Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of  Isl1 gene orchestrates the process to generate cholinergic neurons in the spinal cord and forebrain. Isl1 forms two different types of multi-protein complexes in the spinal cord and forebrain. Both complexes bind the same genomic regions in a group of genes critical for cholinergic signal transmission, and promote their simultaneous expression. These cholinergic genes include enzymes that synthesize acetylcholine and proteins required to package acetylcholine into vesicles. The Isl1-containing multi-protein complexes were able to trigger the generation of cholinergic neurons in embryonic stem cells and neural stem cells. Our study reveals crucial mechanisms to coordinate the expression of genes in the same biological pathway in different cell types. Furthermore, it suggests a new strategy to produce cholinergic neurons from stem cells.Neurons utilize various chemicals to transmit signals to a target cell. Distinct types of neurons in the spinal cord and forebrain, collectively termed cholinergic neurons, utilize the same chemical, acetylcholine, for signal transmission. These neurons play critical roles in controlling locomotion and cognition. In this study, we have found that the  The choice of neurotransmitter is one of the most fundamental aspects of neuronal fate decision. Cholinergic neurons are located in diverse regions of the CNS, which do not share the developmental origin, and regulate complex behaviors. In the spinal cord, cholinergic motor neurons (MNs) control locomotion, whereas in the forebrain, cholinergic neurons regulate cognitive processes vesicular acetylcholine transporter  gene is encoded within an intron of the choline acetyltransferase (ChAT) gene in all metazoans examined thus far, including C.elegans, Drosophila and mammals ChAT and VAChT genes are co-regulated by a single set of transcription factors. Furthermore, in a subset of cholinergic MNs of C. elegans, an Ebf-type transcription factor UNC-3 regulates a battery of cholinergic genes via a shared UNC-3-response motif The cholinergic neurotransmission system requires the function of several key factors that are highly expressed in all cholinergic neurons, termed cholinergic pathway genes  [4], [5]C.elegans? Second, could there be a transcription factor(s) that determines cholinergic fate across different types of cholinergic cells in the vertebrate CNS? While very limited information is available for the first question, it is interesting to note, for the latter question, that a LIM homeodomain (LIM-HD) transcription factor Isl1 is expressed in several cholinergic neurons in the spinal cord, hindbrain, forebrain and retina, such as spinal MNs, hindbrain MNs, some FCNs, and starburst amacrine cells Isl1 gene results in a loss of MNs in the spinal cord and hindbrain Isl1 gene using a Six3-Cre transgene led to a reduction of restricted FCNs in the brain and cholinergic amacrine cells in the retina Two critical questions remain to be answered. First, is a battery of cholinergic pathway genes coordinately regulated by a common transcription factor in vertebrate CNS, similar to UNC-3-directed control of cholinergic genes in In the developing spinal cord, Isl1 directs motor neuron fate specification by cooperating with another LIM-HD factor Lhx3 In this study, we found that the Isl1-Lhx3-hexamer directly activates the expression of a suite of cholinergic genes by binding to cholinergic gene enhancers that were discovered via ChIP-seq experiments. We also found that Isl1 is co-expressed with Lhx8 and NLI in the embryonic ventral forebrain and forms a hexamer complex with Lhx8 and NLI, named Isl1-Lhx8-hexamer. Interestingly, like the Isl1-Lhx3-hexamer in the spinal cord, the Isl1-Lhx8-hexamer directly controls cholinergic pathway gene expression via the same cholinergic gene enhancer in the forebrain. These findings imply that, despite distinct developmental histories and locations within the nervous system, MNs and some FCNs employ a common molecular mechanism that determines their cholinergic neuronal identity.ChAT has a strong peak within an intronic region that lies downstream of the VAChT gene, which is itself encoded within the intron of the ChAT gene. The Acly gene has two strong peaks in intronic regions and one upstream peak, while the CHT gene has a peak ∼100 kb downstream of its coding region. All the peaks have at least one hexamer response element (HxRE)   [20].To test whether the Isl1-Lhx3-hexamer is recruited to Isl1-Lhx3-bound peak regions of the cholinergic genes in vivo, we purified genomic DNA bound by the Isl1-Lhx3-hexamer from E12.5 embryonic spinal cords using ChIP assays with α-NLI, α-Isl1, and α-Lhx3 antibodies. All three components of the Isl-Lhx3-hexamer bound to the peaks in the cholinergic genes, while they did not bind to the genomic regions without the peaks , indicatTo test whether the Isl1-Lhx3-hexamer is capable of inducing the expression of multiple cholinergic genes in embryonic spinal cord, we misexpressed Isl1 and/or Lhx3 in the chick neural tube and monitored the expression of cholinergic genes. Co-electroporation of Isl1 and Lhx3 triggered the ectopic expression of a panel of cholinergic genes, including ChAT, VAChT, Acly and CHT, in the dorsal neural tube, while electroporation of Isl1 or Lhx3 alone did not . Together, these data demonstrate that the endogenous Isl1-Lhx3-hexamer binds to the cholinergic enhancers via the HxRE motifs and triggers the transcription of their target cholinergic genes as MNs are specified in embryonic spinal cords, establishing the Isl1-Lhx3-hexamer as a critical determinant of cholinergic neuronal identity in MNs.To identify in vivo cell types in which the cholinergic enhancers activate gene expression in the developing spinal cord, we electroporated the neural tube of chick embryos with the GFP reporters linked to each cholinergic enhancer in ovo at a time when MNs are being specified. Interestingly, the nal cord , S3A. Innal cord , suggestn in MNs , S3B, den in MNs . Thus, tRE motif . These dcly-HxRE . Similar+ cells were found in the MZ of the lateral ganglionic eminence (LGE), which are likely the cells tangentially migrating from the MGE   , 6. Alth the CPu , correla the CPu . Despiteanalyses . Given tf/f;nestin-CreIsl1 mice die soon after E12.5, precluding us from observing cholinergic neuronal differentiation in the forebrain. To understand the role of Isl1 in FCN specification in the forebrain, we generated f/f;Nkx2.1-CreIsl1 mice, in which Isl1 gene is deleted in cells derived from the Nkx2.1-expressing MGE f/f;Nkx2.1-CreIsl1 mice, the number of Isl1+ cells was greatly reduced in the BMC, but not in the CPu where most of Isl1+ cells were derived from LGE and thus did not express Nkx2.1-Cre  of NLI is fused to LIM domains-deleted Isl1 (DD-Isl1ΔLIM lacks the LIM-interaction domain (LID) of NLI, it cannot bind to LIM-HD factors via typical interaction interfaces between NLI-LID and LIM-domains of LIM-HD factors, which lead to tetramer formation. Co-immunoprecipitation assays revealed that NLIDD-Isl1ΔLIM associated with Lhx8 in cells despite the lack of NLI-LID in this fusion  assay with Isl1, Lhx8, or an Isl1-Lhx8 fusion, in which full-length Isl1 and Lhx8 proteins were linked by a flexible short linker . Isl1-LhConsidering the shared function of Isl-Lhx3-hexamer and Isl1-Lhx8-hexamer in inducing cholinergic genes and the similar features of their binding motifs, it is possible that they bind to the same enhancer regions of cholinergic pathway genes. To test whether, in the developing forebrain, the endogenous FCN-hexamer is recruited to the same cholinergic enhancers identified as targets of the Isl1-Lhx3-hexamer in our ChIP-seq analysis, we performed ChIP assays for the Isl1-Lhx8-hexamer using the dissected E15.5 embryonic forebrains and found that Isl1, Lhx8, and NLI, all components of the Isl1-Lhx8-hexamer, bound to the cholinergic enhancers . These rAcly-HxRE:LUC and ChAT-HxRE:LUC reporter constructs. The co-transfection of Isl1 and Lhx8 activated each cholinergic enhancer, while expression of Isl1 or Lhx8 alone had minimal effect . The co-electroporation of Isl1 and Lhx8 along with the Acly-HxRE:GFP reporter drastically increased the number of GFP+ cells and the levels of GFP expression in the ventral forebrain, whereas expression of Lhx8 or Isl1 alone did not exhibit potent effects on Acly-HxRE:GFP. Likewise, the transfection of cortical progenitors using in utero electroporation revealed that the expression of Isl1-Lhx8, but not Isl1 or Lhx8 alone, strongly activates Acly-HxRE in the developing cortex , namely Isl1-Lhx8-ESCs . Isl1-Lhdetected . Dox tres in EBs , suggests in EBs . In the Establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification, but very little is known about how a battery of genes involved in a specific chemical-driven neurotransmission is coordinately regulated during vertebrate development. In this study, we report that Isl1 directly regulates a battery of genes establishing a cholinergic neurotransmitter characteristic in two developmentally unrelated cell types in vertebrate CNS . FurtherC. elegans, a set of dopamine pathway genes, which encode dopamine synthesizing enzymes and dopamine transporters, are co-regulated through a specific cis-regulatory element that is activated by the ETS transcription factor AST-1 C.elegansChAT, VAChT, CHT and Acly, our ChIP-seq also uncovered the hexamer-binding peaks in other cholinergic pathway genes, such as acetylcholine esterase and a cluster of nicotinic acetylcholine receptors Chrna5/a3/b4 (data not shown). An important area for future study is whether similar Isl1-containing complexes exist to control cholinergic fate decision in other areas of the CNS, such as retina and hindbrain. Isl1 is co-expressed with Phox2a, a paired-like homeodomain transcription factor, in the cranial MNs of the hindbrain ChAT gene, which we identified in this study, when co-expressed with Phox2a In C. elegans MNs, a single transcription factor UNC-3 serves as a key regulator of the cholinergic pathway genes, whereas, in vertebrate CNS, Isl1-containing cell type-specific transcription complexes control the cholinergic gene battery. The combinatorial utilization of transcription factors is beneficial to generate massively divergent cell types in development. It is possible that regulation of the cholinergic genes by a single transcription factor in ancestral species has been diversified to a transcription complex in vertebrates, as the CNS circuitry becomes more complex. Another possibility is that the hexamer complexes and Ebf transcription factors, vertebrate UNC-3 orthologs, function cooperatively and/or redundantly to control cholinergic genes in the vertebrate CNS. Several findings support a possibility that the Isl1-containing hexamers act together with Ebf. Ebf proteins are expressed in differentiating MNs and ventral forebrain during embryonic development Lhx8-deficient mice While the concept that a defined transcription factor controls the cholinergic gene battery is shared between nematodes and vertebrates, a clear difference is also noteworthy. In Hb9, which recruits the Isl1-Lhx3-hexamer in vivo chromatin context may play a role in cell type-specific gene expression. For instance, MN genes, such as Hb9 and Isl2, may possess transcription-permissive chromatin environment in the spinal cord and transcriptionally inactive chromatin in the forebrain, thus allowing the gene activation by the Isl1-Lhx3-hexamer only in the developing spinal cord, but not in the forebrain. In this regard, it is noteworthy that the activation of Acly-HxRE:GFP reporter gene, which is free from chromain-mediated regulation, is cell context-independent. Both Isl1-Lhx3 and Isl1-Lhx8 was capable of activating the Acly-HxRE:GFP reporter in both the developing spinal cord and forebrain  at OHSU.DD-Isl1ΔLIM is a fusion of 1-298aa of NLI containing the self-dimerization domain of NLI and 111-349aa of Isl1, which is a C-terminal region of Isl1 that does not include the LIM domains. Isl1-N230S and Lhx3-N211S are missense mutatnts, which are deficient in their ability to directly bind DNA Rat Isl1, Isl1-N230S, and mouse Lhx3, Lhx3-N211S, Lhx8, Lhx1, Isl1-T2A-Lhx3, Isl1-Lhx3 fusion, Isl1-Lhx8 fusion, NLI, and LacZ genes were cloned into pCS2, pcDNA3 (Invitrogen) containing a HA, Flag or myc-epitope tag, or pCIG for expression in mammalian cells and chick embryos and for in vitro transcription and translation reactions. All of these vectors except Lhx8 were previously described ChAT/VAChT, chr14:33256618–33257117; CHT, chr17:54298028–54298480; Acly, chr11:100381966–100382465, chr11:100379377–100379876, and chr11:100395147–100395645. Mouse genomic regions covering the Acly-enhancer, ChAT-enhancer and CHT-enhancer were amplified using PCR, and two or three copies of these enhancers were cloned into TK-LUC or synthetic TATA-GFP reporter vectors. Primers to amplify these genomic enhancers are Acly-enahncer1, forward 5′- GA AGA TCT TGA TAG CAC ACT ACT TTG CTC TGG, reverse 5′- CG GGA TCC CAG TGA CGC ACG GCG AGC GGG AAG; ChAT-enhancer, forward 5′- GA AGA TCT TAC TAA TTG GAT TAA TTG ATT TGC, reverse 5′- CG GGA TCC GGG AAT TAA TAA CTT AGA ATT TGA; CHT-enhancer, forward 5′- GA AGA TCT TGA GCA GCC TAT GCC ACA AGG ACA, reverse 5′-CG GGA TCC AGG AAT CCA TCA CAA AGC TAA GAC. AAGCTGATTA sequences in Acly-enh1 were mutated to CCGCGCGGCC to generate the Acly-enh1-HxRE-mt reporter. Acly-HxRE:LUC, Acly-HxRE:GFP, ChAT-HxRE:LUC and ChAT-HxRE:GFP reporters were created by cloning multiple copies of the following duplex oligonucleotides into synthetic TATA-GFP or TK-LUC vectors. Acly-HxRE, 5′- CAG AGC TAAT CAG CTTG AGTG GGT-3′; ChAT-HxRE 5′- TGG TAC TAAT TGG ATTA ATTG ATT-3′.The location of Isl1-Lhx3-bound ChIP-seq peaks for cholinergic genes in mouse genome (mm9) is the following; f/fIsl1, Nestin-Cre, and Nkx2.1-Cre mice has been described previously f/fIsl1 mice were crossed with f/+;NesticCreIsl1 mice or f/+;Nkx2.1CreIsl1 mice to generate f/f;NesticCreIsl1 or f/f;Nkx2.1CreIsl1 embryos, respectively, for analyses. Mouse embryos were collected at the indicated developmental stages, and fixed in 4% paraformaldehyde, embedded in OCT and cryosectioned in 12 µm thickness for immunohistochemistry assays or 18 µm thickness for in situ hybridization with digoxigenin-labeled probes.The generation of These assays were performed as described For immunohistochemistry assays, the following antibodies were used; rabbit anti-Hb9 For in situ hybridization analyses, cDNA for mouse ChAT, Acly and CHT and chick CHT, Acly, VAChT and ChAT were cloned to pBluescript vector and these vectors were used to generate digoxigenin-labeled riboprobes.3VO4, 200 mM NaF, 20 mM Na-pyroPO4, and protease inhibitor cocktail). In these studies, precipitations were performed with either α-Flag antibody (Sigma) or glutathione sepharose beads . The interactions were monitored by western blotting assays using α-Flag (Sigma) and α-HA (Babco) antibodies. Following western blotting with fluorescence-labeled secondary antibodies, the bound fractions of proteins were scanned by the Odyssey imaging system (Li-Cor) following western blotting with fluorescence-labeled secondary antibodies.HEK293T cells were seeded onto 10 cm tissue cultures dishes, cultured in DMEM media supplemented with 10% fetal bovine serum, and transfected using Superfect (Qiagen). 48 hours after transfection, cells were harvested and lysed in IP buffer  and 7.5% bovine calf serum. For luciferase assays, P19 cells were seeded and incubated for 24 hours, and transient transfections were performed using Lipofectamine 2000 (Invitrogen). An actin promoter-β-galactosidase plasmid was cotransfected for normalization of transfection efficiency, and empty vectors were used to equalize the total amount of transfected DNA. Cells were harvested 36–40 hours after transfection. Cell extracts were assayed for luciferase activity and the values were normalized with β-galactosidase activity. Data are presented as means of triplicate values obtained from representative experiments. All transfections were repeated independently at least four times. Luciferase reporter data are shown in relative activation fold (mean +/− standard deviation).2. Slices were fixed in 4% paraformaldehyde, washed in PBS and analyzed post-fix using immunofluorescence histochemistry.The overall procedures for ventral forebrain electroporation and organotypic slice culture were previously described 2. Slices were fixed in 4% paraformaldehyde and analyzed for GFP expression.The overall procedures for ex vivo brain electroporation and organotypic slice culture were previously described Each set of mouse brain electroporation experiments was repeated independently three to six times. For each set of mouse brain electroporation, three to four brain slices were electroporated per condition. Reproducible results were presented in the figures. Confocal images were acquired using a Nikon Eclipse Ti inverted microscope with perfect focus and a motorized stage coupled to a 4 laser line A1 scanning confocal system. Representative sets of images were presented.For in utero electroporation, timed-pregnant C57BL/6N females were anesthetized at stage E13.5 with isoflurane , and the uterine horns were exposed by way of laparotomy. 1 µℓ of the expression vector in PBS containing 0.05% fast green  was injected into the lateral ventricle of the embryo using a 100 mm glass capillary . Electroporation was performed using Tweezertrodes  with 5 pulses of 45 V for 50 millisecond duration and 950 millisecond intervals using a square-wave pulse generator . The uterine horns were then returned to the abdominal cavity, the wall and skin were sutured, and embryos were allowed to continue their normal development and collected for the further analyses at indicated stages.Hb9, 5′-GTT GGA GCT GGA ACA CCA GT, 5′-CTT TTT GCT GCG TTT CCA TT; ACLY, 5′-GAA GCT GAC CTT GCT GAA CC, 5′-CTG CCT CCA ATG ATG AGG AT; ChAT, 5′-CCT GCC AGT CAA CTC TAG CC, 5′-GGA AGC CTT TAT GAT GAG AA; CHT, 5′-GTG GTC TAG CTT GGG CTC AG, 5′-GGC AAT GAG TGC AGA GAC AA; VAChT, 5′-TTG ATC GCA TGA GCT ACG AC, 5′-CCA CTA GGC TTC CAA AGC TG; Hb9, 5′-GTT GGA GCT GGA ACA CCA GT, 5′-CTT TTT GCT GCG TTT CCA TT; Isl2, 5′-GCA AAC TCG CTG AGT GCT TTC, 5′-ACC ATA CTG TTG GGG GTG TC; Chodl, 5′-CAG TGG AAT GAC GAC AGG TG, 5′-GGT TCC CAA AGC AAC CAG TA; Isl1, 5′-GAC ATG ATG GTG GTT TAC AGG C, 5′- GCT GTT GGG TGT ATC TGG GAG; Lhx3, 5′-AGA GCG CCT ACA ACA CTT CG, 5′-GGC CAG CGT CTT TCT TCA GT; Lhx8, 5′-CAG TTC GCT CAG GAC AAC AA, 5′-AGC CAT TTC TTC CAA CAT GG; GAPDH, 5′-ACC ACA GTC CAT GCC ATC AC, 5′-TCC ACC ACC CTG TTG CTG TA; and Cyclophilin A, 5′-GTC TCC TTC GAG CTG TTT GC, 5′-GAT GCC AGG ACC TGT ATG CT. RT-PCR experiments were performed with three or four independent sets of samples. Data are represented as the mean of duplicate or triplicate values obtained from representative experiments. Error bars represent standard deviation.Total RNAs were extracted using the Trizol (Invitrogen) and reverse-transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). For quantitative PCR of ChAT, VAChT, Acly and CHT, the following probes and primers predesigned by the TaqMan Gene Expression Assay (Applied Biosystems) for each gene were used with TaqMan Universial Master MixII and 7500 ABI qPCR machine (Applied Biosystems); ChAT (Assay ID-Mm01221882_m1), VAChT (Assay ID-Mm00491465_s1), Acly (Assay ID-Mm01302282_m1), CHT (Assay ID- Mm00452075_m1) and Eukaryotic 18S rRNA Endogenous Control . In addition, the following primers were used with the SYBR green kit  and Mx3000P (Stratagene). The ChIP-seq data used for the analysis in this paper has been deposited in the Gene Expression Omnibus (GEO) database (assession no. GSE50993) 3, 50 mM Tris-HCl, pH 8.0) and decross-linked by incubating at 65°C overnight. Eluate was incubated at 50°C for more than two hours with Proteinase K. Next, DNA was purified with Phenol/chloroform and DNA pellet was precipitated by ethanol and resolved in water. The purified final DNA samples were subjected to quantitative PCR reactions using the SYBR green kit  and Mx3000P (Stratagene). The total input was used for normalization. All ChIP experiments were repeated independently at least three times. Data are represented as the mean of duplicate or triplicate values obtained from representative experiments, and error bars represent standard deviation.To perform the ChIP assays with mouse embryonic tissues, we dissected E12.5 spinal cords or E15.5 forebrains. The microdissected spinal cords from five E12.5 embryos or the forebrains of three E15.5 embryos were combined together for each ChIP reaction with a specific antibody. The tissues were dissociated completely, fixed by 1% formaldehyde for 10 min at room temperature, and quenched by 125 mM glycine. Next, cells were washed with Buffer I  and Buffer II  sequentially. Then, cells were lysed with lysis buffer  and were subjected to sonication for DNA shearing. Next, cell lysates were diluted 1∶10 in ChIP buffer  and, for immunoclearing, were incubated with IgG and protein A agarose beads for one hour at 4°C. Supernatant was collected after quick spin and incubated with appropriate antibodies and protein A agarose beads to precipitate the hexamer/chromatin complex for overnight at 4°C. After pull-down of the hexamer/chromatin complex/antibody complex with protein A agarose beads, the beads were washed with TSE I , TSE II (same components as in TSE I except 500 mM NaCl) and Buffer III  sequentially for 10 minutes at each step. Then the beads were washed with TE buffer three times. The hexamer/chromatin complexes were eluted in elution buffer , rabbit anti-Isl1 The generation of Isl1-Lhx3-ESCs was described previously 3 ESCs per 20 µℓ drop). Hanging drops were transferred to suspension culture in 6 well low attachment dishes and cultured. EBs were cultured without or with doxycycline (2 µg/ml) for 2–5 days in the ESC medium without LIF or in the differentiation medium that contains KnockOut serum replacement (Life technologies). Then, EBs were collected for either RT-PCR or immunohistochemical analyses.To induce cell differentiation, Embryoid bodies (EBs) were formed and cultured for 2 days using the hanging drop method  Schematic representation of the Isl1-Lhx3-hexamer composed of two Isl1, two Lhx3 and two NLI molecules. The Isl1-Lhx3-hexamer binds to HxRE (hexamer-response element) in target MN genes and activates their transcription. (B) The location and sequences of the putative HxRE motifs in each of the cholinergic gene peaks. The bars represent 500 bp-long Isl1-Lhx3-bound ChIP-seq peaks associated with cholinergic genes. The number below each bar shows the relative position of the HxRE within each peak . The core sequences of the HxRE motifs are shown in red.The binding sites of the Isl1-Lhx3-hexamer in the cholinergic pathway genes. ((TIFF)Click here for additional data file.Figure S2The co-expression of Isl1 and Lhx3, but not expression of Isl1 or Lhx3 alone, triggers ectopic expression of cholinergic genes in the dorsal spinal cord. The chick neural tube was electroporated with LacZ, Isl1, Lhx3 or Isl1 plus Lhx3, were analyzed for expression of VAChT or ChAT using in situ hybridization. The efficiency of electroporation was determined by immunostaining with α-LacZ, α-myc, or α-Lhx3 antibodies. + indicates the electroporated side. Brackets mark ectopic induction of cholinergic genes.(TIFF)Click here for additional data file.Figure S3A–C) GFP reporter activity was monitored in chick embryos electroporated with Acly-enh1:GFP (A), Acly-enh1-HxRE-mt:GFP (B), and Acly-HxRE:GFP (C) reporters with LacZ. LacZ expression marks the electroporated cells. Acly-enh1 and Acly-HxRE drove MN-specific expression of GFP, while Acly-enh1-HxRE-mt failed to do so, indicating that the HxRE motif is required for the MN-specific enhancer activity of Acly-enh1.  Co-expression of Isl1 and Lhx3 activated Acly-enh1 (D) and Acly-HxRE (E) in the dorsal spinal cord as marked by brackets, but Isl1 or Lhx3 alone was not sufficient to activate the reporters in the dorsal spinal cord. + indicates the electroporated side.The Isl1-Lhx3-hexamer activates the cholinergic enhancer via HxRE motifs in the developing spinal cord. ((TIFF)Click here for additional data file.Figure S4f/f;Nkx2.1CreIsl1 and littermate control mice at E17.5 (A) or P2 (B). VAChT+ cholinergic neurons in the CPu failed to form in the MGE-specific Isl1-null embryos.Isl1 is required for the formation of cholinergic interneurons in the CPu of the developing forebrain. Immunohistochemical analyses on the CPu of (TIFF)Click here for additional data file.Figure S5The comparison of HxRE motifs for Isl1-Lhx8 or Isl1-Lhx3 complexes. The Isl1-Lhx8-binding motif was identified by SELEX. The Isl1-Lhx3-binding motifs were identified by SELEX or ChIP-seq assays (TIFF)Click here for additional data file.Figure S6Acly-HxRE was activated by both Isl1-Lhx8 and Isl1-Lhx3. (A) GFP reporter activity was monitored in mouse cortices electroporated in utero with Acly-HxRE:GFP along with various constructs indicated above each image. The Acly-HxRE was highly activated by Isl1-Lhx8 or Isl1-Lhx3. (B) GFP reporter activity was monitored in chick embryos electroporated with Acly-enh1:GFP and Isl1-Lhx8. Expression of Isl1-Lhx8 activated Acly-HxRE in the dorsal spinal cord, as marked by brackets, but failed to induce ectopic Hb9+ MNs.(TIFF)Click here for additional data file.Figure S7A, B) Gene expression analyses in mouse cortices electroporated in utero with constructs, as indicated by color bars. In utero electroporation was performed with E13.5 brains and the qRT-PCR analyses were done in E18.5 cortices. Y-axis indicates the relative expression levels of each gene, shown in the x-axis, on the electropoated side over the control side. The expression from the electroporated constructs was detected by qRT-PCR (A). Expression of Isl1 and Lhx3, either alone or in combination, failed to induce MN genes Isl1 and Chodl. The expression levels of Hb9 were below detection level in qRT-PCR analyses in any of these conditions. Error bars indicate standard deviation.  Cell differentiation assays in chick embryos electroporated with constructs as indicated on top. Expression of either Lhx8 or Lhx3 led to the ectopic formation of Chx10+ V2 interneurons in the dorsal spinal cord, which was suppressed by co-expression of Isl1. Among all conditions, only co-expression of Isl1 and Lhx3 resulted in ectopic upregulation of Hb9 or VAChT. + indicates the electroporated side. Brackets indicate ectopic Chx10+ V2 interneurons or Hb9+VAChT+ MNs.Analyses of mouse or chick embryos electroporated Isl1, Lhx3, Lhx8, Isl1-Lhx3 or Isl1-Lhx8. ((TIFF)Click here for additional data file.


Here, we present step-by-step guidelines for the computational analysis of ChIP-seq data. We address all the major steps in the analysis of ChIP-seq data: sequencing depth selection, quality checking, mapping, data normalization, assessment of reproducibility, peak calling, differential binding analysis, controlling the false discovery rate, peak annotation, visualization, and motif analysis. At each step in our guidelines we discuss some of the software tools most frequently used. We also highlight the challenges and problems associated with each step in ChIP-seq data analysis. We present a concise workflow for the analysis of ChIP-seq data in Figure 1 that complements and expands on the recommendations of the ENCODE and modENCODE projects. Each step in the workflow is described in detail in the following sections.Mapping the chromosomal locations of transcription factors, nucleosomes, histone modifications, chromatin remodeling enzymes, chaperones, and polymerases is one of the key tasks of modern biology, as evidenced by the Encyclopedia of DNA Elements (ENCODE) Project. To this end, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is the standard methodology. Mapping such protein-DNA interactions  ChIP-seq first cross-links bound proteins to chromatin, fragments the chromatin, captures the DNA fragments bound to one protein using an antibody specific to it, and sequences the ends of the captured fragments using next-generation sequencing (NGS). Computational mapping of the sequenced DNA identifies the genomic locations of bound DNA-binding enzymes, modified histones, chaperones, nucleosomes, and transcription factors (TFs), thereby illuminating the role of these protein-DNA interactions in gene expression and other cellular processes. The use of NGS provides relatively high resolution, low noise, and high genomic coverage compared with ChIP-chip assays (ChIP followed by microarray hybridization). ChIP-seq is now the most widely used procedure for genome-wide assays of protein-DNA interaction Chromatin immunoprecipitation followed by sequencing (ChIP-seq), first described in 2007 Effective analysis of ChIP-seq data requires sufficient coverage by sequence reads (sequencing depth). The required depth depends mainly on the size of the genome and the number and size of the binding sites of the protein. For mammalian transcription factors (TFs) and chromatin modifications such as enhancer-associated histone marks, which are typically localized at specific, narrow sites and have on the order of thousands of binding sites, 20 million reads may be adequate (4 million reads for worm and fly TFs) Arabidopsis ChIP-seq data, above 70% uniquely mapped reads is normal, whereas less than 50% may be cause for concern. A low percentage of uniquely mapped reads often is due either to excessive amplification in the PCR step, inadequate read length, or problems with the sequencing platform, but with some ChIPed proteins it may be unavoidable . The read mappers are designed to allow a (user-settable) number of mismatches in the reads, and it is important to choose this parameter to be appropriate with the NGS platform being used (consult the manufacturer). A final potential cause of high numbers of “multi-mapping” reads is that the protein binds frequently in regions of repeated DNA. In this last case, using paired-end sequencing to reduce the mapping ambiguity may help. It should be kept in mind that multi-mapping reads will be ignored (filtered out) by most peak-calling algorithms , although they can drive the discovery of novel binding sites Before mapping the reads to the reference genome, they should be filtered by applying a quality cutoff Box 1. Thttps://github.com/najoshi/sickle). Additionally, library complexity is a common quality measure for ChIP-seq libraries , and library complexity is linked to many factors such as antibody quality, over-cross-linking, amount of material, sonication, or over-amplification by PCR. The latter can be corrected by systematic identification and removal of redundant reads, which is implemented in many peak callers as it may improve their specificity. Readers may be interested in the Galaxy toolbox, which offers access to many of the tools described here Preprocessing of ChIP-seq data will, in general, be similar to that of any other sequencing data and will assess the quality of the raw reads to identify possible sequencing errors or biases . Phred quality scores are used to describe the confidence of each base call in each sequence tag, are logarithmically linked to error probabilities, and can be used to filter low-quality reads. After this filtering step, it may also be necessary to trim the end of reads that are of low quality  of the ChIP-seq experiment should be assessed, for example via quality metrics such as strand cross-correlation k) applied to one of the two strands . The cross-correlation typically peaks at the shift corresponding to the fragment length and the shift corresponding to the read length. The ratio between the cross-correlation at the fragment length and the background cross-correlation, referred to as normalized strand cross-correlation coefficient (NSC), and the ratio between cross-correlation at the fragment length and the cross-correlation at the read length, referred to as relative strand cross-correlation coefficient (RSC), jointly reflect signal-to-noise ratio in the ChIP-seq data. Very successful ChIP experiments generally have NSC>1.05 and RSC>0.8 Strand cross-correlation analysis p-value for significance of enrichment according to the percentage allocation of reads in each type of regions. Based on the empirical p-value distribution computed from a set of ENCODE IP-Input and Input-input experiments on human data, it estimates a q-value by treating the two types of experiments as true positives and false positives, respectively. The q-value thus is interpreted as the fraction of comparisons with ENCODE data that show differential enrichment at the level of the user's data but turn out to be technical replicates of the Inputs. The software determines the success of the experiment based on the q-values, and also reports some descriptive quality statistics, such as the percentage increase in mean tag density in IP compared to Input and the percentage of the genome classified as signal region. Because the q-values are computed based on human data, users should be aware that the q-values may not be relevant if their data are generated from other organisms.The software CHANCE CHANCE also provides a graphical visualization of IP strength with genome coverage, by plotting the empirical cumulative percentage of tags covered by the bins that are sorted in an increasing order of read density for both the IP and the Input. By examining and comparing the IP and Input curves, one may identify quality issues, such as insufficient sequencing depth, amplification bias, and weak IP enrichment.–Table S1, and p-value or FDR, could be hugely affected by the statistical model used, the sequencing depth, or the actual number of binding sites in the genome. Thus, using the same p-value or FDR threshold does not ensure that the numbers of peaks called are comparable across libraries and different peak callers A pivotal analysis for ChIP-seq is to predict the regions of the genome where the ChIPed protein is bound by finding regions with significant numbers of mapped reads (peaks). A fine balance between sensitivity and specificity depends on choosing an appropriate peak-calling algorithm and normalization method , and the peak callers appropriate for point-source factors  and Scripture There are also some factors (such as RNA Pol II) that bind to DNA in regions with bigger variation. It is known that some RNA Pol II complexes are stalled while others are moving along with active transcription Table S2). Although many methodologies focus on normalization to a control sample, none of them make the distinction on the type of control samples used. An intuitive and commonly used linear normalization technique is called sequencing depth normalization. In this method the number of reads is multiplied by a scale factor to make the total reads in different samples the same (see Whether comparing one ChIP sample against input DNA (sonicated DNA), “mock” ChIP  in peak calling, or comparing a ChIP sample against another in differential analysis, there are linear and non-linear normalization methods available to make the two samples “comparable”  reads present a challenge because they can arise from independent DNA fragments or by PCR amplification of a single fragment. In the former case, the duplicate reads are signals, in the latter case they are noise . A safe solution is to keep a fixed number of hits per genomic location (considering different strands as different locations) according to sequencing depth, and in this way better specificity  can be achieved p-value) makes the numbers of peaks declared more comparable across experiments To ensure that experimental results are reproducible, it is recommended to perform at least two biological replicates of each ChIP-seq experiment and examine the reproducibility of both the reads and identified peaks p-value, the q-value, or the fold enrichment. Significant peaks generally are ranked more consistently across the replicates than the peaks with low significance. This provides an indicator of the transition from real signal to noise. IDR Figure S2). It assigns each signal a reproducibility index, which estimates its probability to be reproducible, and also reports the expected rate of irreproducible discoveries in the selected peaks (referred to as IDR) in a fashion analogous to that of false discovery rate (FDR). An R package for computing IDR is given in Given a set of peak calls for a pair of replicate data sets, the peaks can be ranked based on a criterion of significance, such as the A major advantage of the IDR method is that it is independent of the peak-calling algorithms and can be applied to a variety of significance criteria, across labs and platforms. It has been shown that it produces a stable threshold that is more consistent across laboratories, antibodies, and analysis protocols  than FDR measures Comparative ChIP-seq analysis of an increasing number of protein-bound regions across conditions or tissues is expected with the steady raise of NGS projects. For example, temporal or developmental designs of ChIP-seq experiments can provide different snapshots of a binding signal for the same TF, uncovering stage-specific patterns of gene regulation Table S3 and Two alternatives have been proposed. The first one—qualitative—implements hypothesis testing on multiple overlapping sets of peaks p-values or q-values linked to read-enrichment fold changes). It is strongly advised to verify that the data fulfill the requirements of the software chosen for the analysis. For instance, DIME Typically, both methodologies assume that significant  and reproducible (see section “Assessment of Reproducibility”) peaks have been found in advance independently for each condition. In order to increase sensitivity for detecting differentially bound regions , more relaxed thresholds can be used to find peaks at each condition. Then, depending on the biological question, the sets of peaks called in any of the conditions can be considered separately, or collapsed into one or more meaningful lists of consensus peak regions. One can use the qualitative approach to get an initial overview of differential binding. However, peaks identified in all conditions will never be declared as differentially bound sites by this approach based just on the positions of the peaks Text S1 and The aim of the annotation is to associate the ChIP-seq peaks with functionally relevant genomic regions, such as gene promoters, transcription start sites, intergenic regions, etc. In the first step, one uploads the peaks and reads  coming from unrealistic statistical models used in most methods The number of false positive peaks can be reduced both experimentally and computationally. Improving antibody specificity is a long-term endeavor, and despite impressive progress, still a quarter of histone modification antibodies fail the specificity test Perhaps the most important novel developments are related to the detection and analyses of distal regulatory regions, which are distant in sequence but brought close in 3-D space by DNA bending. To reveal such 3-D mechanisms of transcriptional regulation, two major techniques have emerged: chromatin interaction analysis by paired-end tags (CHIA-PET) indirect DNA binding by the protein (via another protein or complex), so predicted sites not containing the motif may also be functional. Finally, binding does not necessarily imply function, so it will remain necessary to use additional information (such as expression or chromatin conformation data) to reliably infer the function of individual binding events Biological functions of binding sites are not necessarily indicated by the reproducibility of peaks or FDR/IDR values . This isThe diverse experimental and computational methods discussed here are revolutionizing our understanding of the complex networks that, by regulating transcription, impact translation and almost all biological processes.Text S1Standard graphing track data formats for genome browser visualization.(DOCX)Click here for additional data file.Figure S1Assessment of read quality using strand cross-correlation. Strand cross-correlation is computed as the Pearson correlation between the positive and the negative strand profiles at different strand shift distances, k. The cross-correlation (panel A) usually peaks at two distances of shift, one corresponding to the read length, and one to the average fragment length of the library. The absolute and relative height of the two peaks is useful for assessing IP enrichment. Adapted from Landt et al. (TIF)Click here for additional data file.Figure S2The irreproducible discovery rate (IDR) framework for assessing reproducibility of ChIP-seq data sets. Panel A shows a scatterplot of the significance scores of peaks identified in two replicate ChIP-seq experiments. The IDR method classifies peaks into reproducible (black) and irreproducible (red) groups, and computes for each peak the probability that the peak belongs to the irreproducible group. It ranks and selects peaks according to this probability, and computes IDR, the expected rate of irreproducible discoveries in the selected peaks. Panel B shows the estimated IDR at different rank thresholds when the peaks are sorted by the original significance score.(TIF)Click here for additional data file.Table S1Examples of peak callers employed in ChIP-seq. The list includes tools that allow the processing and post-processing of diverse types of narrow read-enriched regions (peaks), broad enriched regions (domains), and mixed signals such as in RNA Pol II ChIP-seq.(DOCX)Click here for additional data file.Table S2Normalization methods for the comparative analysis of ChIP-seq data sets.(DOCX)Click here for additional data file.Table S3Software packages for the analysis of differential binding in ChIP-seq. The table shows examples of algorithms available for differential binding analysis using ChIP-seq data.(DOCX)Click here for additional data file.Table S4Software tools for motif analysis of ChIP-seq peaks and their uses. The table gives examples of publicly available software tools for performing motif analysis on ChIP-seq peaks or nearby genes. The tools are grouped by the major task (“category”), and checkmarks indicate the specific steps that each tool performs. Web-based motif discovery input size limits—ChIPMunk: unknown; CompleteMOTIFS: 500,000 base pairs; MEME-ChIP: 50,000,000 base pairs; peak-motifs: no limit; Cistrome: 5,000 peaks.(DOCX)Click here for additional data file.


Histone post-translational modifications are critical determinants of chromatin structure and function, impacting multiple biological processes including DNA transcription, replication, and repair. The post-translational acetylation of histone H4 at lysine 16 (H4K16ac) was initially identified in association with dosage compensation of the Drosophila male X chromosome. However, in mammalian cells, H4K16ac is not associated with dosage compensation and the genomic distribution of H4K16ac is not precisely known. Therefore, we have mapped the genome-wide H4K16ac distribution in human cells.We performed H4K16ac chromatin immunoprecipitation from human embryonic kidney 293 (HEK293) cells followed by hybridization to whole-genome tiling arrays and identified 25,893 DNA regions  with average length of 692 nucleotides. Interestingly, although a majority of H4K16ac sites localized within genes, only a relatively small fraction (~10%) was found near promoters, in contrast to the distribution of the acetyltransferase, MOF, responsible for acetylation at K16 of H4. Using differential gene expression profiling data, 73 genes (> ±1.5-fold) were identified as potential H4K16ac-regulated genes. Seventeen transcription factor-binding sites were significantly associated with H4K16ac occupancy (p < 0.0005). In addition, a consensus 12-nucleotide guanine-rich sequence motif was identified in more than 55% of the H4K16ac peaks.The results suggest that H4K16 acetylation has a limited effect on transcription regulation in HEK293 cells, whereas H4K16ac has been demonstrated to have critical roles in regulating transcription in mouse embryonic stem cells. Thus, H4K16ac-dependent transcription regulation is likely a cell type specific process. Saccharomyces cerevisiae (SAS2 and YBF2/SAS3), interactions with human immunodeficiency virus Tat in humans (TIP60), are also known to have transcriptional functions as well as roles in DNA damage repair[GT]GG[CAT][GAT][CG] and their complementary sequence). We found 13,230 unique peaks  with at least one 9-mer CTCF motif. DNase I hypersensitive sites data for HEK293T cells (a cell line derived from HEK293) were used for our correlation analysis obtained form: http://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=302749441&c=chr6&g=wgEncodeOpenChromDnase.For transcription factors enrichment analysis we first generated ten control sequences by randomly picking sequences of same size from the same chromosome. Both the peak sequences and control sequences were given as input into the Match tool to scan for enriched PWMs listed in TRANSFAC professional. The total occurrence of any given TFBS on each peak sequence was considered as the observed frequency. Similarly, the occurrence of a TFBS in control set sequences, gave the randomly expected frequency. The discrepancy between observed and expected frequency was evaluated by determining the statistical variable chi-square (χThe authors declare that they have no competing interests.NH, GGS, and TKP designed research; GGS performed ChIP-chip and MC network analyses; NH, PK, GGS, MC, CH, SC, and TKP performed experiments, provided reagents, and analyzed data; and NH, PK, GGS, MC, CH, KW, SC, and TKP reviewed data analyses and wrote the paper. All authors read and approved the final manuscript.


Enhancers and promoters often contain multiple binding sites for the same transcription factor, suggesting that homotypic clustering of binding sites may serve a role in transcription regulation. Here we show that clustering of binding sites for the transcription termination factor TTF-I downstream of the pre-rRNA coding region specifies transcription termination, increases the efficiency of transcription initiation and affects the three-dimensional structure of rRNA genes. On chromatin templates, but not on free rDNA, clustered binding sites promote cooperative binding of TTF-I, loading TTF-I to the downstream terminators before it binds to the rDNA promoter. Interaction of TTF-I with target sites upstream and downstream of the rDNA transcription unit connects these distal DNA elements by forming a chromatin loop between the rDNA promoter and the terminators. The results imply that clustered binding sites increase the binding affinity of transcription factors in chromatin, thus influencing the timing and strength of DNA-dependent processes. The sequence-specific binding of proteins to regulatory regions controls gene expression. Binding sites for transcription factors are rather short and present several million times in large genomes. However, only a small number of these binding sites are functionally important. How proteins can discriminate and select their functional regions is not clear, to date. Regulatory loci like gene promoters and enhancers commonly comprise multiple binding sites for either one factor or a combination of several DNA binding proteins, allowing efficient factor recruitment. We studied the cluster of TTF-I binding sites downstream of the rRNA gene and identified that cooperative binding to the multimeric termination sites in combination with low-affinity binding of TTF-I to individual sites upstream of the gene serves multiple regulatory functions. Packaging of the clustered sites into chromatin is a prerequisite for high-affinity binding, coordinated activation of transcription and the formation of a chromatin loop between the promoter and the terminator. Drosophila development, regulating early patterning genes in vivo, suggesting that HCTs coordinate the recruitment of transcription initiation factors An intriguing question for understanding protein-DNA recognition is how low-abundant transcription factors recognize their target sites in genomic DNA 1–T10) for the transcription termination factor TTF-I. Binding of TTF-I to the terminator elements is required to stop elongating RNA polymerase I (Pol I) and termination of pre-rRNA synthesis occurring predominantly at the first terminator T10, located 170 bp upstream of the transcription start site Cockayne Syndrome protein B), NoRC (Nucleolar Remodeling Complex), or NuRD (Nucleosome Remodeling and Deacetylation complex), respectively, has been shown to recruit histone modifying enzymes which lead to the establishment of a specific epigenetic signature that characterizes active, silent or poised rRNA genes To assess the functional relevance of homotypic clustering of transcription factor binding sites, we studied the 3′-terminal region of murine rRNA genes (rDNA), which contains ten binding sites (Tin vitro and to link two DNA fragments in trans0 and the downstream terminators T1–T10 to loop out of the pre-rRNA coding region TTF-I has been shown to oligomerize 1–T10) spaced by 18–123 bp, preventing the accommodation of nucleosomes in between the TTF-I binding sites. The consensus sequence of these TTF-I binding sites share almost perfect sequence identity within the core motif GGTCGACCAG, while the surrounding nucleotides vary slightly  yielded long read-through transcripts in the absence of TTF-I. The addition of recombinant TTF-I led to the synthesis of terminated transcripts whose lengths correspond to the distance from the transcription start site to the terminator T1 (1-T10), both read-through transcripts and a heterogeneous population of transcripts randomly terminated at any of the TTF-I binding sites were synthesized due to sub-saturating TTF-I levels in the extract  and DNase I footprinting experiments to compare TTF-I binding to free DNA and chromatin. Consistent with the transcription data on free DNA, EMSAs on terminator DNA fragments containing more than one TTF-I binding sites yielded heterogeneous nucleoprotein complexes, reflecting binding to each binding site with similar affinity . On chroin vitro transcription experiments, TTF-I binds on free DNA to the promoter-proximal terminator T0 and the downstream terminators with similar affinity  . TTF-I bfree DNA . Like inin vivo, we transfected CHO cells with reporter plasmids containing the murine Pol I promoter, an internal ribosomal entry site (IRES), Firefly luciferase cDNA and either no terminator (pTΔ) or one (pT1), two (pT2) or ten (pT10) termination sites. As shown in To study the functional relevance of clustered sites 1 (CHO-pT1) or all ten terminators (CHO-pT10)  . Using tHO-pT10) , B. RNA HO-pT10) . ExpressHO-pT10) , reinforTo decipher the molecular mechanism underlying HTC-driven transcriptional activation, we compared transcription factor occupancy within the stable cell lines, containing single rDNA minigenes with either one (CHO-pT1) or ten terminators  or different DNA binding motifs (heterotypic clustering), is an important regulatory feature of eukaryotic gene expression, about 62% of transcription factor genes and 66% of developmentally regulated genes comprising clustered binding sites in vertebrates 1–T10) prior to TTF-I binding to the promoter-proximal binding site T0in vivo. Sequential binding of TTF-I to the 3′- and 5′-end of the rDNA transcription unit will ensure that transcription initiation will take place exclusively at rRNA genes that are associated with TTF-I and will be properly terminated. In addition, a direct interaction between the promoter and the terminator is only established when the terminator comprises several TTF-I binding sites. This mode of binding and the formation of an intragenic loop may serve two functions. First, it links the terminator with the respective transcription unit to be activated. Second, it enhances transcription at genes associated with TTF-I by forming a highly active ribomotor structure Cooperative binding of proteins has been shown to disrupt nucleosomes, thereby increasing the accessibility of transcription factors to regulatory sites It is well established that gene activation by a distal regulatory element correlates with long-range interactions between enhancer(s) and gene promoters by factor-mediated formation of chromatin loops Active enhancer elements are characterized by eukaryotic histone marks, e.g. H3K27ac or H3K4me1, which are involved in long-range chromatin interactions 2, 10% glycerol and 0.05% IGEPAL CA-630. Affinity measurements were carried out in a Monolith NT.015T (NanoTemper Technologies) as described Histidine-tagged full-length TTF-I and the deletion mutants TTFΔN210 and TTFΔN348 were purified on a heparin column (Bio-Rad), followed by purification with Ni-NTA agarose according to the manufacturer's instructions (Qiagen). For microscale thermophoresis experiments, 50 nM of fluorescently labelled DNA oligonucleotides were incubated with 5 nM–2.4 µM of protein for 10 min at 30°C in 80 mM Tris-HCl (pH 7.6), 80 mM KCl, 0.2 mM EDTA, 5 mM MgClDrosophila extract was digested with 1.5 U of MNase (Sigma) for 40 s in 10 mM Tris-HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2, 10% glycerol, 0.5 mM ATP, and 200 ng/µl BSA. Reactions were then stopped by the addition of 0.2 volumes of 4% SDS, 100 mM EDTA, 1 µg of glycogen, 10 µg of proteinase K. Purified DNA was analysed by a single round of PCR  using radioactively labelled oligonucleotides that hybridize to the rDNA promoter or terminator. Primer extension fragments were resolved on 8% sequencing gels and visualized by autoradiography.300 ng of chromatin reconstituted with 1–T10). The promoter and the terminator elements are separated by 686 bp. Transcription reactions were performed as described Transcription experiments were performed on pMrWT-T, a template comprising the murine rDNA promoter (from −170 to +155 with regard to the transcription start site) fused to a 3.5 kb 3′-terminal rDNA fragment (BamHI/EcoRV Fragment) harbouring all ten terminators. (TCHO and CHO Flp-In cells (Invitrogen) were grown in DMEM (GIBCO) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. For transient transfections, 200.000 cells were transfected with 1 µg of plasmid DNA. Prior to transfection of the CHO Flp-In cells, 100 µg/ml zeocin (Invitrogen) was added to the medium and for transfection 0.25, 0.5 or 1.0 µg of the rRNA reporter construct and the flipase encoding plasmid pOG44 (Invitrogen) in a ratio of 1∶9 were used. During the selection process, 500 µg/ml of hygromycin (PAA) was added to the medium; afterwards the stable cell lines were passaged with 250 µg/ml of hygromycin.10 constructs) in a pGL3-Basic vector (Promega). Plasmids for genomic integration contain in addition the enhancer/promoter regions from position −2148 to +181 cloned into a pcDNA5-FRT vector (Invitrogen).Transiently transfected rRNA minigenes Cells were transfected with Pol I driven firefly luciferase reporter constructs and a Pol II renilla luciferase control plasmid, pRL-TK (Promega). TTF-I co-transfections were performed with the expression vectors TTFΔN348-EGFP or TTF-IΔN470-EGFP in a TTF-I∶reporter ratio of 10∶1. Protein expression was monitored by Western Blot analysis. Reporter gene measurements were performed using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions using a single-tube luminometer .RNA isolation was performed with the NucleoSpin RNA II kit (Macherey-Nagel). Purified RNA (500 ng) was used for cDNA preparation with the iScript Select kit (Biorad).To determine the number of integration sites, genomic DNA was isolated by cells lysis , 20 mM EDTA and 250 µg of RNase A), the addition of proteinase K and incubation at 37°C o.n. The supernatant was precipitated with ethanol and ammonium acetate.Quantitative real-time PCR was performed in a Rotor-Gene cycler (Qiagen) using a HotStar master mix containing SYBR green (Qiagen). Primer sequences and annealing temperatures are listed in the in 3. RNase A was added to a concentration of 100 µg/ml and incubated for 2.5 h at 37°C. Following the Proteinase K digestion , reverse crosslinking was carried out at 64°C o.n. DNA was isolated by phenol/chloroform/isoamylalcohol extraction and precipitated with ethanol and sodium acetate.Cells were transfected with 10 µg of DNA and cross-linked with 1% formaldehyde for 10 min (α-Pol I and α-UBF ChIPs) or 10 mM DMA for 30 min +1% formaldehyde for 10 min (α-TBP) at RT. The reactions were quenched with 125 mM glycine. Cells were washed twice in ice-cold PBS and the cell pellets were lysed in SDS lysis buffer . Chromatin was sheared in a Biorupter sonicator (Diagenode) to fragments of 400–1000 bp in length. The samples were diluted in IP dilution buffer . Paf53 antibody for Pol I detection and the pre-serum were obtained from the Grummt lab 4Cl/1× PBS pH 7.4, and once in 1× PBS. Coverslips were then transferred to 70% ethanol and incubated o.n. at 4°C. Before hybridization, coverslips were rehydrated in 2× SSC/50% formamide for 15 min at RT. Hybridization mixtures were added for o.n. incubation at 37°C. Post-hybridization washes were carried out as follows: 2×25 min at 37°C in 50% formamide/2× SSC and 2×5 min in 2× SSC at RT. The subsequent immunostaining, DNA staining and mounting was performed as in interphase DNA FISH experiments. Nick-translated, biotin-labeled pcDNA5-FRT-rRNA reporter served as hybridization probe in all experiments.Fluorescence in situ hybridizations on metaphase chromosome spreads and on interphase nuclei combined with nucleolar immunostaining were performed as described http://biowhat.ucsd.edu/homer/, A custom build of the mm9 assembly was generated by replacing unsequenced bases at the 5′-end of chromosome 18 with a murine rDNA repeat (GenBank accession no. BK000964). We used Bowtie Figure S1Sequence comparison of the TTF-I binding sites in mouse and human is shown below. (B) MNase digestion of reconstituted chromatin. Chromatin was reconstituted with the Drosophila embryo extract and digested with increasing amounts of MNase. Purified DNA was visualized by agarose gel electrophoresis and ethidium bromide staining. The regular fragment ladder is indicative of an efficiently assembled nucleosomal array (1n through 8n).Related to (JPG)Click here for additional data file.Figure S2In vitro transcription analysis was performed comparatively on free DNA (lanes 1–5) or in vitro assembled chromatin (lanes 6–10) on pMrSB containing a single termination site (T1), either in the absence (lanes 1 and 6) or presence of TTF-I (lanes 2–5 and 7–10). The radioactively labeled transcripts were separated by PAA gel electrophoresis and detected by autoradiography. (B) In vitro transcription using the rRNA minigene pMrBH harboring the first two termination sites (T1+T2). The DNA was analysed for in vitro transcription on free DNA and chromatin with increasing amounts of TTF-I as described in (A). (C) In vitro transcription using the rRNA minigene pMrT5 harbouring the first five termination sites (T1 to T5). The DNA was analysed for in vitro transcription on free DNA and chromatin with increasing amounts of TTF-I as described in (A).Related to (JPG)Click here for additional data file.Figure S30 , T1 to T3  and a control region of the vector  were designed. Primers were mixed to allow simultaneous detection and quantification of the three DNA regions. The plasmid was used as free DNA or reconstituted into chromatin with the Drosophila embryo extract. DNA or chromatin was incubated with TTF-I for 10 min and then partially digested with MNase . TTF-I bound DNA fragments were retained on Ni-NTA material in a batch assay and washed twice in Ex150 buffer. The associated DNA was purified and analysed by PCR using the mixture of primers. (B) Binding of TTF-I to the promoter and the terminator on free DNA. 50 fmoles of free DNA were incubated with increasing amounts of TTF-I  and DNA was partially hydrolysed with MNase. A control digestion revealing the input DNA is shown in lane 14. Purified DNA was amplified with a mixture of primers giving rise to the Promoter (P), Terminator (T) and control (c) PCR fragments. Lanes 1 to 4 show the PCR amplification of increasing amounts of the partially digested pMrEnLT10 plasmid, revealing that the individual fragments were amplified with similar efficiency over a 16-fold concentration difference. Ni-NTA purification of the DNA in the absence of TTF-I gives rise to a background of PCR fragments (lane 5) that remains in the fractions containing increasing amounts of TTF-I (lanes 6 to 12). However, with higher concentrations of TTF-I the promoter and terminator fragments accumulated with similar efficiency  suggesting binding of TTF-I. The promoter and terminator fragments appear with similar TTF-I concentrations, suggesting similar binding affinities of TTF-I with the promoter and terminator sites on free DNA. (C) Binding of TTF-I to the promoter and the terminator in reconstituted chromatin. A control digestion of chromatin revealing the input DNA is shown in lane 15. The same experiment as shown in B) was performed with reconstituted chromatin. Lanes 1 to 4 show the PCR amplification of increasing amounts of the partially digested pMrEnLT10 plasmid reconstituted into chromatin. Incubation of chromatin with increasing concentrations of TTF-I  revealed an amplification of the terminator fragment at lower TTF-I concentrations (starting in lane 7) than for the promoter fragment (starting in lane 10). The result suggests that TTF-I binds with higher affinity to the rDNA terminator reconstituted into chromatin and with lower affinity to the chromatinized rDNA promoter. The result confirms the in vitro transcription experiment (Related to periment  and the periment .(JPG)Click here for additional data file.Figure S4in vivo. (A) Transiently transfected rRNA minigenes contain mouse rDNA (BK000964) sequences from position −217  or −148 (pΔT0T10s) to +181, an IRES, the Firefly luciferase gene, and rDNA terminator regions from position +13169 to +15278 (pΔT0T10s and pT10s) in a pGL3-Basic vector (Promega). The plasmids contain a shorter non-specific insert than the constructs shown in Firefly luciferase reporter constructs and a Pol II Renilla luciferase control plasmid, pRL-TK (Promega). Reporter gene measurements were performed using the Dual Luciferase Reporter Assay System (Promega). Deletion of either the promoter-proximal or the terminator TTF-I binding sites reduces transcriptional activity, complementing the results shown in Related to (JPG)Click here for additional data file.Figure S51 and pT10) were genomically inserted into CHO Flp-In cells and stable single integrants were selected. This resulted in the cell lines CHO-pT1 and CHO-pT10. In all experiments, non-transfected CHO Flp-In cell lines were used as controls. Bars represent the mean of three independent stable transfections and error bars indicate standard deviations. (A) FISH detection of genomically inserted mouse rRNA minigenes on CHO Flp-In metaphase spreads. Chromosomes were stained with DAPI and are illustrated in red. Hybridization signals of reporter probes are shown in green. Arrows indicate the single genomic insertion site. The lower panel shows copy number determination of the integrated rDNA reporter plasmids. qPCR was performed on genomic DNA and comparative quantitation was performed between the luciferase gene and the copy number of two single-copy housekeeping genes, β-actin and PabpnI. Bars represent the mean of two independent experiments, error bars denote standard deviations. (B) The number of termination sites does not influence localization of the rDNA minigenes. 3D immuno-FISH analysis of genomically inserted pT1 and pT10 in interphase nuclei. Nuclear DNA was stained with DAPI (shown in blue in the middle merged panel), nucleoli with an α-B23 antibody and indirect immunofluorescence , and the rRNA minigenes were visualized by FISH . Bars depict the percentage of genomically integrated minigenes associated to the nucleolus, n denotes the absolute number of assayed alleles. Bar: 5 µm. (C) Firefly luciferase reporter gene assay on genomically integrated rRNA minigenes. Relative light units (RLU) were measured in three independent experiments, error bars indicate standard deviations. As control, non-transfected CHO Flp-In cells were assayed.Related to (JPG)Click here for additional data file.Figure S6Related to (JPG)Click here for additional data file.Figure S7In silico comparison of the human and mouse rDNA repeat. The murine terminator region comprising of T1 to T10 does not overlap with E-box elements, the canonical c-Myc binding sites. (B) Enrichment of histone modifications at rDNA in MEL cells. The whole rDNA repeat is plotted from position +1 (the TSS) to position 45.500. The terminator track indicates TTF-I binding sites by black vertical lines. The black box highlights the clustered terminator elements at the 3′ end of the gene. ChIP-Seq tracks of c-Myc and TBP display relative enrichments compared to input.Related to (JPG)Click here for additional data file.Table S1Summary of published NGS data used in this study. The table provides an overview of all next-generation sequencing datasets that have been used in the study. Cell types, accession numbers and respective publications are indicated for each dataset. The number of reads indicates absolute tag counts of sequencing reads mapped to the expanded reference genome.(DOC)Click here for additional data file.Table S2List of qPCR primers used for ChIP analyses. The primer lists contains all primers used for quantitation of ChIP assays. Name, binding site, sequence and annealing temperatures are provided for each primer pair used in the study.(DOCX)Click here for additional data file.


Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify cisBmp4 -regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.To elucidate the transcriptional regulation of  Bmp4 regulates several developmental processes including patterning, proliferation, differentiation and apoptosis (http://omim.org/entry/112262).Bmp4, a member of the TGF-β superfamily, is a secreted signaling molecule essential for embryogenesis Bmp4 or Bmp receptor knockout alleles has shed light on the spatiotemporal functions of Bmp4 in epithelial-mesenchymal interactions during early craniofacial and limb morphogenesis Bmp4 or Bmp4r1a in the facial primordia leads to isolated cleft lip or bilateral cleft lip and palate and deficient tooth development Bmp4 in distal mandibular arch ectoderm results in mandibular truncation and lack of incisors, indicating essential functions of epithelial Bmp4 in these ectodermal organs The study of conditional Bmp4 also plays an important role in regulating limb development, as suggested by its strong expression in the apical ectodermal ridge (AER), an important epithelial signaling center at the distal end of the limb bud, and in the anterior and posterior limb mesenchyme. Moreover, conditional inactivation of Bmp4, alone or in combination with other Bmps, or inactivation of its receptors, in limb bud AER or mesenchymal domains, has revealed roles in AER induction and maintenance respectively, as well as anteroposterior and dorsoventral limb patterning, and digit specification gene regulatory networks (GRNs) that control Bmp4 expression in vivo, we searched for Bmp4 CRMs (cis-regulatory modules) using a transgenic reporter assay. Previously, Chandler et al. (2009) used a BAC reporter-based transgenic approach and identified a Bmp4 lateral plate mesoderm (LPM) enhancer ∼46 kb upstream of the Bmp4 transcription start site. We independently identified the same highly conserved, developmentally active Bmp4 regulatory region, but have extended the prior characterization by Chandler et al. (2009) of a 4.3 kb CRM and of a smaller 467 bp subregion to reveal several important new attributes As a step towards defining the Bmp4 enhancer and identified a high-affinity binding sequence for Pitx homeobox TFs, which are strongly expressed in the developing mandible, tooth and limb, and which have been implicated in human and mouse odontogenic defects and lower limb malformations Pitx1 may activate gene expression in dental epithelium Pitx2 null mutants exhibit an early stage arrest in tooth development −/−Pitx1 and −/−Pitx2 double mutants exhibit altered signaling molecule expression in the AER, which is proposed to account for the proximal limb reduction defect in these mutants Pitx1, Pitx2 and Bmp4 remains unclear. We show here that Pitx homeoprotein family members bind a specific site in the Bmp4 incisor epithelium limb bud (“IE/LB”) enhancer that is necessary for its activity in vivo. These results define a minimal, highly conserved Bmp4 enhancer and identify Pitx homeoproteins as key TFs that regulate its embryonic expression.We refined this CRM to an essential 396-bp minimal enhancer that confers reporter gene expression in developing distal mandibular and incisor epithelium and the limb bud, tissues that require Bmp4 for proper morphogenesis. We also used TF (transcription factor) DNA binding specificity motif data from the UniPROBE database Bmp4 enhancers, we conducted a phylogenetic footprinting analysis Bmp4 gene. The mouse Bmp4 transcription unit is located on chromosome 14qC1, and spans ∼7 kb with five exons and two alternative TATA-less promoters Bmp4, and the 7 kb Bmp4 transcription unit itself. We compared this 159 kb mouse genomic sequence, obtained from the UCSC genome database and devoid of other known genes and ESTs, with homologous genomic sequences of human and pufferfish  using the local alignment program BLASTZ which displays homology as blocks of sequence conservation Fugu sequence, although distantly related, was used in the analysis based on the assumption that non-coding sequences that have tolerated selective pressure for hundreds of million years of evolution are likely to be functionally significant and to play important roles in gene regulation Bmp4 plays a critical role  are likely to be fully conserved in fish, we reasoned that certain core regulatory sequences might be. Moreover, we hypothesized that the compactness of the Fugu genome could further filter the relatively high degree of conservation between human and mouse, thereby prioritizing putative regulatory regions for further analysis.To identify candidate Bmp4 exons IA, IB, II, III and IV, 87 blocks of sequence conservation (31 blocks located 5′ and 56 blocks located 3′ to the Bmp4 transcription start site) between mouse and human were identified and considered as candidate Bmp4 regulatory sequences  11.5 and compared them to E11.5 embryos heterozygous for a lacZneoBmp4 reporter allele Bmp4 expression domains at E11.5  of embryos exhibited transgene expression in the oral epithelium overlying the maxillary process and mandibular arch, a pattern similar to that of endogenous Bmp4  and CONS3.7 (424-bp) respectively, into reporter vectors and tested their ability to confer expression. Although a few CONS3.7 embryos exhibited reporter gene activity in the proximal limb, neither construct gave detectable reporter activity in the AER, mandibular arch or incisor dental lamina ; hence these were scored as negative  enhancer previously characterized by Chandler and coworkers Thus, the 396-bp CONS3.8 sequence represents the minimal enhancer element necessary for Bmp4 enhancer, we searched the UniPROBE database (http://thebrain.bwh.harvard.edu/uniprobe/) for putative transcription factor (TF) binding sites within the minimal 396-bp Bmp4 enhancer (CONS3.8) sequence. To further restrict the list of candidate TF regulators, we generated and analyzed microarray gene expression datasets from tissues with CONS3.8 enhancer activity. Epithelial gene expression analysis, using laser capture microdissection (LCM) of E11.5 mouse incisor tooth germs . This list of expressed genes was then intersected with the list of TF families having conserved putative binding sites in the minimal CONS3.8 Bmp4 enhancer  and Msx (5′-GTAATTG-3′) TF families within the minimal 396-bp enhancer (Bmp4 enhancer (WTPitxBSCONS3 or WTMsxBSCONS3) and their canonical sequences  (Bmp4 enhancer oligonucleotide competitor (WTPitxBSCONS3 or WTMsxBSCONS3)  score >0.45).To ascertain the necessity of these intact DNA sites for protein binding, we introduced mutations into each site that were predicted to abolish binding see . As expein vivo, we assayed the activity of CONS3 enhancer sequences that contained clustered point mutations in the respective binding sites in transient transgenic mice. The introduced mutations fulfilled the criteria of completely abolishing TF binding in EMSAs, and insofar as could be determined, did not generate an adventitious site that could bind other TFs.To test the functionality of the Pitx and Msx binding sites in CONS3 MutPitxCONS3 and MutMsxCONS3 reporter transgenes with mutations in Pitx and Msx binding sites respectively, displayed significantly reduced reporter activity compared to wild type  when compared to the 55% of wild type CONS3 transgenics that were lacZ-positive in all domains  exhibited very weak and spotty expression in the AER and limb mesenchyme and only 1 of these embryos showed faint transgene expression in the oral epithelium (n = 1/18)  . Thus, sBmp4 enhancer in living cells, we performed Chromatin Immunoprecipitation (ChIP) assays in LS8 mouse dental epithelial cells. LS8 cells were previously derived from the developing mouse enamel organ Pitx2Bmp4 enhancer sequence (CONS3.8), DNA purified from crosslinked LS8 chromatin after immunoprecipitation with an anti-Pitx2 antibody yielded a 4.7-fold increase in amplicon abundance, relative to an IgG control, by PCR and qPCR  element described by Chandler and coworkers We identified a 396 bp minimal ‘incisor epithelium/limb bud’ (IE/LB) Bmp4 expression in tooth and limb development. First, the presence of a required Pitx1/2 binding site in the Bmp4 IE/LB enhancer supports a direct, positive regulatory relationship between Pitx1/2 and Bmp4 gene expression. Interestingly, however, Bmp4 expression is expanded in Pitx2-deficient mandibular ectoderm at E10.5, suggesting a repressive regulatory relationship Bmp4 craniofacial expression is not observed in Pitx1 null mutants Pitx1 expression at the bud and cap stages of tooth development, including its exclusion from the enamel knot at the cap stage, mimics the persistent ectopic expression of the Bmp4 IE/LB enhancer at these stages of tooth development , Pitx1 or Pitx2 may activate the ithelium . NonetheBmp4 expression and Pitx1/2 in developing limb bud and lateral plate mesoderm, although similarly complex regulatory relationships may exist. Pitx1 and 2 are differentially expressed in posterior and left sided lateral plate mesoderm, respectively, where Pitx1 specifies hindlimb identity in LPM and Pitx2 helps determine laterality Pitx1 and Pitx2 expression overlap in early LPM and they cooperate in hindlimb specification Bmp4 expression is unaffected in the phenotypically affected hindlimbs of Pitx1 null embryos Bmp4 expression in this tissue.Less is known about the relationship between BMP4 gene. This region was compared against various epigenomic profiles in ENCODE human cell lines Finally, since mouse CONS3 has strong sequence homology with human, we performed bioinformatic analyses on the human sequence orthologous to mouse CONS3 to determine if it exhibits features of a regulatory enhancer. After aligning the mouse CONS3 to human sequence, we identified a 243-bp region of sequence homology located ∼45 kb upstream of the human Bmp4 in craniofacial, limb and potentially lateral plate mesoderm tissues. This result furthers the view that while the fine details of gene regulatory networks may differ from case to case, the central features of these regulatory circuits are conserved, not only in evolution, but also in multiple developmental contexts within an organism.In sum, one of the most interesting aspects of the present work is that a single enhancer can regulate the expression of a key developmental signaling gene such as Bmp4 gene (76 kb upstream and 76 kb downstream of the Bmp4 gene) from public database (GenBank accession X56848.1) was compared against orthologous human and pufferfish sequences using BLASTZ (http://bio.cse.psu.edu/pipmaker/), a local alignment program that generates graphical outputs by PipMaker as blocks of sequence conservation http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker).To generate human, mouse and pufferfish alignments, mouse genomic sequence surrounding the The blocks of conserved non-coding sequences (CONS regions) were amplified by polymerase chain reaction (PCR) using either MasterAmp PCR amplification or Extralong PCR amplification kit with proofreading DNA polymerase . The PCR primers were designed to contain a restriction site and an additional 3–6 nucleotides at their 5′ ends for subsequent restriction enzyme digestion and directional cloning of the PCR product. All constructs CONS3.1, 3.2, 3.3, 3.4, 3.5 were subcloned directly from CONS3 construct using restriction enzyme digestion.E. coli lacZSV40pA reporter cassette under the control of a minimal human β-globin promoter The PCR products were purified using QIAquick PCR Purification Kit , restriction enzyme-digested and subcloned into a multiple cloning site of GLKS plasmid which contains an lacZ transgene using PCR with primers wlacZ-F 5′-TTCACTGGCCGTCGTTTTACAACGTCG-3′ and wlacZ-R 5′-ATGTGAGCGAGTAACAACCCGTCGGA-3′. Permanent transgenic lines were established by crossing the founder animal with FVB/N wild type mice. Age-matched lacZneoBmp4 knockin heterozygous embryos were used as controls. The control embryos were derived from crossing lacZneoBmp4 heterozygous male mice with wild type CD-1 or ICR female mice. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard Medical School.The constructs were diluted in microinjection buffer to a concentration of 1–2 ng/µl and injected into fertilized mouse oocytes derived from FVB/N matings using standard procedures Staining for β-galactosidase activity in whole embryos and on cryosections was performed according to standard protocols p-value less than 0.05. The E11.0 tooth germ microarray data is deposited in the GEO database (http://www.ncbi.nih.gov/geo/).Embryonic tissue was dissected in ice-cold RNase-free phosphate buffered saline (PBS) and the E11.0 heads were frozen immediately in Tissue Tek OCT . Fresh-frozen tissue was cryosectioned and collected on PEN membrane slides . The slides were immediately refrozen and maintained on dry ice before staining and dehydrating with Histogene Staining Kit . Discrete incisor epithelial tissues were isolated using a Leica laser capture microdissection (LCM) LMD 6000 microscope. The tissue was isolated directly into the extraction buffer provided with Pico Pure Isolation Kit . RNA purification was performed according to the Pico Pure Isolation Kit and included an on column treatment with RNase-free DNase I . Eluted RNA quality was determined using the Agilent Bioanalyzer 2100. 10 to 25 ng of total RNA was then amplified to yield 7–10 µg of single-stranded DNA using a poly-dT based Ovation RNA Amplification System V2 . The quality and size distribution of amplified DNA were confirmed using an Agilent Bioanalyzer 2100. Biotinylation was achieved through abasic site creation in the single stranded DNA with Uracil N-Glycosylase , and reaction with Aldehyde-Reactive Probe (ARP) N-(aminooxyacetyl)-N′-(D-biotinoyl) hydrazine, trifluoroacetic acid salt (Invitrogen). 1.5 µg of biotinylated DNA was hybridized according to the NuGEN manufacture note to the Illumina Mouse Ref-6 whole genome expression array. For expression profiling of the developing limb, Affymetrix microarray datasets were obtained from Gene Expression Omnibus (GEO) reference series GSE2560. Probe datasets were called “Present” if at least two of the three replicates had a detection http://thebrain.bwh.harvard.edu/uniprobe/) to the CONS3.8 CRM. The Mouse July 2007 (NCBI37/mm9) assembly was used along with the 46-way multiz vertebrate alignment from Galaxy (http://main.g2.bx.psu.edu/). A putative binding site in CONS3.8 was selected for further analysis when at least two consecutive overlapping ungapped 8-mers scored above an enrichment score (ES) of 0.35 (medium affinity binding site) and 0.45 (high affinity binding site). We focused on TF families whose members had binding sites that were conserved in at least 90% of the vertebrate alignment within CONS3.8. TFs were grouped into families based on their names and their common DNA binding domain (DBD) annotation from the InterPro database e.g., since the TFs Msx1, Msx2, and Msx3 each have the same name, Msx, and the same three DBD annotations of IPR000047 (helix-turn-helix), IPR001356 (homeodomain), and IPR009057 (homeodomain-like), we grouped these three TFs into the family “Msx IPR000047; IPR001356; IPR009057”). We further restricted our candidate TF families based on the expression of at least one of their members in the tissues of interest. Microarray datasets were used from mouse tissue of E11.5 tooth germ (this study) and E12.5 fore-  and hindlimb . Transcription factors were retained if they were called present in at least 2 out of the 3 replicates, in all three tissues, and had conserved binding sites in CONS3.8.Custom MATLAB scripts were written to map ungapped 8-mers from the UniPROBE database for mouse TFs . We purified the PCR products using Gel Extraction kit . Approximately 1 µg of the purified PCR products was subjected to double digestion. The digested products were then ligated into the expression vector (pGEX3X). Both Pitx1 and Msx2 constructs were directly sequenced with forward and reverse primers designed from plasmid sequences flanking the cloning site (pGEX3X-F: 5′-ATGGCCTTTGCAGGGCTGGCAAGC-3′ and pGEX3X-R: 5′-TCTCCGGGAGCTGCAT-GTGTCAG-3′) to ensure the absence of mutations.Full-length Pitx1 and Msx2 expression plasmids were constructed in pGEX3X by PCR-based cloning of the Pitx1 and Msx2 coding regions. Each reaction contained template, 4 pmoles of each primer, 2.5 U of PfuTurbo® DNA polymerase , and 2× FailSafe PreMix A  which contains 1.5 mM MgEscherichia coli strain BL21 (DE3) was transformed using the clones selected after confirming the sequences. Bacterial cultures were induced with 1.0 mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG) for 3 hours. Cells were collected after centrifugation at 5000xg for 30 minutes at 4°C. The cultures were resuspended in ice-cold phosphate buffered saline (PBS). Cell lysis was performed by adding lysozyme to a final concentration of 1 mg/ml and incubation on ice for 30 minutes. Triton X-100, DNase I and 5 µg/µl RNase A and 1 mM PMSF were added to the lysates. After incubation at 4°C for 30 minutes, lysates were spun at 3000xg for 30 minutes. Supernatants were removed and adjusted to 1 mM DTT and 1 mM PMSF. Fusion proteins were purified using glutathione agarose beads  according to standard protocols.Cells of 5′-TCATGCCTGTAATCCCAGCACTCAG-3′ and MBS: 5′-GATCCACTAATTGGAGG-3′, respectively 2) followed by cooling. They were then labeled in a 20 µl reaction volume using 20 ng of annealed oligonucleotide in polynucleotide kinase (PNK) buffer , 10 units of T4 polynucleotide kinase  and 5 µCi of γ-32P-ATP. Reactions were incubated at 37°C for 1 hour. To remove unincorporated oligonucleotides, the labeled probe reactions were loaded onto Micro Bio-Spin P-30 Tris Chromatography columns  and centrifuged according to the manufacturer’s instructions and diluted with protein binding buffer to ∼2×104 cpm/µg.Two control target DNA sequences used for Pitx1 and Msx2 EMSA were Bicoid: 32P]-labeled annealed probe on ice for 20 minutes. One microliter of loading dye was added to a free probe reaction (no protein) to assist in locating free probe in the gel. Protein-DNA complexes were separated on a pre-cooled 6% non-denaturing polyacrylamide gel. Electrophoresis was performed at 4°C with 1x Tris-Borate-EDTA (TBE) buffer at 200 volts for 2 hours. Gels were transferred to Whatman paper and dried at 80°C for 2 hours before being subjected to autoradiography at –80°C.Protein-DNA binding reactions of 20 µl total volume were performed by adding increasing amounts of purified fusion proteins in final reaction of 1X phosphate buffer, 10% glycerol, 0.3 mg/µl BSA and 0.1 µg/µl DNaseI digested poly[dG-dC], incubated on ice for 10 minutes and then further incubated with addition of 1 µl of [5′-GGGCCTGTTACTCCTTCAGGAATG aggccggATAGGGTCAAATA-AAACATG-3′, SDMMsxBS-R: 5′-CATGTTTTATTTGACCC TATccggcctCATTCCTGAAG-GAGTAACAGGCCC-3′. The mutated constructs were sequenced to confirm the introduced mutation prior to purification for pronuclear microinjection. E11.5 transgenic embryos were collected and tested for reporter activity as described above.We used a QuikChange Lightning Site-Directed Mutagenesis Kit  and the following primer pairs to perform mutagenesis of Pitx or Msx binding sites in the CONS3 construct - SDMPitxBS-F: 5′-GAGGGCTCTTCACGGTAAatgcgtTGTAGGGAA CTTAAAAAGAAG-3′, SDMPitxBS-R: 5′-CTTCTTTTTAAGTTCCCTACAacgcatTTACCG TGAAGAGCCCTC-3′ and SDMMsxBS-F: Bmp4 CONS3 are as follows: forward 5′-CCACCCACAGATTCAGACCT-3′ and reverse 5′-CAGGAAGGAAT- TCGAAGCAG-3′ . The two control primers are as follows: forward 5′-AGCAAACAGGCGATCTCATT-3′ and reverse 5′- GGAGTGGTGAA- GGTCTTGGA-3′ ; forward 5′-TGCATGTGGTCAGTCAGTCA-3′ and reverse 5′-TGCTTCACCACAGGTCTCAG-3′ . All the PCR products were evaluated on a 2% agarose gel in 1X TBE for appropriate size. Quantitative real-time PCR was performed using the same annealing temperature but extending the number of cycles. Identical amounts of the IP DNA and IgG DNA were loaded as template. All of the regular PCRs and real-time PCR products were confirmed by sequencing.The ChIP assays were performed as previously described Figure S1lacZneoBmp4 and CONS3Tg β-gal activity in initiation stage incisor. Whole mount and sagittal sections of lacZneoBmp4 and CONS3Tg transgenic upper incisors. Abbr: NP, nasal pit; MNP, medial nasal process; MxP, maxillary process; MdP, mandibular process; OC, oral cavity; UI, upper incisor; M, mesenchyme; DL, dental lamina; D, dorsal; V, ventral; A, anterior; P, posterior.(TIF)Click here for additional data file.Figure S2lacZneoBmp4 and CONS3.5Tg β-gal activity in cap-stage incisors. Sagittal sections of cap stage lacZneoBmp4 and CONS3.5Tg transgenic upper and lower incisors. Note persistent inappropriate expression of CONS3.5Tg in non-enamel knot dental epithelium and surrounding ectoderm, and its failure to be expressed in the dental papilla and adjacent mesenchyme, as is observed in the control lacZneoBmp4 heterozygote. Abbr: EK, enamel knot; DP, dental papilla; EO, enamel organ; T, tongue; UL, upper lip; LL, lower lip; VL, vestibular lamina.(TIF)Click here for additional data file.Figure S3Laser capture microdissection (LCM) of epithelial incisor region. Sagittal sections of an E11.5 initiation stage incisor pre- and post-LCM. The captured epithelium was identified by bright field microscopy. Scale bar: 100 µm.(TIF)Click here for additional data file.Figure S4Pitx and Msx binding sites in the Bmp4 IE/LB enhancer. Sequence of the 758 bp region  containing the 396 bp Bmp4 minimal enhancer double underlined . The Pitx1/2 binding site (5′-TAATCC-3′) and Msx1/2 binding site (5′-GTAATTG-3′) are indicated, and the location of the 467 bp enhancer  described by Chandler and coworkers (TIF)Click here for additional data file.Figure S5In vitro binding of Msx to the minimal Bmp4 enhancer. Electrophoretic Mobility Shift Assay (EMSA) exhibits robust binding of Msx2 protein to both a positive control MBS DNA sequence and to the consensus Msx1/2 binding site in the CONS3 sequence. Competition with specific or non-specific cold probes indicates sequence-specific binding of GST-Msx2 fusion protein to the consensus Msx1/2-binding motif.(TIF)Click here for additional data file.Figure S6Transgenic mutational analysis of Pitx and Msx binding sites. Graphical representation of the transgenic mutational results presented in (TIF)Click here for additional data file.Figure S7Integrative epigenomic analysis suggests that the human orthologous sequence of mouse CONS3 likely functions as an enhancer. Using public epigenomic data from the University of California at Santa Cruz (UCSC) Genome Browser, we analyzed the human homolog of CONS3 . This region is enriched for an enhancer associated histone mark H3K4me1, is DNaseI hypersensitive, is a binding site for transcription factors Max and c-Fox in human embryonic stem cells, and is annotated to be in “enhancer state” in multiple human cell lines by ChromHMM (TIF)Click here for additional data file.Table S1Families of Transcription Factors (TFs) with their InterPro (IPR) DNA binding domain annotation number that have potential binding sequences in the minimal Bmp4 enhancer. Transcription factors families with binding sites (BS) in the minimal Bmp4 enhancer in >75% and >90% of aligned vertebrates are indicated.(PDF)Click here for additional data file.