3 cells were co-transfected with 50 ng of Tbx1-pCDNA3.1 and 50 ng of either F148Y, H194Q or G310S Tbx1-pCDNA3.1 as we described earlier. Although there was a slight increase in activation in the presence of the WT protein with the G310S mutant protein, these values were still lower for the F148Y mutations when compared to the WT protein. Again, there was no change observed when the WT TBX1 protein was co-transfected with the H194Q mutant protein. (Human mutations in protein. . Becauseprotein. . We concwww.informatics.jax.org) to ascertain whether any could be a putative TBX1 downstream transcriptional target (data not shown).A series of bioinformatic approaches were undertaken to identify potential direct downstream transcriptional target genes by examining annotated mouse genome sequence data . The first screen was done to detect binding sites in blocks of evolutionarily conserved sequences. A total of 235,414 sequences matching half sites were found. Among them, 12,659 (5.4%) half sites were found to overlap with conserved elements  These weTbx1 or with phenotypes similar to that in -/-Tbx1 mutant embryos were of particular interest to pursue. To further narrow the list of possible genes regulated by Tbx1, we focused on those containing ½SPS or TR sites. We first checked whether the expression of any were altered in previous gene expression arrays in experiments where +/+Tbx1 versus -/-Tbx1 embryonic tissues were compared Candidate TBX1 binding sites near genes with known expression patterns in mouse embryos similar to that of Fgf8, Bmper and the Otog-MyoD locus.Electrophoretic mobility shift assays were performed on motifs near candidate downstream target gene loci to determine if GST-TBX1 could bind to them. GST-TBX1 formed protein-DNA complexes with 19 of the 27 motifs with three distinct intensities of protein-DNA complexes referred to as high (similar to TBX1-TR), medium and low . The posFgf8 gene encodes a secreted fibroblast growth factor (FGF) that is required for craniofacial and heart development Tbx1, a genetic interaction between Fgf8 and Tbx1 has been found Fgf8 locus has a ½ SPS located 4 kb downstream of the transcriptional stop site  (data not shown). As expected from the EMSA, the transcription reporter assay in Jeg3 cells showed activation at a level similar to the ½ SPS consensus (5 fold change)  . Whole mendoderm  as previBmper gene encodes a secreted protein that inhibits bone morphogenetic protein (BMP) function. The Bmper locus has a TR binding site in the intron lying between exons 13 and 14, sharing evolutionary conservation only with rat and opossum . The Otog gene encodes an N-glycosylated protein present in the acellular membranes of the sensory epithelia patches of the inner ear, important for hearing Otog and MyoD are neighboring genes, however the TR motif within the regions tested in EMSA and luciferase assays, is 70 kb from the MyoD TSS  to identify the preferential binding site of mammalian TBX1. We found two classes of binding sites; a perfect direct repeat (TR), consisting of two classic Brachyury half-sites and a second, imperfect direct repeat (½SPS), in which the 5′ site is similar to the Brachyury half site, but the 3′ half is different. One important possibility is that there are differences in the amino acid constitution of TBX1 that confers a different binding preference as some of the other T-box proteins. For example, TBX1 appears to bind strongly to the TR, but weakly to the Brachyury palindrome and not at all to the half site motif. Although the DNA binding domain is highly conserved amongst different T-box proteins, some differences may contribute to specificity of binding and the sequences might affect the orientation in which various T-box proteins bind to DNA. Interestingly, a few amino acids that are important for Brachyury dimers to bind to the palindrome are not conserved in TBX1 The T-box family of transcription factors is important in vertebrate development and human disease. The preferential binding site of a number of T-box proteins, including Brachyury, TBX2, TBX5, TBX6, TBX15 and TBX18 were previously identified by taking either in vitro, it had the least effect on transcription (2.3 fold decrease). One possible reason is that we used only the DNA binding domain of TBX1 for EMSAs but used the full-length protein for luciferase assays. There are multiple examples where there is a lack of direct correlation between relative binding affinity and transcriptional activation of a cis-acting motif We found some inconsistencies between apparent binding affinities to DNA in EMSAs versus transcriptional activity in luciferase assays in cell culture. For example, although the P13 mutation in the ½SPS led to the greatest loss of binding TBX1 mutations have an impact on transcriptional regulation of reporter constructs, but these used palindromic sequences as the binding motif, but these had conflicting results TBX1. Previous studies did not perform any in vitro binding assays to DNA. We found that the F148Y, H194Q and G310S mutant proteins could strongly bind to the two consensus sequences we identified (TR and ½SPS sites). In contrast to what has been previously reported, where the F148Y and G310S mutations showed no effect on transcription Previous studies have tested whether Fgf8, Bmper and Otog-MyoD.In addition to understanding the effects of mutations on gene function, one major goal is to identify direct transcriptional target genes required for embryonic development. Using various bioinformatic selection methods, we identified and validated DNA binding to 19 different motifs present in gene loci of interest, and confirmed transcriptional activation for 11 of them (data not shown), including Tbx1 is expressed in mouse embryos in the endoderm and mesoderm of the pharyngeal arches as well as the ectoderm of the distal pharyngeal apparatus, with some localized expression in the somites Tbx1 and Fgf8 are coexpressed in the pharyngeal endoderm and a subset of the pharyngeal mesoderm and they genetically interact in mouse embryos, implicating them in the same genetic pathway Fgf8 gene locus and drives expression of a reporter in forebrain, somites and tail bud but not the pharyngeal apparatus Tbx1 and Fgf8 are also expressed in the somites and presomitic mesoderm (PSM). However, inactivation of Tbx1 does not result in loss of Fgf8 expression in these tissues nor does it affect development of these structures. The most parsimonious explanation is that Tbx1 acts redundantly with other T-box genes, upstream of Fgf8. There are several T-box genes expressed in the somites and tail bud The pharyngeal apparatus is an embryonic structure that becomes remodeled to form the face, neck and cardiac outflow tract Bmper and Otog-MyoD gene loci, however, they are not in regions of high evolutionary conservation. There have been a number of reports examining regulatory regions that are not in conserved elements. For example, many of the p300 sites found by ChIP-Seq (chromatin immunoprecipitation followed by next-generation sequencing) were not in evolutionarily conserved blocks, however, they did drive expression of reporters in vivopax9 and otx1b in zebrafish Bmper and the Otog-MyoD loci.Although many known regulatory regions show evolutionary conservation, not all follow this pattern. We found putative TBX1 protein binding sites in the Bmper gene, encoding a BMP antagonist Tbx1-/- embryos. We found that Bmper mRNA expression is lost in the first pharyngeal arch in Tbx1-/- embryos.The core mesoderm of the pharyngeal arches form the muscles of the craniofacial region and neck, required for chewing and swallowing Bmper, another gene, MyoD is also expressed in the core mesoderm of the pharyngeal arches and it is also reduced in expression in Tbx1-/- embryos. Inactivation of MyoD and other basic helix-loop-helix regulatory transcription factors, results in loss of craniofacial muscle formation Tbx1 results in loss of development of craniofacial muscles Otog gene body, but near the 3′ end of the gene. Otogelin (Otog) encodes a glycoprotein present in the acellular gelatinouses structures covering the sensory epithelia of the inner ear Otog is expressed in the inner ear as early as E10. Mutations in Otog lead to autosomal-recessive sensorineural nonsyndromic hearing loss, showcasing the importance of this protein in inner ear development and hearing Otog to determine if it is co-localized withTbx1. On the other hand, it is possible that this site is important for regulation of MyoD expression in vivo, that these sequences are important for regulation of Otog, or neither. Nearby enhancers within neighboring genes have been found to regulate genes at a distance. This is the case for Dlx5, with two enhancers being exons of the neighboring gene Dync1/1in vivo reporter assays in transient transgenic mouse models can validate this hypothesis. In conclusion, we used an in vitro SELEX selection process to identify two novel TBX1 consensus sequences, the TBX1-TR and the TBX1-½SPS. We found that TBX1 can activate reporter constructs harboring the newly identified binding sites in tissue culture. In addition, we have also demonstrated that in the presence of the F148Y human mutation in TBX1, activation of reporter constructs was strongly diminished. This was only possible having a highly active consensus site for transcription reporter assays in cell culture. Finally, as a prelude to future ChIP-seq and other biochemical studies, we provide an in silico list of possible direct downstream target genes, some of which may be biologically relevant to TBX1 function, such as Fgf8, Bmper and Otog-MyoD.Similar to Figure S1Cloning of GST-Tbx1 (T-box) Construct. A: The T-box region (amino acids 90–303) of mouse Tbx1 was PCR amplified from cDNA with flanking EcoRI and XhoI restriction enzyme sties. These sites were used to subclone the fragment into the pGEX4t3 vector  to generate a GST-TBX1 fusion protein. B: GST-TBX1 was detected via western blot with an α GST antibody, with an approximate molecular weight of 52 kD. C: EMSA with recombinant GST-TBX1 (90–303) binds to published palindromic Brachyury palindrome motif CTAGATTTCACACCTAGGTGTGAAATCTAG.(TIF)Click here for additional data file.Figure S2Testing the binding specificity of TBX1 binding motifs. Gradients of both poly dI-dC (left) and specific cold-competitor (right). As the concentration of the poly dI-dC increased (0.5–2 µg), the binding decreased in intensity but the creation of protein-DNA complexes still occurred at the same position. Increasing amounts of specific cold-competitor (25x-200x) was used to demonstrate the specificity of binding.(TIF)Click here for additional data file.Table S1Sites and nearest genes with T half site. The sites listed below are half-sites that lie in evolutionarily conserved regions across the mouse genome (mm9).(XLS)Click here for additional data file.Table S2Sites and nearest genes with tandem repeat site. The sites listed below are TR sites across the mouse genome (mm9), irrespective of evolutionary conservation.(XLSX)Click here for additional data file.Table S3TR and ½ SPS endogenous sites in the mouse genome. The sites listed above are the endogenous sites that were bound by GST-TBX1 in EMSA experiments. Listed as well is the distance to the TSS and comparative gel shift band intensity. *Gel shift bands were compared to the binding of TBX1 to the TR TBX1 site (High).(TIF)Click here for additional data file.