Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions.NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of  In eukaryotes DNA is folded and compacted into manageable units by wrapping around a protein spool of histone proteins to form nucleosomes. By varying the position and dynamics of nucleosomes using energy-dependent chromatin remodeling enzymes, genes can be selectively turned off or on in cells, controlling development and cellular function. Distinct sub-families of ATP-dependent chromatin remodeling enzymes have been characterised. However, their specific nucleosome targets in the genome and how they are recruited to these are not completely defined. Here we have identified nucleosome targets of the conserved higher eukaryotic chromatin remodeling enzyme NURF. Our data indicate three distinct functions for NURF during transcription. NURF organizes nucleosome positions at gene enhancer elements to regulate transcription initiation, but is also required to maintain nucleosome position downstream of the transcription start site of active genes. In addition, we detect NURF remodeling and recruitment at distal insulator sites that are required for functional organisation of the genome. We postulate that NURF function at insulators as well as promoters reflects functional interaction between distant insulators and active promoters, with functional clustering of regulatory elements providing a solution to how chromatin remodeling enzymes engage multiple targets in the genome. The organization of DNA in nucleosomes has a major function in controlling accessibility of DNA to the protein complexes that process genetic information. By altering nucleosome dynamics, targets for the transcription, replication and repair machineries can be rendered inaccessible or made available. A number of mechanisms exist by which chromatin states can be altered. Post-translational modification of the histone tails (HPTMs) can change associations between histones and DNA, altering chromatin flexibility and conformation (reviewed in Tessarz and Kouzarides ). Howevenucleosome remodeling factor (NURF) is one of the founding members of this family. Although chromatin remodeling complexes based on ISWI type catalytic subunits are present in all metazoa, NURF is an innovation of the bilateria. NURF complexes are built around a large, bilaterian-conserved, NURF-specific subunit, in Drosophila Nurf301/Enhancer of bithorax (E(bx)), in humans BPTF (Bromodomain and PHD finger Transcription Factor) [Nurf301/E(bx) were initially identified as regulators of the bithorax-complex [The imitation switch (ISWI) family of ATP-dependent chromatin remodeling factors mediate energy-dependent nucleosome sliding , 4. The  Factor) , 6. Like Factor) , 6, allo-complex , and sub-complex –9.Current models for NURF function propose activity at defined enhancers leading to regulation of a restricted set of gene targets. We and others have shown that Nurf301/E(bx) can directly interact with sequence-specific TFs that include the GAGA factor (Trithorax-like (Trl)), ecdysone receptor (EcR), and the repressor Ken , 8, 9, sNurf301E(bx) mutants [The core promoter regulates transcription initiation by serving as the recognition site for the basal transcription apparatus and determines specificity for upstream enhancers. In bilateria marked diversification of core promoter architecture occurs, with complexes including the canonical TATA-binding protein (TBP) containing complex TFIID, as well as complexes containing TBP-related factors (TRFs) such as the DREF/TRF2 complex, binding distinct core sequences –12. Prev mutants , suggestDrosophila based on enhancer blocking function which can be mediated by combinations of three DNA-binding proteins—Boundary Element Associated Factor (BEAF), CCCTC-Binding Factor (CTCF) and Suppressor of Hairy wing (Su(Hw))–and two associated proteins, Centrosomal Protein 190 (CP190) and Modifier of mdg4 (Mod(mdg4)) [In turn, intra- and inter-chromosomal communication between the core promoter and enhancer regions can be controlled by insulator elements. These were first defined in d(mdg4)) .in vivo nucleosome targets of a metazoan chromatin remodeling enzyme. Our data indicate NURF action at three distinct transcription regulatory elements. In addition to upstream enhancer elements, we identify a novel function of NURF in orchestrating nucleosome spacing downstream of the +1 nucleosome on active genes regulated by DREF/TRF2. Moreover, we also detect NURF remodeling and recruitment at distal insulator sites, where NURF interacts with DREF and known insulator proteins including CP190 to establish nucleosome-depleted domains.To provide an unbiased assessment of regulatory elements at which NURF acts, we have used whole genome MNase-sequencing to map at base pair resolution nucleosomes that require NURF for positioning. In parallel, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) of the Nurf301/E(bx) specificity subunit to identify sites of stable recruitment of NURF. To our knowledge this is the first comprehensive base pair resolution map of Drosophila WT and NURF deficient primary macrophages (hemocytes). As a first step normalized tag densities of uniquely-mapped reads were determined in a sliding 50 bp window across the genome and log2 fold changes between mutant and wild-type (WT) were used to define regions of altered read densities, we term shifts. Using this approach, we identified 47,000 shifted nucleosomes in Nurf301/E(bx) deficient larval hemocytes. Assuming an averaged female Drosophila genome size of 175 Mb [To identify sites of NURF activity in the genome we profiled nucleosome distribution in f 175 Mb , this coThe distribution of NURF nucleosome shifts relative to known gene features was then determined. Consistent with the hypothesis that NURF regulates transcription by remodelling enhancer nucleosomes, we observed significant enrichment of nucleosome shifts on 5’ regulatory elements and under-representation on coding exons and gene bodies , S2 Fig.Nurf301/E(bx) mutant backgrounds, nucleosomes that require NURF to maintain position could be discriminated. The validity of this mapping procedure was confirmed by analysing nucleosome positions on heat-shock loci, the targets originally used to identify NURF [Nurf301/E(bx) mutants this nucleosome position was not maintained and new nucleosome positions were detected  mutants corresponded to sites of NURF localisation determined by ChIP-seq. Nurf301/E(bx) ChIP peaks were ordered according to strength of associated signal, and sub-divided into quintiles from highest (1st) to lowest (5th) Nurf301/E(bx) signal. Analysis of nucleosome position flanking these sites demonstrated consistent changes in nucleosome position in Nurf301/E(bx) mutants (Nurf301/E(bx) mutant hemocytes. Analysis of expression of 667 genes that have assigned GO (gene ontology) molecular functions related to chromatin and transcription  mutants these positions were lost and chromatin flanking the site was more accessible, suggesting these TFs potentially could tether NURF to mediate directional nucleosome sliding. In contrast, class IV predicted TF-binding sites, exemplified by Trl (Nurf301/E(bx) mutants. The final mode was observed flanking consensi for DNA-binding proteins including Su(Hw) (Nurf301/E(bx) mutants.Predicted binding sites for class I TFs were independent of NURF and located within nucleosomes, . Bindingd by Trl , were log Su(Hw) . BindingNurf301/E(bx) mutants  mutant backgrounds. This showed the expected nucleosome distribution in WT samples with a nucleosome-depleted region flanking the TSS, a well-positioned +1 nucleosome and a regular downstream array of nucleosomes (Nurf301/E(bx) mutants, a superficially similar distribution was observed, with a regular array of nucleosomes downstream of the TSS. However absolute nucleosome position was shifted towards the TSS for the first six nucleosomes following the TSS (+1 to +6 nucleosome positions).However, NURF function was not restricted to remodelling nucleosomes at enhancers. Our initial comparative analysis of nucleosome tag densities detected potential nucleosome shifts not only upstream but also downstream of the TSS . To elucleosomes . In NurfNurf301/E(bx) mutants (Nurf301/E(bx) mutants were propagated over the next five nucleosomes after which the nucleosome array reset and limited differences between mutant and WT arrays were observed.This analysis included both active and inactive TSSs. To discriminate if NURF differentially affected transcriptionally active versus inactive promoters, we categorized active and inactive TSSs in hemocytes by profiling the hemocyte distribution of the H3K4me3 HPTM, which decorates the +1 nucleosome of active genes . ProfiliCG10699 gene showed a 10 bp change in position of the +1 nucleosome in Nurf301/E(bx) mutants  mutants. In contrast, on inactive genes, the extrapolated WT average nucleosome repeat length of 185 bp was unchanged in Nurf301/E(bx) mutants. Similar analysis of nucleosome positions at other genomic regions, for example flanking exon-intron boundaries, also showed no change in nucleosome spacing in Nurf301/E(bx) mutants. Taken together we conclude that NURF has a specific nucleosome positioning and spacing function downstream of active TSSs.Calculating average nucleosome repeat length downstream of the TSS on active and inactive genes in both genetic backgrounds showed that nucleosome repeat length on active genes decreased from 175 bp in WT samples to 170 bp in Nurf301/E(bx) mutant hemocyte gene expression levels were determined by Affymetrix microarray profiling. TSSs were binned into quintiles based on normalized WT transcript level from high (1st) to low (5th), and averaged nucleosome position flanking TSSs of each quintile determined in both backgrounds. We observed that, while the WT +1 nucleosome dyad location was shifted downstream as expression level increased (from +125 bp in the lowest expression quintile to +136 bp in the highest quintile), for all quintiles +1 nucleosome positions were relocated towards the TSS in Nurf301/E(bx) mutants  mutants. Surprisingly, real-time RT-PCR (Nurf301/E(bx) mutant hemocytes. Furthermore, using whole genome expression profiles to categorize TSSs with increased or decreased expression in Nurf301/E(bx) mutants, and profiling averaged nucleosome densities flanking the corresponding TSSs, indicated that only TSSs up-regulated in Nurf301/E(bx) mutants demonstrated signature NURF-dependent +1 nucleosome shifts  mutants. This showed clear NURF activity on promoters containing DREs and Ohler motifs 1, 5, and 7 (Nurf301/E(bx) mutants. NURF activity on DREF-dependent promoters was confirmed by comparison of Nurf301/E(bx) and published DREF ChIP-Seq profiles [We demonstrated a specific NURF nucleosome spacing function downstream of active TSSs. To discriminate if NURF promoter targeting was mediated by core promoter components we classified TSSs based on associated core promoter elements. Nucleosome organisation flanking these distinct categories of TSSs was then determined in WT and 5, and 7 , S7 Fig,profiles . HeatmapNurf301/E(bx) mutants  mutants  ChIP-Seq profiles revealed NURF localisation to isolated elements that corresponded to previously defined insulator elements, including the Fab8 and Mcp boundaries in the bithorax complex . This coNurf301/E(bx) mutants, nucleosome-depleted domains at CP190-containing insulator sites were reduced in size and nucleosomes repositioned. Consistent with the discriminating role of CP190, reciprocal co-immunoprecipitation revealed that NURF and CP190 were physically associated in S2 cell extracts  and BEAF  showed textracts .Nurf301/E(bx) mutants, nucleosome-depleted domains at DREF-containing insulator sites were reduced in size. Consistent with DREF action at insulators, co-immunoprecipitation showed that the DREF/TRF2 subunit Wash and CP190 were physically associated in S2 cell extracts  and CP190 at insulator elements in the bithorax complex  and predextracts . In addiextracts , a hallmin vivo was confirmed using the known enhancer blocking function of gypsy retrotransposons. These contain binding sites for Su(Hw), which can recruit insulator components to establish a functional insulator. When integrated between the wing enhancer and promoter of the cut gene in the ct6 mutation, gypsy elements suppress ct wing expression and disrupt the adult wing margin. This normally is decorated with approximately 90 mechanosensory bristles. Loss of bristles in ct6 mutants can be used as a sensitive assay for gypsy insulator function. In the absence of other mutations ct6 alleles reduce mechanosensory bristle number to 14  , 25, sug domains . Consist domains , and inv domains .Finally, as Nurf301/E(bx), DREF and CP190 colocalize at insulators, and Nurf301/E(bx) and DREF also overlap at active promoters, we tested whether insulator components may also be similarly localized to active promoters. Comparison of CP190 and Nurf301/E(bx) ChIP-Seq profiles in S2 cells reveals that CP190 was present at TSSs. However, CP190 was only detected on active genes .CP190 binding to both active promoters and distant insulator elements resembles the reported association of TAF3 with CTCF that mediates looping between distal enhancer elements and the proximal promoter , raisingin vivo targets and mechanisms by which ATP-dependent chromatin remodelling activity is deployed. Here we have used mononucleosome mapping of WT and Nurf301/E(bx) mutant cells to discriminate nucleosomes remodelled by NURF in vivo. We detect interactions with transcription factors and insulator components that direct NURF activity to both gene promoters and distant regulatory elements. To our knowledge this is the first comprehensive base pair resolution map of in vivo nucleosome targets of a metazoan chromatin remodeling enzyme. Our data indicate NURF remodelling occurs at discrete sites in the genome, but we speculate that by mediating local chromatin reorganization NURF can profoundly impact genome organization and long-range regulatory interactions.Modulation of nucleosome dynamics by ATP-dependent chromatin remodelling enzymes has the potential to regulate all chromatin templated reactions. Key to understanding functions in diverse processes ranging from transcription to repair and replication is to elucidate Drosophila ISWI abundance [Nurf301/E(bx) mutants [Nurf301/E(bx) mutants suggest more localized effects on nucleosome positioning [A central question in the ATP-dependent chromatin remodelling field is whether remodelers are highly abundant, affecting all nucleosomes and genome function generally, or more restricted factors that are recruited to discrete sites to regulate specific nuclear processes. Early estimates of bundance , and dis mutants , 27, weritioning . Data geitioning .Nurf301/E(bx) mutants. Nevertheless, these results are broadly consistent with in vitro experiments demonstrating for example how GAL4 provides a “barrier” that competes with nucleosomes for occupancy at DNA targets [NURF targets defined here include upstream enhancers, consistent with the original isolation of NURF as a TF cofactor and initial transcriptome analysis –9, 17. O targets , 30 or,  targets .Nurf301/E(bx) mutants.However, our data reveal an additional nucleosome spacing function for NURF downstream of active TSSs including house-keeping targets of TRF2/DREF. The consequences of this are distinct from remodelling at enhancers, which mediates high-level changes in transcription of specific developmental/signal transduction pathways, instead exerting a general dampening effect on active gene transcription. Our data indicate that NURF shifts nucleosomes in the 3’direction away from the TSS, similar to yeast ISW1b and CHD1 remodelers  and alsoNURF localisation and activity has parallels with yeast ISWI remodelling complexes, which show recruitment to TSSs but action on gene bodies , 36. We in vitro [Promoter localization of NURF is likely further stabilized by NURF binding HPTMs that decorate the +1 nucleosome , 39 but,in vitro . A precein vitro . It has in vitro . In our in vitro , 39.Nurf301/E(bx) mutants are not the consequence of loss of NURF ATP-dependent nucleosome-sliding activity, but rather that NURF acts stoichiometrically and non-catalytically at some sites, binding and physically occupying linker DNA in a manner incompatible with nucleosome formation. Changes in nucleosome position could thus potentially be due to the lack of NURF physical presence at some sites. The ability of remodelers to engage in either “traditional” catalytic ATP-dependent nucleosome sliding versus and non-catalytic modes would allow diversification of remodeler function, and offer the potential that allosteric modulation (by for example histone modifications) could switch complexes between different modes of remodeling to program distinct local chromatin architectures. The use of strains containing catalytically inactive NURF induced by expressing mutant forms ISWI, in which ATPase activity is eliminated but complex assembly unaffected, could offer one approach to experimentally investigate this.It is formally possible that some changes in nucleosome positioning observed in Nurf301/E(bx) mutants. Indeed initial analysis of yeast remodeling enzymes required depletion of three remodeling enzymes  to observe substantial effects on nucleosome organisation [It is also important to consider that these experiments were performed in cells in which function of other remodeling complexes was unaffected. Systematic profiling of yeast chromatin remodeler distributions indicates overlapping domains of function. As such it is possible that NURF is functionally redundant with other chromatin remodeling enzymes . Thus, tnisation Nurf301/E(bx) mutants modify bithorax mutations and that Nurf301 corresponds to Enhancer of bithorax E(bx) [bithorax mutations are caused by gypsy transposons that bind Su(Hw) and act as ectopic insulators to disrupt expression. NURF remodeling at insulator elements is also consistent with results showing that NURF is required for Drosophila cell-based insulator/enhancer blocking assays [Finally, our data reveal that NURF interacts with the insulator elements and interacts with components including CP190 and DREF. Activity of NURF at insulators is consistent with our initial studies showing that ax E(bx) . The bitg assays . This apg assays  and integ assays .Interestingly, colocalization between Nurf301/E(bx) and insulator proteins is not only detected at distal insulator regions but also at active promoters. We postulate that overlap of insulator components and NURF at insulators as well as promoters reflects functional interaction between distant insulators and active promoters as has been speculated for CTCF at mammalian promoters , 47. It Nurf3012, Cp1901 and Cp1902 alleles were as described [Nurf3012 allele is an EMS-induced mutation that encodes a glutamine to stop codon substitution (aa 545) that truncates Nurf301 after the first PHD finger and which behaves genetically as a null allele. Unless stated flies were raised at 25°C.escribed , 48. TheNurf3012 mutant female third instar larvae as described previously [g for five minutes. Cells were washed three times with ice cold 1XPBS containing protease inhibitors and stored as pellets at -80°C until required.Third instar larvae were sexed and primary hemocytes collected from wild-type and eviously . BrieflyS2-DRSC cells were cultured at 25°C in Insect-XPRESS medium (Lonza) containing 10% FCS. S2 cells for standard ChIP were fixed with 1% formaldehyde and washed as described above.2 was added to a final concentration of 1 mM, 800U MNase (Worthington) added, and the sample incubated for 12 minutes at 16°C to liberate mononucleosomes. Digestion was stopped by adding an equal volume of Stop buffer (0.1 M Tris (pH 8.5), 0.1 M NaCl, 50 mM EDTA, 1% SDS) and samples centrifuged at 17,400g for 10 minutes to purify soluble chromatin. Formaldehyde cross-links were removed by addition of NaCl to 150 mM, one-tenth volume Proteinase K (Roche) and incubation at 65°C overnight. DNA was purified by phenol/chloroform extraction and ethanol precipitation, treated with RNase (Promega) at 37°C for 1 hour and then run on 2.2% recovery FlashGels (Lonza). Mononucleosomal DNA was pipetted from recovery wells in FlashGel Recovery Buffer and DNA subjected to further round of phenol/chloroform extraction and ethanol precipitation. Libraries were generated from two biological replicates for each genotype, sequenced, and reads pooled for nucleosome mapping.Hemocyte preparations from 1000 larvae were thawed, pooled and resuspended in buffer A (15 mM Tris (pH 7.4), 15 mM NaCl, 60 mM KCl, 0.34 M sucrose, 1 mM DTT, 25 mM sodium metabisulfite, 0.5 mM spermidine, 0.15 mM spermine). Cells were homogenized with a pellet pestle. CaClFor MNase-ChIP experiments, hemocytes from 1000 larvae were processed as above but soluble chromatin after Stop buffer addition was diluted in ChIP dilution buffer (16.7 mM Tris (pH 8.1), 167 mM NaCl, 1.2 mM EDTA, 0.1% SDS, 1.1% Triton X-100), and processed for ChIP as described below.3) for 15 minutes at room temperature. Cross-links were reversed as described above and ChIP DNA purified using 1.8 volumes Agencourt AMPure XP beads (Beckman Coulter). The following antibodies were used: rabbit anti-NURF301, rabbit anti-H3K4me3 , rabbit anti-CP190 [ChIP was performed as described in  with theti-CP190 . DNA forDrosophila genome (BDGP R5/dm3 Assembly) in color space using Bowtie [Drosophila genome (BDGP R5/dm3 Assembly) using the bioscope mapping tool (Life Technologies). Reads were then filtered for high quality reads where read quality was greater than 15 using Samtools [SOLiD reads for nucleosome mapping were mapped to the g Bowtie  and filtg Bowtie . To deteSamtools . ChIP peSamtools  and MACSSamtools . PublicaSamtools  were usew1118 and Nurf3012 wandering third instar larvae were staged using the blue-gut method and hemocytes isolated in batches of 50 larvae as described previously [http://www.R-project.org) and the gcrma and limma libraries of Bioconductor version 3.0 (http://www.bioconductor.org). Expression values were computed using gcrma [eviously , 38. mRNeviously , 38 and ng gcrma . Array dDrosophila S2 cells using a modification of the protocol of Wysocka and colleagues [2, 5% Glycerol, 0.5 mM DTT, 1 mM Sodium Metabisulphite, 1x Protease inhibitors ) for 1 hour on ice with gentle swirling. Extracts were clarified by centrifugation at 100,000 g for 1 hour at 4°C. SNF was dialysed against extraction buffer adjusted to 100mM KCl. Protein G-conjugated Dynabeads (Invitrogen) were blocked by incubation in blocking buffer ) and antibodies bound by incubation overnight at 4°C. SNF was diluted into bind buffer  incubated with antibody-coated Protein G-conjugated Dynabeads for 2.5 hrs with rotation at 4°C. Beads were washed four times, 10 minutes each, using wash buffer . Bound proteins were eluted by boiling in 1XSDS-PAGE sample buffer. 5% input run as a loading control for all IPs.Soluble nuclear fraction (SNF) was prepared from lleagues  to prepa2, 0.5mM CaCl2 0.1% Tween-20) containing the respective enzymes at 37°C for 10 minutes, followed by three further washes in wash buffer. Bound proteins were eluted as described above.Additional IPs were performed using soluble chromatin prepared according to the protocol of Wysocka and colleagues . Nuclei Antibodies used were rabbit anti-NURF301, rabbit anti-CP190 , mouse aFor confirmation of microarray expression data, mRNA was isolated from wild-type and mutant hemocytes using μMacs columns according to manufacturer’s instructions  and reverse transcribed by Superscript II (Invitrogen) at 42°C. Primer sets used are listed in S1 Fig(A) Schematic illustrating a Hilbert plot. These are continuous space-filling curves in which one-dimensional data  is mapped in two dimensions allowing visualization of all data simultaneously. In this procedure a unit square is progressively divided into smaller quadrants and the one-dimensional line corresponding to each chromosome arm folded such that it passes through the center of each quadrant. The iterative folding has the advantage of allowing all data to points to be observed simultaneously and preserves locality, points that co-localize on the line typically co-map on the curve. In the example two features are plotted—in red (closely juxtaposed arrays of peaks), and in green peaks that are well separated from their neighbours. When the entire chromosome arm is viewed as in a genome browser window, it is impossible to discriminate between these two features due to the low resolution of the whole chromosome arm view. However, if these features are plotted as a Hilbert plot, as the linear arm is folded through smaller and smaller quadrants resolution increases and it becomes possible to discriminate closely juxtaposed and well separated features. In this context, extended domains of nucleosome re-organization would result in clusters of shifts being detected as extended clusters/domains on the Hilbert plot. (B) Hilbert plots of NURF-dependent nucleosome shifts on all chromosome arms.(TIF)Click here for additional data file.S2 Fig(A) Distribution of nucleosome shifts relative to chromosome arms compared with genome average of features. (B) Promoter distribution of nucleosome shifts compared with genome average of features. (C) Gene body distribution of nucleosome shifts compared with genome average of features. (D) Distribution of nucleosome shifts downstream of terminators compared with genome average of features.(TIF)Click here for additional data file.S3 FigNurf301 mutation affects expression of transcription factors or chromatin regulators that could indirectly affect nucleosome organisation in Nurf301 mutant hemocytes GO Molecular Function assignments (Flybase) were used to identify 667 targets of interest. Venn diagram indicates respective sub-categories of molecular function. (B) Expression of this set of 667 genes was analysed in Nurf301 mutant hemocytes relative to control wild-type hemocytes. Eight genes display elevated expression in Nurf301 mutant hemocytes, twelve show reduced expression. Of these, nine are male-expressed genes and, as nucleosome profiles were analysed in female hemocytes, these could be excluded from the analysis. None of the remaining eleven genes have known functions in control of global chromatin organisation or nucleosome positioning suggesting that the effects of Nurf301 mutants on nucleosome organisation are unlikely to be mediated through indirect effects on expression of other factors. (C) No change in expression of transcription factors previously analysed for effects on nucleosome organisation was observed. Expression of selected genes previously identified to be up-regulated (up-regulated controls) or down-regulated (down-regulated controls) in Nurf301 mutant hemocytes are displayed as controls. Change in expression is listed as log fold change in Nurf301 mutant hemocytes relative to wild-type hemocytes.(A) To distinguish whether (TIF)Click here for additional data file.S4 FigDrosophila TFs Asense, BEAF, Biniou, Broad, Brinker, Caudal, Crocodile, Deformed, Engrailed, Ets were determined using MEME and averaged nucleosome probability plots flanking predicted TF sites generated for both wild type (WT) and Nurf301 mutant populations. Five categories of nucleosome organization around TF sites could be distinguished. Dyad position of the +1 nucleosome is labeled. (B) Averaged profile plots of predicted TF-binding sites relative to the TSS.(A) Predicted binding sites for (TIF)Click here for additional data file.S5 FigDrosophila TFs Ftz, Hr46, Mef2, Repo, Runt, Scute and Zerknult were determined using MEME and averaged nucleosome probability plots flanking predicted TF sites generated for both wild type (WT) and Nurf301 mutant populations. Five categories of nucleosome organization around TF sites could be distinguished. Dyad position of the +1 nucleosome is labeled. (B) Averaged profile plots of predicted TF-binding sites relative to the TSS.(A) Predicted binding sites for (TIF)Click here for additional data file.S6 FigNurf301/E(bx) mutants.To discriminate whether nucleosome profiles were affected by clustering of TF binding sites, sites for (A) Snail, (B) Antp, (C) Stat and (D) Su(Hw) were divided into those with at least 2 sites within 50 bp (clustered sites) or those that did not possess neighbouring sites (isolated). Averaged nucleosome probability plots flanking these sites generated for both wild type (WT) and (TIF)Click here for additional data file.S7 FigNurf301 mutants. (B) TSSs were categorized based on core promoter motifs associated with DREF/TRF2 targets, the DRE and Ohler Boxes 1,5,6,7,8. Averaged nucleosome probability plots flanking the TSS of each category generated for both wild type (WT) and Nurf301 mutants. Dyad position of the +1 nucleosome is labeled.(A) TSSs were categorized based on core promoter motifs associated with developmentally regulated transcripts, TATA-box, initiator (INR) element, TCT motif, Motif Ten Element (MTE) and the downstream core promoter element (DPE). Averaged nucleosome probability plots flanking the TSS of each category generated for both wild type (WT) and (TIF)Click here for additional data file.S8 FigNurf301 mutants at (A) all TSSs, and TSSs with (B) paused polymerase or (C) active not stalled polymerase. Dyad position of the +1 nucleosome is labeled.TSSs were categorized as stalled or active . Averaged nucleosome probability plots flanking the TSS were generated for both wild type (WT) and (TIF)Click here for additional data file.S9 FigNurf301 mutant hemocytes at all CTCF sites ordered according to CTCF signal. CP190 signal is shown for comparison. (B) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at CTCF sites that contain CP190 ordered according to CP190 signal. CP190 and CTCF signal is shown for comparison. (C) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at CTCF sites that lack CP190 ordered according to CTCF signal. CTCF signal is shown for comparison. Graph shows averaged nucleosome probability at CTCF sites that either contain (+CP190) or lack (-CP190) in wild-type and Nurf301 mutant backgrounds.(A) Heatmap of nucleosomes in wild-type and (TIF)Click here for additional data file.S10 FigNurf301 mutant hemocytes at all Su(Hw) sites ordered according to Su(Hw) signal. CP190 signal is shown for comparison. (B) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at Su(Hw) sites that contain CP190 ordered according to CP190 signal. CP190 and Su(Hw) signal is shown for comparison. (C) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at Su(Hw) sites that lack CP190 ordered according to Su(Hw) signal. Su(Hw) signal is shown for comparison. Graph shows averaged nucleosome probability at Su(Hw) sites that either contain (+CP190) or lack (-CP190) in wild-type and Nurf301 mutant backgrounds.(A) Heatmap of nucleosomes in wild-type and (TIF)Click here for additional data file.S11 FigNurf301 mutant hemocytes at all BEAF sites ordered according to BEAF signal. CP190 signal is shown for comparison. (B) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at BEAF sites that contain CP190 ordered according to CP190 signal. CP190 and BEAF signals are shown for comparison. (C) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at BEAF sites that lack CP190 ordered according to BEAF signal. BEAF signal is shown for comparison. Graph shows averaged nucleosome probability at BEAF sites that either contain (+CP190) or lack (-CP190) in wild-type and Nurf301 mutant backgrounds.(A) Heatmap of nucleosomes in wild-type and (TIF)Click here for additional data file.S12 FigNurf301 mutant hemocytes at Mod(mdg4)2.2 sites that contain CP190 ordered according to Mod(mdg4)2.2 signal. Mod(mdg4)2.2 and CP190 signals are shown for comparison. (B) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at Mod(mdg4) sites that contain CP190 ordered according to Mod(mdg4) signal. CP190 and Mod(mdg4) signal is shown for comparison. (C) Heatmap of nucleosomes in wild-type and Nurf301 mutant hemocytes at Mod(mdg4) sites that lack CP190 ordered according to Mod(mdg4) signal. Graphs show averaged nucleosome probability at Mod(mdg4)2.2 and Mod(mdg4) sites that either contain (+CP190) or lack (-CP190) in wild-type and Nurf301 mutant backgrounds.(A) Heatmap of nucleosomes in wild-type and (TIF)Click here for additional data file.S13 FigNurf301 mutant hemocytes at CP190 sites ordered according to CP190 signal. (B) CTCF, Su(Hw), Mod(mdg4) signals are shown for comparison. (C) BEAF and Mod(mdg4)2.2 signal is shown for comparison. Graph shows averaged nucleosome probability at CP190 sites in wild-type and Nurf301 mutant backgrounds.(A) Heatmap of nucleosomes in wild-type and (TIF)Click here for additional data file.S1 TableList of primers used in RT-PCR analysis of gene expression levels.(DOCX)Click here for additional data file.


Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.  •Comprehensive genome-scale resource for studying embryonic blood cell specification•cis elements driving differential gene expressionGenome-scale definition of •A gene regulatory network model for hematopoiesis aiding reprogramming experiments•Analysis suggests a role for TEAD factors in hematopoietic specification Goode, Obier, Vijayabaskar et al. isolate cells at six different stages of hematopoietic differentiation, starting from embryonic stem cells, and perform a comprehensive multi-omics analysis of this developmental pathway. The data identify regulators of hematopoietic specification and highlight the minimum requirements for the reprogramming of non-blood cells to blood. Cellular identities in multicellular organisms are defined by their individual gene expression programs and are established in a series of cell fate changes starting from pluripotent cells of the embryo. The information on the balanced and coordinated up- and downregulation of gene expression is encoded in our genome and is read by transcription factors (TFs), which interact with the epigenetic regulatory machinery to program the chromatin of lineage-specific genes into active and inactive states. To understand the mechanisms by which TFs establish and maintain specific transcriptional programs, it is essential to investigate developing biological systems, as illustrated by studies in non-vertebrate models .Spi1), and its target, the Csf1r growth factor receptor gene  , which pIn this study, we surveyed the global transcriptional journey from the ESC to the terminally differentiated state of macrophages via blood precursor cells by generating data for RNA sequencing (RNA-seq), DNase sequencing (DNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) for histone marks and 16 different TFs across six sequential developmental stages. To facilitate access across the wider scientific community, we have integrated all genome-scale datasets into an online resource with advanced browse, search, and analysis capabilities. We have exploited our datasets to assemble a core regulatory network model that was able to inform the design of TF-mediated reprogramming strategies for the production of blood cells from fibroblasts. Furthermore, computational analysis of regulatory elements revealed the nature of TFs involved in stage-specific priming of distal elements, and informed functional validation experiments identifying TEAD/YAP interaction as a stage-specific regulator of early murine blood specification in vitro and in vivo. Finally, we identified TEAD target genes and their associated pathways, thus significantly enhancing our understanding of the signaling processes driving embryonic blood cell development.Brachyury GFP reporter (+/Flk1−), which then progress to the hemangioblast (HB) stage (Bry:GFP+/Flk1+) with smooth muscle, endothelial, and hematopoietic potential, followed by the hemogenic endothelium (HE) stage that has both endothelial and hematopoietic potential (CD41−/Tie2+/Kit+). HE cells then undergo the endothelial-hematopoietic transition (EHT) involving a shape change, after which they are fully committed to blood (CD41+ cells). CD41+ cells were further differentiated to generate CD11b+ macrophages (MAC). From purified cells we determined global gene expression profiles by RNA-seq and mapped the full set of cis-regulatory elements at each developmental stage by global DNaseI hypersensitive site (DHS) mapping (DNaseI-seq). We used ChIP-seq to generate global maps of TF binding for key regulators across this entire developmental pathway as well as global patterns of H3K4me3, H3K9ac, H3K27ac, and H3K27me3 histone modifications to investigate how TFs programmed the chromatin landscape. TFs were chosen according to the cell type in which they were expressed  for dynamic alterations in promoter state are shown in Runx1). The promoters of some genes highly expressed in macrophages indeed transit through a poised state, which in many cases is already evident in ESCs . PU.1 binding shows significant overlap with DHS patterns in HPs but is also found at sites that only become hypersensitive in MACs. This suggests that PU.1 can prime MAC-specific regulatory regions already in early multi-potent progenitors, lending weight to the finding that it is capable of opening chromatin .We next correlated the statistical significance of the dynamics of distal DHS patterns with dynamic gene expression patterns B. This aOur next analysis determined the combinatorial pattern of TF-DNA interactions driving target gene expression at key stages of blood development. We therefore interrogated the 31 expression clusters F to asceTo uncover the hierarchy of transcription factors driving blood specification forward, we generated gene regulatory network (GRN) representations connecting all 16 TFs analyzed by ChIP-seq, with separate representations for all six stages of development. To visualize different features, we illustrated multiple different data types within a single GRN representation at each locus . AnnotatCebpb, Elk4, Gata2, Lmo2, Meis1, Runx1, and Tal1, which display open or poised chromatin at their promoters, but also bind Gfi1b, Gata1, and Spi1, whose promoters are organized in closed-unmarked/repressed chromatin. As early as the HB stage, several hematopoietic regulator genes are upregulated, including Tal1 and Lmo2, which exhibit autoregulation and co-regulate multiple genes. These include Fli1 and Meis1, both of which are upregulated upon differentiation into HE. This stage is characterized by the involvement of LMO2, TAL1, and FLI1 (and in some cases MEIS1) in co-regulating genes for multiple hematopoietic TFs, revealing a densely connected GRN composed of potential feedback loops, which is likely to set the stage for the next step of hematopoietic commitment.In ESCs all four pluripotency TFs participate in a highly connected core network circuit and already at this stage bind loci for hematopoietic TFs, including Gfi1, Gfi1b, and Runx1, and nine out of ten bind to Cebpb and Tal1 . LMO2, TAL1, and to some extent FLI1 continue to co-bind and at this stage all target genes are shared with GFI1, consistent with the results shown in Lmo2 or Meis1, both of which are strongly upregulated. While Gata1 is upregulated by a combination of GFI1/LMO2/TAL1, Gata2 is bound by C/EBPβ, TAL1, LMO2, FLI1, GATA1, GFI1, and GFI1B and is downregulated, uncovering a potential feedback mechanism regulating this TF within the network.The HP stage shows highest expression for many of the key hematopoietic TFs, with binding events being complex and combinatorial. All ten TFs tested at this stage bind to Gata2 and Tal1 and a strong increase in the expression of PU.1 and C/EBPβ, which dominate global binding patterns. Within the GRN both TFs already share many target genes in HPs, including all genes encoding for experimental TFs . Nine of these loci  are also bound by FLI1 at this stage where Lmo2 and Tal1 are downregulated. Taken together, our datasets provide deep insights into the regulatory processes that control the dynamic rewiring of network connections during blood cell specification and differentiation. In the remaining part of this article we provide examples of how these data can be used to gain insights into the regulation of hematopoietic specification.In macrophages, part of the HP-specific network is deconstructed with the further downregulation of early hematopoietic regulator genes such as Fli1 and Gata2. Moreover, these factors synergize in driving hematopoietic development in zebrafish . These experiments demonstrate that the integrated analysis of time course TF binding, chromatin accessibility/modification, and expression data enables to highlight those factors that are on top of the hierarchy of tissue specification and are involved in the activation of the majority of genes governing lineage-specific gene expression programs. We believe that this principle will be applicable in multiple developmental settings.A number of recent publications reported a variety of TF combinations capable of generating blood cells via the reprogramming route . We reasebrafish . We ther factors A and ens factors B. We the factors B and mea factors C. These We next used our dataset to identify transcriptional regulators of blood cell specification. We reasoned that cell-stage-specific regulators would leave their mark in the epigenome by occupying their respective binding motifs within cell-stage-specific DHS. To capture all relevant regulatory regions at the genome-wide scale, we used our chromatin accessibility data to perform a pairwise comparison of distal DHS from one cell type with all others as outlined in Drosophila  culture when the emergence of blood cells occurs  were the most likely to succeed in this task and which ones would not. However, the actual molecular mechanism by which this occurs requires further investigation. E-Box motifs are widespread in the genome and are used by a variety of factors; we therefore suggest that the interaction with the bridging molecule LMO2  is cruciould not , and wheOur study provided an example of how our data resource can be used to advance our understanding of hematopoietic specification, in this case by using the differential analysis of enriched motifs in DHS. This type of analysis confirmed the role of known factors (such as RUNX1) but also identified a number of potential cell-stage-specific regulators of hematopoietic specification, leading to the identification of motifs for the Hippo-regulated TEAD TFs enriched specifically in MES, HB, and HE. Immunostaining analyses confirmed the presence of YAP in the nucleus of cells at stages prior to the EHT in both ESCs and embryo-derived cells, but not thereafter. Our functional studies show that the interaction of TEAD factors and the YAP co-factor in the nucleus at this early stage is strictly required for the formation of hematopoietic precursor cells from both sources.cis-regulatory elements may respond to Hippo signaling before, but not after the EHT. We have previously shown that genes associated with TAL1 and FLI1 binding in the HE are highly enriched for genes regulating cell shape and focal adhesion. During the EHT RUNX1 is strongly upregulated and relocates TAL1 and FLI1 to new binding sites after the EHT , HB cells were (Bry+/Flk1+), HE cells were Tie2+/cKit+/CD41−), and HPs were CD41+. Macrophages were isolated by differentiation of CD41+ cells to CD11b-expressing cells.A mouse ES cell line carrying a brachyury (Bry) GFPFor each stage of differentiation cells were sorted, crosslinked, and stored either as frozen cells (histone modification ChIP) or nuclei (TF ChIP) for subsequent ChIP assays, performed as described previously . A full DNaseI and ChIP samples were amplified and sequenced according to the manufacturer's instructions. Some samples  were processed using an ABI SOLiD 4 sequencer, and subsequently either the Illumina 2G Genome Analyzer or the Hi-Seq 2000 were used.RNA preparation and sequencing were performed as described previously .One to three million freshly sorted cells were digested with DNaseI enzyme as described in detail previously  and sizeE14.5 murine wild-type fibroblasts (MEFs) or from iTal1-2A-GFP transgenic mice carrying rtTA and TRE-TAL1 cassettes allowing inducible expression of TAL1 upon addition of doxycycline were prepared as described previously . Reprogr+/FLK1− mesoderm-enriched population was cultured for 48 hr with or without verteporfin. Expression of Fgf5, Bry, and Flk1 was measured by RT-PCR. FLK1+/CD41− HE-enriched cells were sorted from E7.5 embryos by fluorescence-activated cell sorting (FACS) and cultured for 4 days on irradiated OP9 in HE media, and hematopoietic differentiation of HE cells was measured by staining with a CD41-PE antibody. CD45+ committed hematopoietic cells were sorted from E10.5 embryos and cultured on OP9 with verteporfin or DMSO. Growth and survival of hematopoietic cells was assessed by FACS after staining with a CD45-fluorescein isothiocyanate antibody.Verteporfin was added to day-1, -2, -3, or -4 EB cultures at a final concentration of 9.6 μM, and EB-derived cells were stained with CD41-PE and CD45-brilliant violet 421 (Becton Dickinson) and analyzed by flow cytometry. BRYE7.5 embryos were fixed in 4% paraformaldehyde, mounted, and stained with rabbit Pan-TEAD  (1:100), rabbit YAP  (1:100), or purified anti-mouse Tie2  (1:100). Secondary antibodies were Alexa Fluor 488 goat anti-rat immunoglobulin G (IgG)  and Alexa Fluor 647 F(ab′)2 fragment of goat anti-rabbit IgG (H + L) .A detailed description of all bioinformatics methods can be found in D.K.G., N.O., M.L., M.L.A.L., A.J.L., M.F., K.B., M.C., K.W., J.G., and M.H. performed experiments; M.S.V., H.R., S.A.A., and P.C. analyzed data. The senior authors  jointly obtained funding, conceived, and directed the study, and, together with D.K.G., wrote the paper. Within the consortium, D.R.W. directed data analysis/integration and creation of the website; G.L. and V.K. oversaw cell differentiation and purification, RNA-seq data generation, reprogramming and the in vivo and inhibitor studies; B.G. oversaw the transcription factor ChIP data generation and further data analysis; and C.B. was the project coordinator and oversaw chromatin data generation as well as further data analysis and integration.


Transcription termination is a fundamental process in which RNA polymerase ceases RNA chain extension and dissociates from the chromatin template, thereby defining the end of the transcription unit. Our understanding of the biological role and functional importance of termination by RNA polymerase II and the range of processes in which it is involved has grown significantly in recent years. A large set of nucleic acid-binding proteins and enzymes have been identified as part of the termination machinery. A greater appreciation for the coupling of termination to RNA processing and metabolism has been recognized. In addition to serving as an essential step at the end of the transcription cycle, termination is involved in the regulation of a broad range of cellular processes. More recently, a role for termination in pervasive transcription, non-coding RNA regulation, genetic stability, chromatin remodeling, the immune response, and disease has come to the fore. Interesting mechanistic questions remain, but the last several years have resulted in significant insights into termination and an increasing recognition of its biological importance. Saccharomyces cerevisiae, where one process operates to terminate short non-coding transcripts and another involves components of the polyadenylation (polyA) machinery and nucleases such as Xrn2 to terminate the synthesis of mRNA precursors. Recent reviews have described some of these fundamental mechanisms and factors involved in termination by RNA polymerase II (pol II)6. Here, we will focus on the role of the termination of transcription by pol II in a variety of biological contexts and describe how new discoveries have helped elucidate longstanding questions as well as contributed to the establishment of new paradigms. It has become clear that transcription termination occupies a critical role as a regulator of cellular processes. This process and the termination machinery occupy an increasingly important place in human health and disease.Transcription termination is the complex and tightly regulated process in which polymerase stops RNA chain elongation and dissociates from the end of transcription units. A multiplicity of termination factors, which assemble into a number of complexes, govern the biogenesis of various types of transcripts including messenger RNA (mRNA), small nuclear RNA, small nucleolar RNA (snoRNA), and long non-coding RNA (lncRNA). Much of what we know has been learned from studies inallosteric andtorpedo models. In its simplest form, the first posits that as elongating pol II encounters a polyA signal, a physical change in the complex is triggered that provokes termination7. The torpedo model suggests more specifically that termination is facilitated by a nuclease brought to the transcriptvia the polyA signal and associated RNA processing machinery. After the co-transcriptional endonucleolytic cutting of the primary transcript in preparation for its polyadenylation, a nuclease engages the 5’ end of the 3’ portion of the RNA that remains polymerase associated, and digestion of this RNA ‘stump’ in the 5’ to 3’ direction enables the nuclease to chase down pol II, whereupon termination is triggered by an undetermined mechanism8. The difference between these two models may be reduced to the following question: what are the changes in pol II that persuade it to go from its default mode of continuing elongation into stopping and departing from the template? Although there has been some controversy over the relative acceptance of these two hypotheses, the two models are not mutually exclusive, and indeed a combination of both mechanisms has been proposed11.Two prevailing hypotheses for how pol II terminates transcription have guided experiments for almost three decades; these are the so-calledin vitro, a polyA signal is sufficient to induce elongation complex disassembly independent of transcript cleavage12. These authors propose that there is an important conformational shift that can be blocked by α-amanitin, implicating pol II itself in this transition. These findings seemingly support an allosteric component to the model,i.e. something happens to pol II following synthesis of the polyA signal in the nascent RNA, but cutting of the RNA is not needed. However, identifying that change in pol II has remained elusive. In an almost reciprocal study, it had been shown that exonucleolytic degradation of the transcript by the Rat1/Rai1 nuclease (the two subunits that compose yeast Xrn2) was found to be insufficient to dislodge pol II from its templatein vitro13. So, even if a nuclease torpedo is involved in termination, it is not sufficient to complete the process.A recent test of the concept provides new insight showing thatet al. showed that Xrn2 was necessary for proper termination at thousands of genes14. The requirement was not absolute in that loss of Xrn2 delayed, rather than prevented, termination, resulting in longer transcripts. This supports the idea of a kinetic competition in termination, as the Xrn2 torpedo chases the elongating polymerase while Xrn2 digests the transcript from 5’ to 3’. In cells with mutant pol II that was either abnormally slow or fast, it should take Xrn2 less and more time to catch pol II, respectively. Consistent with this prediction, slow pol II led to shorter terminated transcripts, and fast pol II produced longer terminated transcripts. This result can be taken as strong evidence in favor of the torpedo model. Curiously, Xrn2 was found to be important for termination, even for non-coding transcription units whose miRNA and lncRNA products are not acted upon by the cleavage and polyA machinery.The conserved Xrn2 nuclease has been considered a strong candidate to be the torpedo. Recently, Fonget al. mapped the chromatin locations of pol II in aS. cerevisiae strain depleted for Ysh1, the endonuclease that generates the 3’ end that becomes polyadenylated11. The analysis revealed that pol II stops elongation but remains template bound, presumably because the lack of cleavage prevents a nuclease torpedo from gaining access to the elongation complex11. This gives rise to the idea that both an allosteric change resulting from crossing the polyA site and the torpedo effects of Rat1 are necessary for efficient pol II release in yeast.In another test of the need for cleavage of the primary transcript for termination, Schaughencyet al.15 employed a chemical genetic screen introduced by Shokat and co-workers17 to look for substrates of the Cdk9 kinase, an enzyme implicated in transcription as a subunit of the pTEFb elongation factor18. By mutating its active site to accommodate a bulky ATP analog, these investigators could label kinase substrates specific for Cdk9 that could then be tracked by their covalently attached thiophosphate group. One such substrate was Xrn215. The phosphorylated form of Xrn2 could be found on chromatin and the modification was associated with a modest enhancement of its nuclease activity. Inhibition of Cdk9 kinase activity and mutation of the phosphorylated threonine in Xrn2 led to the expected defects in termination predicted by the torpedo model. This new finding incorporates Xrn2 into a cellular signaling pathway. It will be important to learn what cellular events activate and reverse phosphorylation-based control of the torpedo’s activity.While implicated as an essential part of the torpedo model, there is also a growing body of research on Xrn2’s post-translational modification. SansóSchizosaccharomyces pombe20. In this case, heterochromatin formation was dependent on transcription and termination, which was coupled to the nucleolytic elimination of the resulting RNAs. Chalamcharlaet al. showed that the Xrn2 homolog Dhp1 was necessary for premature termination of non-coding RNAs that marked sites of repression of facultative heterochromatin at meiotic genes19. Loss ofdhp1 in an exosome-deficient strain resulted in compromised RNAi-mediated gene silencing, similar to the changes found inago1 mutants. Tuckeret al. also found a similar effect for Dhp1 in silencing and also showed that loss of Dhp1 resulted in defects in meiotic chromosome segregation20. Interestingly, the two groups have different mechanistic explanations for their observations. Chalamcharlaet al. postulate that termination-coupled degradation by the exosome triggers recruitment of the heterochromatin machinery. Tuckeret al. consider the functions of the Dhp1 nuclease in termination and gene silencing as separable, with the termination role not an important aspect of its silencing duty. These reports, and that of Kowaliket al.21, have been productive investigations into the role of transcription termination factors inS. pombe gene silencing, a phenomenon that had been explored previously inS. cerevisiae24 but which seems mechanistically distinct between the yeast species considering the absence of post-transcriptional gene silencing inS. cerevisiae.Xrn2 was also recently shown to regulate chromatin structure to promote meiotic gene silencing during vegetative growth inIn recent years, it has been repeatedly shown that transcription initiation is promiscuous and widespread, with much of the genome capable of being copied into RNA. A corollary to this is the recognition that the termination reaction is an important governor over the transcriptome in its capacity to partition or funnel transcription products into a degradative ‘clean up’ pathway for RNA elimination (such as seen for cryptic unstable transcripts), a maturation pathway for limited processing (such as snoRNAs), or an option to yield spliced and polyadenylated mRNAs.29. Non-coding transcripts that extend in the ‘wrong’ (antisense) direction are terminated, and their degradation is tightly coupled to this process34. Interestingly, the genomic DNA extending in that direction tends to be enriched in polyA signals and hence termination sequences. In contrast, those in the sense or mRNA-yielding direction are depleted of termination potential because they are enriched in splicing signals, which protect transcription from premature termination36. Thus, termination enforces promoter directionality.One form of pervasive transcription is seen as divergent initiation from promoters. Recent work has gone into characterizing transcripts that radiate in both directions from bidirectionally firing promoters37, something that might be expected given the imperfect processivity of pol II engaged in transcribing megabase-long genes.A recent genome-wide study showed that prematurely terminated transcripts are one class of RNA that is cleaned up by nuclease surveillance. Mutation of the human nuclear ribonuclease complex, known as the exosome, revealed the surprising frequency with which prematurely terminated RNAs are generated from the genomeS. pombe suggests that the exosome complex can capture a specific paused and backtracked form of pol II and take it to the termination pathway38. Here, the exosome is thought to directly recognize the 3’ end of the extruded nascent transcript in arrested (‘backtracked’) complexes. Yet unresolved is whether the protein sets employed in conventional (polyA-coupled) pol II termination or short non-coding termination (such as the Nrd1-Nab3-Sen1 system inS. cerevisiae) are required for this mode of stopping transcription.In an interesting flipping of the conventional way of thinking that termination feeds transcripts to the degradation machinery, a study inet al. found that herpes virus inhibits the termination of host, but not viral, transcription39. The resulting readthrough RNAs became observable in a manner similar to those seen in other systems when the exosome or termination machineries become incapacitated by experimental manipulation37. These aberrant, even polygenic, transcripts were not spliced or translated properly, thus resulting in a form of host shut-off that may favor viral gene expression. How widespread the phenomenon of viral corruption of the integrity of the host’s transcription units is, and its mechanistic basis, remains to be elucidated.Not only is the regulation of termination important for cellular genomes but also viruses can antagonize normal transcription termination during infection. Using 4-thiouridine labeling of RNA and ribosome profiling of newly made RNA following infection, Rutkowskiet al. in a human cancer40. Through transcriptome profiling of various renal cell carcinoma samples, they found widespread transcriptional readthrough in many of the samples from patients with some of the worst prognoses. Again, chimeric transcripts spanning transcription units could be identified. TheSETD2 gene, which encodes a histone methyltransferase, was frequently inactivated in these cancer lines. Knockdown ofSETD2 could recreate the readthrough phenotype. Ectopic expression of the wild-type protein could reverse it, thus implicating chromatin modifications in disease-related failure to terminate effectively. Interestingly, Bcl2, an anti-apoptotic protein often elevated in cancer, was upregulated, suggesting a possible mechanism by which mutation leads to loss of proliferative control.Another case of a pathologically defeated transcription termination system was observed by Grossodownstreamofgenes containing transcripts) has been identified42. These transcripts have a 5’ end corresponding to an upstream gene and an aberrant 3’ extension of up to tens of kilobases. Hundreds of DoGs were detected following KCl treatment of neuroblastoma cells whereupon their length and abundance increased. These readthrough products, only some of which became polyadenylated, encountered fewer polyA sites downstream of the coding sequence, which could explain less efficient termination for the DoG transcript. It is not clear what functional role the DoGs play; their strong association with chromatin suggests they become incorporated into the nuclear scaffolding during stress responses. Intuitively, this is an appealing model, as osmotic stress shrinks the cell nucleus and collapses chromatin. DoGs may mitigate such deleterious effects, as recently suggested for a set of non-coding, repeat-containing RNAs43. This finding emphasizes that terminator override plays an important role across physiological states and reveals plasticity in our definition of a transcription unit.Yet another version of a hybrid readthrough transcript called DoGs , is thought to track with elongating pol II and is associated with premature termination of transcription. This mechanism is proposed to link transcription to a specific hotspot for physiologically important mutations. A recent report extends this model and proposes that pervasive transcripts are functionally important for proper class switch recombination and somatic hypermutation in B lymphocytes46. The suggested model is that transcripts in paused, divergently facing elongation complexes become stabilized, offering the complex more of an opportunity to form short DNA-RNA hybrids called R-loops. These loops are part of the transcription termination reaction in which RNA is hybridized to the DNA from which pol II has just departed and can be considered an intermediate on the pathway to disintegration of the transcription bubble and the digestion of the short, anti-sense RNA. B lymphocytes appear to have evolved a mechanism to capture this nucleic acid framework with its exposed single-stranded DNA and make it a target for the AID mutagenesis machinery. In this manner, B cells can focus the genetic changes on the very specific region of Ig proteins that need to vary. We will come back to a discussion of the importance of R-loops in termination in the next section.The transcription elongation complex has been of interest to immunologists for many years owing to transcription-dependent mutations introduced into immunoglobulin (Ig)-encoding genes during somatic hypermutationet al. also present evidence in support of the idea that premature termination provides an opportunity for the single-stranded DNA of the transcription bubble to become a substrate for localized mutation by AID47. They showvia chromatin immunoprecipitation that pol II concentrates in the region that undergoes hypermutation. Subsequent manipulation of elongation factor Spt5 by knockdown, which should facilitate premature termination, revealed that DNA strands in the region acquire a single-stranded character and are mutated at higher frequency. Early termination was suggested by the molar abundance of RNA from the 5’ variable region of the heavy chain locusversus the downstream sequences.Wang48. Typically, a precursor transcript is synthesized that encodes the μ heavy chain constant region exons and, further downstream, the corresponding δ constant region exons. Alternative splicing appends one or the other sets of exons to the VDJ-encoding exons, thereby yielding mRNAs for complete IgM or IgD heavy chains. This regulatory event has been known for some time, although the responsibletrans-acting factors have been elusive50. By engineering a conditional Zfp318 deficiency into the bone marrow lineage of mice, Pioliet al. showed that Zfp318 normally serves to repress an apparent transcriptional termination event at the end of the μ exon series. Thus, there is normally some readthrough into the δ exons, thereby generating a primary transcript that can yield either μ- or δ-encoding mRNA, depending on the splicing pattern48. In the absence of Zfp318, it is suggested that the termination event becomes so strong that transcripts hardly extend into the δ exons, leaving cells with a pool of precursor transcripts ending near the polyA site for the upstream μ exons, and giving B cells no option of making IgD. This was one of the few shifts observed in that transcriptome, showing a very specific consequence following the loss of Zfp318. An independent set of experiments confirmed that incapacitating Zfp318 resulted in a shift from IgD and IgM production to mainly IgM production51. Enderset al. attributed this effect to the rate of pol II elongation and a change in the efficacy of competition between polyadenylation at the end of the μ exonsvs. alternative splicing around them to append the δ exons. It will be interesting to learn how the Zfp318 protein, which may bind nucleic acids and could be a key regulator of this process, operates and if it directly influences a true transcription termination event.In another immunologically interesting system, the Zfp318 protein has been postulated to regulate termination site selection during the transcription of the gene that encodes both the μ and δ heavy chains in B lymphocytes52. However, these structures may also be involved in coordinating chromatin remodeling, as elucidated in a recent study in which repressive chromatin marks were induced over terminators53. In one example, an RNA duplex is suggested to form from anti-sense transcription of the R-loop, which recruits dicer to the region, leading to trimethylation of H3K9 at the termination zone, thereby linking the termination system to chromatin and the RNAi system in human cells.The role of the R-loop in somatic hypermutation and recombination associated with heavy chain class switching is a specialized use of a common feature of the transcription termination zone. One hypothesized function of the senataxin protein, which is the human orthologue of the Sen1 termination factor initially discovered in yeast, is to provoke termination by unraveling the R-loop using a helicase activityet al. show a role for Dicer1 in termination for all three nuclear polymerases, but this function appears to be independent of the rest of the RNAi machinery inS. pombe54.dcr1Δ cells show increased polymerase occupancy at the end of genes, implying stalling of polymerase. Deletion of other RNAi machinery or generating a catalytically dead Dicer1 mutant did not give similar results, suggesting the process is not RNAi mediated. The genes regulated by this mechanism seem to be restricted to those at sites of replicative stress, where high transcription levels result in collision of the transcription bubble and the replication fork. Since DNA-RNA hybrids at these sites are recombinogenic, Dcr1 may be important for maintaining genome integrity, similar to the way senataxin resolves the problem in mammalian cells, although the latter is thought to do so through a helicase activity56. The role for dicer in termination could also be related to prior findings that Rnt1, another RNAse III-type enzyme inS. cerevisiae, can provoke termination57.Interestingly, Castel59. Mutated frataxin exhibits features of heterochromatin, H3K9 methylation, and decreased acetylation of H3 and H4, thus a reduced level of expression is to be expected. However, the altered gene also shares many characteristics of canonical R-loop-terminated genes, including a polyA-signal-like sequence upstream of the expansion, followed by a GU-rich sequence similar to the downstream element of polyA signals. This has led to a proposal that the mutated frataxin allele is the victim of premature termination, which contributes to its low level of expression in patients59. While experimental verification is still needed, this is an interesting model for a role of termination in disease.R-loop-mediated termination is also proposed to play a role in Friedrich’s ataxia. It was suggested that R-loops aberrantly form in the frataxin gene as a result of expansion of GAA repeats in its first intronet al. studied the modification of the repeat region of pol II’s largest subunit60. They found that dimethylation of a specific arginine serves to recruit the survival of motor neuron (SMN) protein to the elongation complex. This protein, in turn, associates with senataxin, which resolves R-loops and recruits the torpedo nuclease Xrn2 during termination. Both SMN and senataxin have been found to suffer mutations in neurodegenerative diseases, again highlighting the importance of the termination reaction in human health and the imperative to understand its molecular basis.In a final case of R-loop involvement in termination, ZhaoOur understanding of the role of transcription termination across biological systems has expanded significantly with recent advances in experimental techniques and with growing interest in the topic across biological systems. New findings have yielded fresh insight into the longstanding questions of termination mechanism and somatic hypermutation and have provided new paradigms with the identification of cases in which the stringency of termination is relaxed during specific regulatory events or accidentally in pathological examples of dysfunction.AID, activation-induced cytidine deaminase; DoGs, downstream of genes containing transcripts; Ig, immunoglobulin; lncRNA, long non-coding RNA; mRNA, messenger RNA; pol II, RNA polymerase II; polyA, polyadenylation; SMN, survival of motor neuron protein; snoRNA, small nucleolar RNA.


Kdm6 gene family, Utx  and Jmjd3 . Male embryos null for active H3K27 demethylation by the Kdm6 gene family survive to term. At mid-gestation, embryos demonstrate proper patterning and activation of Hox genes. These male embryos retain the Y-chromosome UTX homolog, UTY, which cannot demethylate H3K27me3 due to mutations in catalytic site of the Jumonji-C domain. Embryonic stem (ES) cells lacking all enzymatic KDM6 demethylation exhibit a typical decrease in global H3K27me3 levels with differentiation. Retinoic acid differentiations of these ES cells demonstrate loss of H3K27me3 and gain of H3K4me3 to Hox promoters and other transcription factors, and induce expression similar to control cells. A small subset of genes exhibit decreased expression associated with reduction of promoter H3K4me3 and some low-level accumulation of H3K27me3. Finally, Utx and Jmjd3 mutant mouse embryonic fibroblasts (MEFs) demonstrate dramatic loss of H3K27me3 from promoters of several Hox genes and transcription factors. Our results indicate that early embryonic H3K27me3 repression can be alleviated in the absence of active demethylation by the Kdm6 gene family.The early mammalian embryo utilizes histone H3 lysine 27 trimethylation (H3K27me3) to maintain essential developmental genes in a repressive chromatin state. As differentiation progresses, H3K27me3 is removed in a distinct fashion to activate lineage specific patterns of developmental gene expression. These rapid changes in early embryonic chromatin environment are thought to be dependent on H3K27 demethylases. We have taken a mouse genetics approach to remove activity of both H3K27 demethylases of the  Utx and Jmjd3 survive to term and appear phenotypically normal at mid-gestation. We utilize several cell culture models to demonstrate that H3K27me3 is lost from repressed promoters in the absence of active KDM6 demethylation. Our data indicate that KDM6 H3K27me3 demethylation is not essential in the early embryo and that H3K27me3 loss from developmental genes occurs via novel mechanisms.H3K27me3 represses developmental genes at initial embryonic stages. The KDM6 family, comprised of UTX and JMJD3, are the only known proteins that demethylate H3K27me3 and they are hypothesized to catalyze the rapid removal of repressive chromatin in early mammalian development. However, we report that male embryos carrying mutations in both  The mammalian embryo undergoes drastic changes in cellular specification and gene expression programs throughout development. These changes are facilitated by post-translational modifications to histones, which provide an epigenetic mechanism to coordinate initiation and maintenance of lineage specific transcriptional profiles that can be inherited through multiple cellular divisions. In embryonic stem cells and other pluripotent progenitors, crucial developmental genes are maintained in a quiescent state. A bivalent epigenetic signature defines this large class of genes. These promoters are maintained in a repressive chromatin state through histone H3 lysine 27 trimethylation (H3K27me3), however the presence of an active chromatin modification (H3K4me3) suggests that these genes are poised for rapid induction as development dictates H3K27me3 demethylases are members of the KDM6, Jumonji-C (JmjC) domain family of histone demethylases. The three KDM6 proteins, JMJD3 , UTX , and UTY (Y-chromosome), all share a well-conserved JmjC histone demethylation domain −/−Jmjd3 homozygotes exhibit post-natal lethality due to neonatal respiratory deficits −/yUtx hemizygous males survive to adulthood and exhibit a normal lifespan −/−Utx homozygous females and −;Uty−Utx hemizygous males are both mid-gestational lethal with developmental delay and defects in embryonic heart development UTX and JMJD3 are individually involved in early embryonic specification events in cell culture −/y;Jmjd3−/−Utx embryos devoid of KDM6 H3K27 demethylation survived to term. Mid-gestational −/y;Jmjd3−/−Utx embryos appeared phenotypically normal with characteristic features of embryonic day 10.5 (E10.5) embryos. We utilized several model systems  to demonstrate that H3K27me3 can be removed from the promoters of repressed genes in the absence of active KDM6 demethylation. We conclude that KDM6 demethylases are not essential for early embryonic development and that H3K27me3 repression can be alleviated in the absence of active KDM6 demethylation.To study the role of the KDM6 family in early embryonic development we generated mutations designed to eliminate all KDM6 H3K27me3 demethylase activity in the developing mouse embryo. Male Utx and Jmjd3. We previously characterized the generation of the flUtx allele that flanks exon 3 with loxP sites UtxΔ) created a frameshift in the coding sequence and is null for UTX protein. We now characterize a targeted allele, tm1MagJmjd3 (flJmjd3) that integrates loxP sites 5′ to exon 14 and 3′ to exon 20 (Jmjd3Δ) removed the JmjC catalytic H3K27 demethylase domain , prominent fore and hind-limb buds, and developed branchial arches  background ΔUtx and ΔJmjd3 alleles. On a B6 background, Δ/yUtx;Δ/ΔJmjd3 embryos remained viable at both E14.5 and E18.5 timepoints  were crossed with fl/yUtx;fl/ΔJmjd3;VasaCre male mice (with sperm carrying deletion of Utx and Jmjd3) and resulting E10 Δ/yUtx;Δ/ΔJmjd3 embryos  differentiation as outlined in controls . Utxfl/ff the EB . In contf the EB . EmbryoiH3K27me3 . FluoresH3K27me3 . TherefoUtx and Jmjd3 expression was absent, and Uty expression was not diminished in TX treated cells , mesoderm (Flk1), and endoderm , but failed to specify meso-endoderm , mesoderm (Flk1), and endoderm , KO ES , WT RA , and KO RA . Two of the 4 replicates of each group were pooled together and the resulting 2 replicates of each group were subject to high throughput sequencing. H3K4me3 ChIP-seq was also performed on 2 replicates of WT RA and KO RA. The model-based analysis for ChIP-seq (MACS) algorithm identified enrichment peaks of H3K27me3 and H3K4me3 in each group, and edgeR statistical analysis software identified genes undergoing H3K27me3 demethylation in WT (WT ES vs. WT RA) and KO (KO ES vs. KO RA) RA differentiation. Overall, 1044 WT ES promoters (Transcription Start Site: TSS +/−1 KB) and 1141 KO ES promoters demonstrated H3K27me3 peaks. Of these promoters, 945 and 1055 (WT and KO respectively) also had a RA H3K4me3 peak, signifying that the majority of these promoters are bivalent.To study the role of KDM6 in H3K27me3 demethylation, we utilized Retinoic acid (+RA) differentiation of ES cells. As Hox genes lost H3K27me3 in both WT and KO RA differentiation. Tracks of H3K27me3 and H3K4me3 ChIP-seq were uploaded into the UCSC genome browser. With RA differentiation, the proximal Hoxa cluster , Hoxc (Hoxc4-Hoxc6), and Hoxd (Hoxd1-Hoxd4) clusters  and 109 KO promoters (103 genes) that demonstrated significant loss of H3K27me3 with RA differentiation  with man cluster  from Hoxclusters . In addireatment . H3K27mereatment . NotablyKO cells . Utxfl/fcription . H3K27 dcription . One altcription , the proCrabp2, Pax6, Wnt6, Ccnd2, Hoxb1, Foxa1) experienced normal KO RA H3K4me3 up-regulation relative to the ES cell state . EdgeR comparison of WT RA to KO RA identified 161 promoters (147 genes) that exhibited significant (FDR<0.05) reduction in KO H3K4me3 . The majll state . Howevere levels .To better analyze the distribution of H3K27me3 and K3K4me3 we performed a meta-analysis examining the overall distribution of these histone modifications across all promoters with a corresponding MACS peak. These meta analyses  were ploMeta-analysis of promoters exhibiting H3K4me3 reductions in KO RA cells verified that this dataset was significantly deficient in H3K4me3 downstream of the TSS . The H3Kfl/flUtx;fl/flJmjd3;ERCre embryos and treated with tamoxifen as indicated in Hox genes  demonstrated elevated expression levels in MEFs  demonstrated a slight but significant reduction in expression with loss of UTX and JMJD3. Hox H3K27 methylation in fl/flUtx;fl/flJmjd3;ERCre MEFs +TX matched their expression profile as the few distal genes that demonstrated mild expression deficiencies (Hoxa13 and Hoxd13) also exhibited a significant increase in H3K27me3 . Primary MEFs were cultured from E13.5  in MEFs . Followicontrols . Only thH3K27me3 .Δ/yUtx;Δ/ΔJmjd3 embryos to assay function in both establishment and maintenance of a H3K27 demethylated state. Similar to transient TX induced KDM6 loss, Δ/yUtx;Δ/ΔJmjd3 MEFs had significantly reduced expression of more distal Hox genes  in both control and Δ/yUtx;Δ/ΔJmjd3 MEFs , but this may be an artifact of the decreased growth rate of these cells as the H3K27me3 profile of these genes is unaffected. Furthermore, several other transcription factors  also exhibited near-complete loss of H3K27me3 even though all KDM6 demethylases had been removed throughout embryonic development. Notably these genes experience loss of localized H3K27me3 even though total H3K27me3 protein levels  survive through gastrulation and albeit smaller in size, can develop E10.5 features. Therefore, KDM6 is not crucial for alleviating the H3K27me3 repression of genes needed for early embryonic gastrulation events.A tremendous dichotomy exists in the field of H3K27 demethylases. H3K27me3 results in gene repression throughout the early embryo, yet enzymes that catalyze its removal are individually not essential for male embryonic viability. These findings are unexpected given that numerous genes crucial for early embryonic gastrulation events Utx mutant male mice are fertile Hox H3K27me3 demethylation and gene activation Hox H3K27me3 and demonstrate normal transcriptional activation Δ/yUtx;Δ/ΔJmjd3 embryos, KDM6 mutant male ES cells could differentiate normally into all EB germ layers. Mutant female ES cells null for any KDM6 demethylation demonstrated dramatic reduction in both global levels of H3K27me3 (with EB differentiation) and local levels of H3K27me3 from proximal Hox clusters and from promoters of other transcription factors (with RA differentiation). While there was a low level significant H3K27me3 accumulation upstream of these promoters in RA KO cells, these genes were largely cleared of H3K27me3 and experienced normal transcriptional activation, similar to findings in Utx male knockout studies alone Utx and Jmjd3 mutant MEFs demonstrated mild H3K27me3 accumulation and reduced expression only within the most distal regions of the Hox cluster. Similarly, Zebrafish UTX loss of function produces modest deficiencies in distal Hox expression Kdm6 mutant MEFs and may account for altered distal Hox gene regulation. Relative to ES cells, Δ/yUtx;Δ/ΔJmjd3 MEFs exhibited substantial reductions in promoter H3K27me3 of several Hox genes and developmental transcription factors. Thus in the absence of KDM6 H3K27 demethylation, H3K27me3 loss can be both initiated and maintained in developmental situations.Even within individual cellular and organismal models, several studies have produced conflicting reports. UTX mediated H3K27 demethylation is reported to function in cellular reprogramming and germ cell development Our study raises intriguing questions regarding early embryonic removal of H3K27me3. Thus far, only UTX and JMJD3 have demonstrated the ability to demethylate H3K27me3. It is unlikely that another JmjC protein can demethylate H3K27me3. The KDM7 family including JHDM1D and PHF8 can demethylate both H3K9 and H3K27 dimethyl residues, but does not demethylate trimethyl residues Alternatively, H3K27me3 in the early embryo may be replaced by passive mechanisms, as histones can be turned over multiple times within each cell cycle The role of the KDM6 family in development is not clear. Passive H3K27me3 removal may dominate in rapidly dividing cells such as in the early embryo. Active H3K27me3 demethylation may prove more essential for rapid response to specific environmental or developmental cues, particularly in more static cellular populations. Alternatively, rather than facilitating drastic gene induction, H3K27 demethylases may act to fine-tune transcriptional activity to promote accurate robust temporal and spatial patterns of gene expression. Accordingly, UTY associates with a wide array of chromatin and transcriptional machinery that may promote proper gene expression output flUtx allele is described tm1MagJmjd3 (flJmjd3) allele was derived by targeting E14 ES cells. The Jmjd3 targeting construct was generated by BAC recombineering to insert a LoxP site in intron 13 and a FRT-Neomycin-FRT-LoxP cassette in intron 20 of Jmjd3. After successful targeting, the Neomycin cassette was removed by electroporation of a pCAGG-FLP construct. The resulting +/flJmjd3 ES cells were injected into C57BL/6J blastocysts. Resulting chimeras were mated to CD1 females to assess germline transmission, and were maintained on either a mixed CD1 background or were backcrossed to C57BL/6J. ΔUtx and ΔJmjd3 alleles were generated by crossing floxed alleles to the VasaCre transgene that restricts Cre activity to the germline ΔUtx and ΔJmjd3 alleles. To generate higher proportions of desired genotypes in the Utx Jmjd3 genetic interaction cross, +/Δ;Jmjd3+/ΔUtx female mice were mated with either fl/y;Jmjd3fl/Δ;VasaCreUtx or +/y;Jmjd3fl/Δ;VasaCreUtx males. Embryos were PCR genotyped from yolk sac samples for Utx or Jmjd3 and were sexed by a PCR genotyping scheme to distinguish Utx from Uty. All primer sequences are listed in All mouse experimental procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee. The fl/flUtx;fl/flJmjd3;ERCre and fl/yUtx;fl/flJmjd3;ERCre ES cell lines were generated from E3.5 blastocysts in crosses utilizing CAGGCre-ER transgenic mouse line 6 ES cells were cultured in 10 mL of ES culture media lacking LIF on agarose coated Petri dishes. Hanging drop EBs were set up with 25 µL drops of ES cells at a concentration of 20,000 cells per mL. In RA differentiation experiments, ES cells were plated following tamoxifen recovery, and the next day were cultured without LIF in 1 µM Retinoic Acid. E13.5 MEFs were generated as described Gapdh expression and graphed relative to control samples. All primer sequences are listed in 6 cells were sonicated by a Branson Sonifier at 15% duty cycle (0.7 s on 0.3 s off). For some experiments, chromatin was sonicated in a chilled water bath by a Bioruptor sonifier on high setting for 30 s with 60 s rest. Rabbit IgG , anti-H3K27me3 , anti-H3K4me3 , or EZH2  antibodies were used for ChIP. All ChIP primers are listed in RNA was isolated with Trizol and cDNA was synthesized with Multiscribe reverse transcriptase. Gene expression was analyzed by qRT-PCR . All RT-PCR was normalized to http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were then mapped to the B6 genome using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml). Significant enrichment (peaks) were called using MACS (http://liulab.dfci.harvard.edu/MACS/index.html), with the input ChIP-seq datasets for the background model, on pooled replicates. Peak lists were filtered using an FDR cutoff of 0.05. We then identified peaks that were within 1 Kb of transcriptional start sites (TSSs), as annotated in the UCSC genome browser for mm9. Read counts for a particular locus were normalized to the total number of sequence reads generated for each sample. We used edgeR (http://www.bioconductor.org/packages/2.12/bioc/html/edgeR.html) to determine if there was a bias in the number of H3K27me3 or H3K4me3 reads at MACS positive promoters between various samples. Metaplots were drawn using custom Python scripts and R, and t-tests were performed using the average read count per gene within the ranges specified in the text. ChIP-seq datasets were submitted to GEO (accession GSE58391: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58391).ChIP DNA and Input DNA were ligated to Truseq adapters as described Figure S1Jmjd3. (A) Jmjd3 was targeted to introduce a LoxP site in intron 13 and another in intron 20. The FRT site remains after the Neo cassette was removed by FLP recombinase. A schematic of the JMJD3 protein is illustrated above to denote the region that will be removed with Cre recombinase. Splicing from exon 13–21 will introduce a frameshift and stop codon. (B) Verification of the flJmjd3 allele. Southern blotting of WT and targeted +/neoflJmjd3 ES cells with a 3′ probe and XmnI digest demonstrated the expected shift in banding due to a novel restriction site. The neo cassette was removed from targeted ES cells by transfection of a Flp recombinase to create +/flJmjd3. (C) flJmjd3/fl mice were crossed to a germline Cre recombinase to create Δ/ΔJmjd3. A PCR genotyping scheme was designed to distinguish WT, flJmjd3, and ΔJmjd3 alleles in resulting mice. (D) The Jmjd3 deletion is verified by RT-PCR of E18.5 whole embryo RNA. Δ/ΔJmjd3 embryos lack the exons 14–17 product and amplification across exons 13–22 produces a smaller band that corresponds to the expected product lacking exons 14–20. Therefore, the mutant transcript is stable and is expected to produce JMJD3 lacking the JmjC histone demethylase domain. (E) Homozygous Jmjd3 mutant embryos are born, but fail to breathe as indicated by their blue color. (F) Δ/ΔJmjd3 embryos appear phenotypically normal at E10.5.Targeted mutation of (TIF)Click here for additional data file.Figure S2fl/ΔUtx;fl/ΔJmjd3;VasaCre female mice (with oocytes carrying deletion Utx and Jmjd3) were crossed with fl/yUtx;fl/ΔJmjd3;VasaCre male mice (with sperm carrying deletion of Utx and Jmjd3) and embryos from the cross were dissected at E10. Δ/yUtx;Δ/ΔJmjd3 (A′) and Δ/ΔUtx;Δ/ΔJmjd3 (A″) embryos from this cross phenocopy embryos derived from crosses with maternal contribution of UTX and JMJD3 (Utx and Jmjd3 of embryos derived in the cross in Utx and Jmjd3 floxed (fl) alleles to deletions (Δ).Maternal deletion of UTX and JMJD3 does not enhance embryonic mutant phenotypes. (A) nd JMJD3 . (B) Gen(TIF)Click here for additional data file.Figure S3Δ/yUtx;Δ/ΔJmjd3 male embryos.  Quantitative RT-PCR of KDM6 members  and indicated Hox genes  in all indicated combinations of E10.5 male Utx and Jmjd3 mutant embryos relative to +/yUtx;+/ΔJmjd3 controls (N≥4 samples per genotype). (C) Histones were extracted from +/yUtx;+/ΔJmjd3 or Δ/yUtx;Δ/ΔJmjd3 embryos and fluorescent western blots are illustrated for H3K27me3 relative to total H3 loading control. (D) Quantitation of the western blot in part C, H3k27me3 values were normalized to total H3 to calculate H3K27me3%. (E) H3K27me3 immunofluorescence of E10.5 +/yUtx;+/ΔJmjd3 or Δ/yUtx;Δ/ΔJmjd3 myocardium. (F) H3K27me3 immunofluorescence of E10.5 +/yUtx;+/ΔJmjd3 or Δ/yUtx;Δ/ΔJmjd3 ISL1 positive motor neurons in the proximal spinal chord. (G) Histones were extracted from +/yUtx;+/ΔJmjd3 or Δ/yUtx;Δ/ΔJmjd3 MEF lines and fluorescent western blots are illustrated for H3K27me3 relative to total H4 loading control. (H) Quantitation of western blots for H3K27me3%, from +/yUtx;+/ΔJmjd3 (grey) or Δ/yUtx;Δ/ΔJmjd3 (black) MEFs relative to total H4. Significant increases in protein levels are indicated .Analysis of mid-gestation (TIF)Click here for additional data file.Figure S4ERCre induction in Utx and Jmjd3 floxed ES cell lines. (A) PCR genotyping of a fl/flUtx;fl/flJmjd3;ERCre ES cell line left untreated (−TX) or treated with tamoxifen for 2 days (+TX) for Utx, Jmjd3, Sex, and presence of Cre. Controls PCR reactions included are Utx or Jmjd3 floxed alleles (fl), Utx or Jmjd3 deleted alleles (Δ), Male DNA , Female DNA , Cre negative DNA (Neg), Cre positive DNA (Pos), water (H). (B) Western blot of a fl/yUtx;fl/flJmjd3;ERCre ES cell line left untreated (−TX) or treated with tamoxifen for 2 days (+TX) and the GT1/yUtx ES line for UTX (αUTX) or a loading control (αGAPDH). The GT1/yUtx line should trap all Utx products. Therefore, the band present in all lanes at 140 KD is a non-specific band rather than an alternative product because it is not reduced in the GT1/yUtx sample. (C) RT-PCR for the 3 lines described in part B across the Utx region deleted by the floxed allele (Exon 3) and the JmjC domain. Note that all transcripts containing the JmjC domain are reduced in the GT1/yUtx RNA. (D) Western blot using a second independent UTX antibody.Efficiency of tamoxifen (TIF)Click here for additional data file.Figure S5Utx and Jmjd3. (A) As an alternative to formation of EBs in mass culture of ES cells, hanging drop EBs were generated from a defined cell number in a defined drop volume. (B) Fluorescent western blot analyzing loss of H3K27me3 with EB differentiation. H3K27me3 is blotted relative to total H4. (C) Quantification of the blot in Utx and Jmjd3. fl/yUtx;fl/flJmjd3;ERCre ES cells were treated with TX for 3 days, then plated at low density to allow picking of single cell colonies. A clone of Δ/yUtx;Δ/ΔJmjd3 cells was propagated over 3 weeks and several passages, then differentiated into a typical day 4 EB structure relative to the parental fl/yUtx;fl/flJmjd3;ERCre line. (E) Expression analysis of the ES cells (diff −) and day 8 EBs (diff +) described in Utx, Jmjd3, Gata6 (endoderm), and Flk1 (Mesoderm). (F) fl/flUtx;fl/flJmjd3;ERCre ES cells were also treated with TX to generate single cell clones  were differentiated with RA for 2 days (diff +) and RT-PCR compared expression of Utx, Jmjd3, Hoxa1, and Hoxb1 relative to the parental fl/flUtx;fl/flJmjd3;ERCre line.Hanging drop EB analysis and constitutive deletion of (TIF)Click here for additional data file.Figure S6Hoxb, Hoxc, and Hoxd clusters. (A–C) UCSC genome browser view of Hoxb (A), Hoxc (B), and Hoxd (C), clusters. Illustrated are Input (black), WT ES H3K27me3 ChIP (dark green), KO ES H3K27me3 ChIP (dark red), WT RA H3K27me3 ChIP (light green), KO RA H3K27me3 ChIP (light red), WT RA H3K4me3 ChIP (blue), KO RA H3K4me3 ChIP (orange), and MACS defined enrichment peaks are illustrated as black bars underneath each track.UCSC genome browser view of (TIF)Click here for additional data file.Figure S7Utx and Jmjd3 mutant cells. (A) Western blot analyzing levels of MLL complex members ASH2L and RBBP5 in fl/flUtx;fl/flJmjd3;ERCre ES cells −TX or +TX. (B) Western blot levels of PRC2 component EZH2 and RBBP5 in WT ES cells, 2 day RA differentiated ES cells, or 2 day differentiated EBs. (C) EZH2 ChIP-qPCR of fl/flUtx;fl/flJmjd3;ERCre ES cells  or after 2 days of RA treatment  left untreated (green) or pre-treated with tamoxifen (red). An IgG control ChIP is illustrated as white bars. Quantitative PCR of an EZH2 negative locus  was utilized for comparison to RARE, Hoxa1, Hoxa2, Hoxb1, Hoxb2, and Hoxd9.Analysis of MLL and PCR2 complexes in (TIF)Click here for additional data file.Figure S8Utx and Jmjd3 mutant MEFs. (A) RT-PCR of Hoxa3, Hoxc4, Hoxa13, or Hoxd13 in ES cells, retinoic acid treated ES cells, or MEFs. (B) Western blot of EZH2 relative to RBBP5 loading control from +/yUtx;+/ΔJmjd3 or Δ/yUtx;Δ/ΔJmjd3 MEFs. (C) Quantitation of western blots for EZH2 or H3K4me3 from +/yUtx;+/ΔJmjd3 (grey) or Δ/yUtx;Δ/ΔJmjd3 (black) MEFs. Significant increases in protein levels are indicated .Analysis of (TIF)Click here for additional data file.Table S1Promoters experiencing H3K27me3 loss in ES to RA differentiation. EdgeR statistical comparison for significant (FDR<0.05) loss of H3K27me3 normalized sequence reads in WT RA relative to WT ES (sheet 1) and KO RA relative to KO ES (Sheet 2) across the TSS +/− 1 KB. Some genes have multiple alternative promoters, so genes that are duplicated within the list are highlighted in red. Also denoted is whether the promoter has a corresponding RA H3K4me3 MACS peak (listed as 1).(XLSX)Click here for additional data file.Table S2Promoters experiencing H3K4me3 reduction in KO RA differentiation. EdgeR statistical comparison for significant (FDR<0.05) loss of H3K4me3 normalized sequence reads in KO RA relative to WT RA across the TSS +/−1 KB. Some genes have multiple alternative promoters, so genes that are duplicated within the list are highlighted in red. Also denoted is whether the promoter has a corresponding WT or KO ES H3K27me3 MACS peak (listed as 1).(XLSX)Click here for additional data file.Table S3KO RA H3K27me3 MACS peaks that have enrichment in H3K27me3 sequence reads over WT RA. EdgeR statistical comparison for significant (FDR<0.05) increase in H3K27me3 normalized sequence reads in KO RA MACS peaks relative to WT RA across the KO RA peak center +/−0.5 KB. MACS peaks are listed with the nearest neighboring gene. Some genes are associated with multiple MACS peaks, so genes that are duplicated within the list are highlighted in red. Also denoted is whether the promoter of that gene has a corresponding reduction in KO RA H3K4me3 (listed as 1).(XLSX)Click here for additional data file.Table S4Primers used in the study.(XLSX)Click here for additional data file.