Differentiation of one life-cycle stage to the next is critical for survival and transmission of apicomplexan parasites. A number of studies have shown that stage differentiation is a stochastic process and is associated with a point that commits the cell to a change over in the pattern of gene expression. Studies on differentiation to merozoite production (merogony) in T. annulata postulated that commitment involves a concentration threshold of DNA binding proteins and an auto-regulatory loop.In this study ApiAP2 DNA binding proteins that show changes in expression level during merogony of T. annulata have been identified. DNA motifs bound by orthologous domains in Plasmodium were found to be enriched in upstream regions of stage-regulated T. annulata genes and validated as targets for the T. annulata AP2 domains by electrophoretic mobility shift assay (EMSA). Two findings were of particular note: the gene in T. annulata encoding the orthologue of the ApiAP2 domain in the AP2-G factor that commits Plasmodium to gametocyte production, has an expression profile indicating involvement in transmission of T. annulata to the tick vector; genes encoding related domains that bind, or are predicted to bind, sequence motifs of the type 5'-(A)CACAC(A) are implicated in differential regulation of gene expression, with one gene (TA11145) likely to be preferentially up-regulated via auto-regulation as the cell progresses to merogony.We postulate that the Theileria factor possessing the AP2 domain orthologous to that of Plasmodium AP2-G may regulate gametocytogenesis in a similar manner to AP2-G. In addition, paralogous ApiAP2 factors that recognise 5'-(A)CACAC(A) type motifs could operate in a competitive manner to promote reversible progression towards the point that commits the cell to undergo merogony. Factors possessing AP2 domains that bind (or are predicted to bind) this motif are present in the vector-borne genera Theileria, Babesia and Plasmodium, and other Apicomplexa; leading to the proposal that the mechanisms that control stage differentiation will show a degree of conservation. Babesia, Plasmodium and Theileria) to change from one life-cycle stage to the next is critical for establishment of infection and transmission to new hosts. Stage differentiation steps of both Plasmodium and Theileria are known to involve stochastic transition through an intermediate form to a point that commits the cell to generate the next stage in the life-cycle. In this study we have identified genes encoding ApiAP2 DNA binding proteins in Theileria annulata that are differentially expressed during differentiation from the macroschizont stage, through merozoite production (merogony) to the piroplasm stage. The results provide evidence that the ApiAp2 factor in Theileria that possesses the orthologue of the Plasmodium AP2-G domain may also operate to regulate gametocytogenesis, and that progression to merogony is promoted by the ability of a merozoite DNA binding protein to preferentially up-regulate its own production. In addition, identification of multiple ApiAP2 DNA binding domains that bind related motifs within and across vector-borne Apicomplexan genera lead to the proposal that the mechanisms that promote the transition from asexual to sexual replication will show a degree of conservation.The ability of vector-borne Apicomplexan parasites ( Plasmodium and Theileria differentiation systems has provided evidence for an intermediate position, with progression towards or reversal from a point that commits the cell to generate the next life-cycle stage CACA[C/T][A/T] [T. annulata genome, enrichment analysis found that there is a depletion of the motif in the 400 bp upstream region of IGRs of genes down-regulated from macroschizont to the merozoite stage, 13% vs 25% (P < 0.005). There was also evidence of enrichment 400 bp upstream of the ATG start codon in IGRs of genes up-regulated in merozoite, 31% vs 24% (P = 0.06) and piroplasm stages, 46% vs 24% (P < 0.05). No significant enrichment or depletion was obtained on analysis of the region 100 bp upstream of the translation initiation ATG codon. A positive Pearson Correlation (R = 0.93) was observed for expression of the TaAP2-me1 gene (TA11145) and the average profile of macro-mero most up-regulated gene set enriched for ACACAC in their upstream IGRs (−9) with a conserved core motif of AATGTGTAA. This motif shows similarity with the reverse complement of the ACACAC motif, and identity with the motif previously identified by MEME in 5' IGRs of T. parva and T. annulata [P. falciparum conserved TGTGT(G/A)(A/T) motif, and like its Plasmodium counterpart has a widespread distribution in non-coding regions of the genome. A role as a binding site for regulatory nuclear proteins other than transcription factors was proposed [The orthologue of the AP2 domain of TaAP2.me1 , TaAP2.me1 (TA11145D), TaAP2.me2 (TA12015D) and TaAP2.me3 (TA16485D). The fusion proteins were then used in electrophoretic mobility shift assays against biotinylated double-stranded motif probes. As shown , as a shift was obtained using a probe containing the core motif TCTACA identified for the orthologous m domain . The EMSle shift .P. falciparum orthologue of the AP2 domain of TaAP2.me1 (TA11145) has been shown to bind the motif ACACAC [TA11145). The TaAP2.me1 AP2 domain fusion protein (TA11145D) generated a strong shift with this probe (TA11145).The f ACACAC . To testis probe . To confTA11145 gene that is up-regulated during merogony.To determine if native nuclear factors could bind to the probe representing the double ACACAC type motif,, EMSA was performed using extracts from parasite-enriched nuclear fractions derived from macroschizont-infected cells (Day 0) and infected cells undergoing differentiation to the merozoite (Day 9). TA11145 gene is recognised by the AP2 binding domain encoded by the gene indicates that its expression may be auto-regulated. This is of interest as the stochastic model of merogony in T. annulata predicted that the commitment to differentiate is reached via the capacity of regulators of differential gene expression to auto-regulate. It was investigated, therefore, whether there is a greater occurrence of the motif in the IGR upstream of the domain encoding TA11145 gene relative to the other 21 AP2 encoding genes. Seven ACACAC type motifs were found in the IGR upstream of TA11145 (including the double motif separated by five nucleotides) and six were conserved upstream of the T. parva orthologue (TA05055 and TA08375) and an average of 0.95 motifs per AP2 gene IGR was obtained. Both ApiAP2 genes with three motifs were classed in the same expression profile as TA11145 (up from macroschizont (Day 0) to merozoite (Day 9)).Demonstration that the ACACAC motif in the IGR upstream of the thologue . In contPlasmodium AP2G factor  [TA13515), including a double motif separated by 6 bp (core A to core G). These three motifs were conserved in the upstream region of the T. parva gene encoding the orthologous domain (TP02_0497) [Auto-regulation has also been predicted for genes encoding the _143750) ,14. Scre02_0497) .P. falciparum, multiple AP2 domains have been shown to bind to motifs rich in CA di-nucleotides, two variants being ACACAC and CACACA [Theileria orthologues of Plasmodium AP2 domains that bind (A)CACAC(A) can be identified in the phylogenetic analysis performed by Balaji et al., [TA11145, TA07100 and TA02615 were found to be orthologues of the domains encoded by PF3D7_0802100 (MAL8P1.153), PF3D7_0420300 (PFD0985w.D1) and PF3D7_1305200 (PFL13_0026), while the domain encoded by TA19920 was placed in a position in the tree intermediate between TA07100 and TA11145 but without a clear domain orthologue indicated in Plasmodium. To analyse this group of domains in more detail, a maximum likelihood tree was constructed with the four P. falciparum domains and the putative orthologous domains from T. annulata, T. parva and T. orientalis CACAC(A) binding domain, encoded by PF3D7_1456000 (PF14_0533), did not fit into an orthologous group. This was supported by reciprocal BLAST analysis with no clear orthologue identified for the TA19920 AP2 domain in Plasmodium or the PF3D7_1456000 domain in Theileria.In d CACACA . We term et al., . Thus doientalis . The treBabesia, Theileria and Plasmodium. Thus, there are, at least, two phylogenetically related AP2 domains conserved in vector-borne Apicomplexa that bind (A)CACAC(A) type motifs, with orthologous members of a third ApiAP2 domain (represented by TA02615) possibly binding to this, or a closely related, motif.An alignment of AP2 domains in the orthologous groups represented by TA11145 and TA07100 domains, respectively Figs  and S8 iTA11145) encodes the domain shown to bind (A)CACAC(A) and is up-regulated during merogony; while the gene (TA07100) encoding the other AP2 domain in T. annulata that is strongly predicted to bind (A)CACAC(A) did not show a significant difference. To validate the difference in expression qRT-PCR was performed for the TA11145, and TA01700 genes using RNA derived from D7 and D7B12 cells cultured at 37°C (Day 0) and during progression to merogony at 41°C (Day 4 and Day 7). The results indicate that RNA levels of TA11145 were significantly higher  in D7 vs D7B12 cells at the Day 0 time-point and that this difference is exacerbated following culture at 41°C: 8.7 fold (log2) at Day 4 and 11 fold (log2) at Day 7  higher in D7 cells at Day 0, Day 4 and Day 7, respectively. This validates that the TaAP2.me1 gene (TA11145) is expressed at a higher level in a cell line competent for differentiation and that up-regulation is independent of a heat shock response. Thus, the expression level of TA11145 relative to TA07100 is clearly altered in favour of TA11145 in the D7 cell line and this bias is increased during progression towards merogony (Day 4 and Day 7).Cell line D7B12 is severely attenuated in its ability to undergo differentiation to the merozoite stage . Howeverat Day 7 . In contWithin the life cycles of Apicomplexan parasites, transition from stages that undergo multiple rounds of asexual replication to stages that promote life-cycle progression and parasite transmission are regulated by critical cellular differentiation events. Evidence generated across apicomplexan genera indicates that these transition points operate on a stochastic basis and that stage-differentiation steps can be programmed to occur in a time-dependent manner ,13,14,42T. annulata in vitro it was proposed that the stochastic differentiation mechanism involves the build up of DNA binding protein(s) relative to their DNA template to generate a commitment point involving an auto-regulatory loop [From previous investigation of merogony in ory loop . The aimTo search for motifs and DNA binding factors associated with stage differentiation, expression data representing the sporozoite stage, a macroschizont (Day 0) to merozoite stage (Day 9) differentiation time-course, and piroplasm stage was generated. Comparative analysis produced two sizeable lists of genes up- or down-regulated during differentiation to the merozoite . Both lists contained genes predicted from previous studies. For example, genes encoding rhoptry proteins, Tams1  and cystT. parva [Theileria. The data also indicated differences in the pattern of expression for distinct genes across the differentiation (merogony) time-course, implying regulation of gene expression via multiple factors that operate in a temporal order. Similar results have been reported for related Apicomplexa [T. annulata.Several general observations could be made from the temporal expression patterns. Firstly, genes expressed at a high level in one stage were often indicated as expressed at a low level in the preceding or subsequent stage. This supports previous studies reporting that merozoite genes are expressed at the macroschizont stage , and thaT. parva . Thus, rcomplexa ,33,45 anT. annulata microarray data was therefore screened for predicted DNA binding proteins that showed differences in expression level between macroschizont and merozoite stage. Genes encoding four putative AP2 domain DNA binding proteins were found to show elevation of mRNA levels between macroschizont and merozoite.Recent studies on DNA binding proteins have provided strong evidence of their involvement in the regulation of Apicomplexan stage-differentiation events. The T. annulata AP2 domain amino acid sequences with the orthologous domains in Plasmodium or Cryptosporidium (TA12015) showed that there was greater identity for orthologues across genera than between these four paralogous domains within T. annulata. This allowed prediction that each of the four domains bound different target motifs. In addition, consensus motifs bound by the AP2 domains in Plasmodium or Cryptosporidium were predicted for the orthologous domain in T. annulata. EMSA performed with fusion protein domains encoded by TA11145, TA13515 and TA16485 demonstrated that this prediction was valid. Based on previous studies [TA13515 and TA11145, however, do show parallels with data obtained for their orthologues in Plasmodium.Comparison of the four up-regulated  studies ,39,40 thTA13515 in Plasmodium is the domain of the AP2-G factor that is essential for commitment to gametocyte production [TA13515 (92% identity) and binds the motif GxGTACxC. As expected, the Theileria AP2.g domain specifically bound the GxGTACxC motif. This motif is enriched in the upstream region of genes up-regulated from merozoite to piroplasm stage in T. annulata, with no enrichment in any other subset of stage-regulated genes. Furthermore, in a similar manner to Plasmodium AP2-G, a GTAC core motif is present in three copies (one double motif) in the upstream region of the TaAP2.g gene (TA13515) and T. parva orthologue (TP02_0497), indicating putative auto-regulation of gene expression. A role in regulating gene expression as the parasite differentiates into the piroplasm stage is highly likely. The piroplasm stage has been postulated to be equivalent to gametocytes and it is known that a sexual phase occurs within the tick [Plasmodium [T. annulata, however, indicates that up-regulation is primarily liked to developmental events. This conclusion is supported by demonstration of significantly elevated expression in the D7 cell line vs D7B12 cell line when both lines were cultured for 7 days at 41°C  is the orthologue of the PF3D7_0802100 (MAL8P1.153) domain in P. falciparum and TGME49_071030 in T. gondii [Babesia CACAC(A) type motifs detected by its orthologous domain in Plasmodium [Plasmodium and Toxoplasma genomes [P. falciparum [T. gondii [TA07100 in T. annulata and PF3D7_0420300 (PFD0985w.D1) in P. falciparum [The AP2 domain encoded by . gondii , and dom[Babesia . Our fin genomes ,49,50, b. gondii , one of lciparum . The motPlasmodium, four AP2 domains have been shown to recognise (A)CACAC(A) type motifs, three of which are closely related to each other [PF3D7_0802100 and PF3D7145600) are predicted to auto-regulate [Theileria, with at least two domains (encoded by TA11145 and TA07100) displaying a level of similarity to their Plasmodium orthologues that indicates binding to the same or similar motif. Auto-regulation is predicted for TA07100 and TA11145 . However, since there is only one (A)CACAC(A) motif upstream of TA07100 relative to seven in TA11145 there is a much stronger prediction of auto-regulation for TA11145. Auto-regulation of this gene was supported by demonstration that the encoded AP2 domain binds specifically to a probe representing a double (A)CACAC(A) type motif present in the upstream IGR. Multiple auto-regulatory sites were also reported for P. falciparum AP2-G [In ch other . Expressregulate . Based oum AP2-G . One posum AP2-G .et al. [T. annulata encoding AP2 domains that are predicted to bind (A)CACAC(A) motifs overlap during differentiation to the merozoite (see T. annulata [TA11145. Thus expression of TA11145 at the macroschizont stage may be influenced by a stoichiometric relationship between competing factors that bind to (A)CACAC(A) motifs and promote low (repressed) or elevated (activated) gene expression. Following placement at 41°C, an elevation in protein levels relative to DNA template occurs and generates a skewed increase in TA11145 expression over time via an auto-regulatory loop. This loop would be promoted by preferential binding of the AP2 domain encoded by TA11145 to multiple (A)CACAC(A) sites in its own upstream region. One prediction of the model is that the relative level of competing factors would differ between parasite lines attenuated or competent for a stage-differentiation event. This appears to be the case for genes encoding AP2 domains that bind the (A)CACAC(A) motif, with a significant increase in the level of TA11145 expression, relative to TA07100, in an infected cell line that is able to undergo differentiation to the merozoite compared to a line which has lost this ability (Multiple domains that recognise (A)CACAC(A) type motifs allow speculation that different AP2 factors could bind to the same promoter and potentially compete for binding if co-expressed. Indeed, the TA11145 AP2 domain can bind to the motif predicted for the TA07100 domain . In addiet al.  indicateoite see , with TA ability .Further experimental data are required to validate, refute or modify the above model. Nevertheless, it could account for a number of findings common across stage-differentiation events of different Apicomplexan genera. These include, low level expression of genes in the life-cycle prior to the stage where they are expressed at a high level ,12–14; aRecognition of closely related binding motifs by multiple DNA binding proteins shared across genera operates to regulate developmental gene expression in higher eukaryotes, with auto-regulation and competition for binding sites evident . For exaS1 TableT. annulata AP2 binding domains are in bold, mutated motifs are underlined.Motifs predicted for (PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableDifferential expression between cell lines was identified by RP analysis of microarray data (FDR < 0.05). Genes encoding AP2 domains predicted to bind (A)CACAC(A) motifs are highlighted with a blue bar.(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig2 using three independent secondary structure prediction programs: Psi-Pred [Regions of predicted secondary structure are indicated above the alignment and were predicted with PhyrePsi-Pred , SSPro [Psi-Pred  and JNetPsi-Pred . * ident(PDF)Click here for additional data file.S3 FigP. falciparum orthologous domain are shown in blue, a significant Pearson correlation coefficient value is indicated for each plot.ApiAP2 gene expression profile is shown in red, while the average profile of putative target genes possessing the motif bound by the (PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 FigLetters denote shift positions detected in both infected and uninfected cells at Day 0 (37°C), shift B was only detected in extracts derived from infected cells undergoing merogony .(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 FigP. falciparum has been shown to bind an (A)CACAC(A) type motif. Regions of predicted secondary structure are indicated above the alignment and were predicted with Phyre2 using three independent secondary structure prediction programs: Psi-Pred [The domain in Psi-Pred , SSPro [Psi-Pred  and JNetPsi-Pred . * ident(PDF)Click here for additional data file.S9 FigFold-change in expression between cell lines was computed at Day 0 (macroschizont) and Day 4 and Day 7 points of a time-course of differentiation to the merozoite; * denotes significant  fold-change elevated expression in D7 vs D7B12.(PDF)Click here for additional data file.S10 FigArrow denotes the shift position obtained with GST-TA11145D, the more minor shift obtained with GST-TA13515D may be indicative weaker/partial recognition of the probe.(PDF)Click here for additional data file.


FLO1, is representative of a distinct subset of subtelomeric genes that are robustly repressed by the Cyc8–Tup1 complex. We have examined Cyc8–Tup1 localisation, histone acetylation and long-range chromatin remodelling within the extensive FLO1 upstream region. We show that Cyc8–Tup1 is localised in a DNase I hypersensitive site within an ordered array of strongly positioned nucleosomes around − 700 base pairs upstream of the transcription start site. In cyc8 deletion mutant strains, Tup1p localisation is absent, with concomitant histone hyperacetylation of adjacent regions at the FLO1 promoter. This is accompanied by extensive histone depletion across the upstream region and gene activation. The yeast histone deacetylases, Hda1p and Rpd3p, occupy the repressed FLO1 promoter region in a Cyc8–Tup1 dependent manner and coordinate histone deacetylation, nucleosome stabilisation and gene repression. Moreover, we show that the ATP-dependent chromatin remodelling complex Swi–Snf occupies the site vacated by Cyc8–Tup1 in a cyc8 mutant. These data suggest that distinctly bound Cyc8–Tup1 cooperates with Hda1p and Rpd3p to establish or maintain an extensive array of strongly positioned, deacetylated nucleosomes over the FLO1 promoter and upstream region which inhibit histone acetylation, block Swi–Snf binding and prevent transcription.We demonstrate that the yeast flocculation gene,  •Cyc8–Tup1 repression activity is enriched at chromosome subtelomeric regions.•FLO1 gene is subject to chromatin-mediated repression by Cyc8–Tup1.The subtelomeric •Cyc8–Tup1 promotes long-range nucleosome positioning and histone deacetylation.•Hda1p and Rpd3p cooperate with Cyc8–Tup1 to facilitate this repressive chromatin.•Swi–Snf directs extensive nucleosome remodelling when Cyc8–Tup1 is absent. Through association with DNA-bound transcription factors in the nucleus, these often large complexes determine the repressed or active state of gene promoters by altering the chromatin structure or the recruitment and competence of the RNA polymerase II holoenzyme. One of the earliest co-repressors of gene regulation identified in the yeast, Cyc8–Tup1 is involved in many pathways in yeast such as glucose, starch and oxygen utilisation, osmotic stress, DNA repair, mating, sporulation, meiosis and flocculation However, the mechanism of Cyc8–Tup1 repression is still unclear. Cyc8–Tup1 can interact directly with several components of the transcriptional machinery such as the kinase-cyclin pair Srb10/11p, the essential holoenzyme component Srb7p, and the Mediator subunit Med3p in vitro, and the domain that is required for this interaction overlaps with the transcription repression domain in vivoThe Cyc8–Tup1 complex also interacts with chromatin and can influence nucleosome positioning, histone acetylation and deposition of the histone variant, Htz1p FLO1 gene is regulated by the TUP1 and CYC8 (SSN6) gene products and is the dominant member of the FLO family of genes FLO1 gene expression causes flocculation which is the Ca2 +-dependent, nonsexual aggregation of yeast cells FLO1 transcription start site. Thus, the chromatin in which the yeast FLO1 promoter resides has a dynamic structure controlled by remodelling events which form the background in which promoter regulation takes place FLO1 gene represents a paradigm for chromatin-mediated regulation of gene transcription, and offers an amenable model system in which to investigate the mechanism of action of Cyc8–Tup1.The yeast FLO1 gene represents a specific subset of genes subject to robust repression by Cyc8–Tup1 working cooperatively with the histone deacetylases, Hda1p and Rpd3p. We demonstrate that Cyc8–Tup1 localisation is focused at a hypersensitive site in the FLO1 upstream nucleosome array. Removal of Cyc8–Tup1 leading to activation of the FLO1 gene is coincident with long-range modulations of the histone H3 and H4 acetylation pattern and opening of the chromatin structure. Additionally, in the absence of Cyc8–Tup1, the occupancy of the HDACs, Rpd3p and Hda1p decreases in parallel with an increase in the occupancy of Swi–Snf at the site formerly occupied by Cyc8–Tup1. We show that Rpd3p and Hda1p are co-ordinately required for full repression but do not do so by enhancing Cyc8–Tup1 binding.In this study, we propose that the FLO1 promoter and upstream chromatin requires distinctly bound Cyc8–Tup1and Swi–Snf and is mediated by histone acetylation.These data suggest that the long-range remodelling of the 22.1S. cerevisiae strains were in the S288C background , INT-V (H3), or STE6  sequences. The STE6 site was used as a negative Cyc8–Tup1 binding site control. The STE6 gene promoter is bound by Cyc8–Tup1 in Matα cells, but is free of Cyc8–Tup1 in Mata cells. All strains used in this study were Mata. Primers used are listed in ChIP was performed as previously described 2.3HindIII) and − 2177 to − 1781 bp (BsrBI). Chromatin restriction enzyme accessibility was assayed as previously described SpeI) and − 1670 to − 1878 bp (SpeI and RsaI).Chromatin DNase I analysis was performed as previously described 2.4Protein lysates were prepared using a TCA lysis buffer 2.5ACT1 and the complete FLO1 coding region FLO1 values were normalised to ACT1. For RT-qPCR analysis of FLO1 mRNA, RNA was extracted from cells by the hot phenol method ACT1 RNA. Primers used are listed in Northern analysis of mRNA expression was performed as previously described 2.6Exponentially growing cells were resuspended to an equal cell density in YPD or YPD containing 100 mM EDTA (control). Equal volumes of cells were aliquoted into a tissue culture plate and agitated by shaking 2.7S. cerevisiae ORF and chromosomal locations (SGD), to calculate fold changes between wild-type and TUP1 deletion strains. Tup1 protein binding sites were derived from ChIP-seq data generated by Wong and Struhl (NCBI accession no. SRA044839.1) using the algorithms and parameters described by the authors − 4 (in one case compared with 10− 2) were assigned to the closest translational start codon (ATG) of annotated ORFs. Combination of ChIP and expression data, as well as plotting of graphs, was carried out in Excel using VBA custom scripts. For the Hda1p ChIP data analysis, lists of genes scoring above the False Discovery Rate (FDR) for each of three probe locations, as well as those shared between probes, were collated from Venters et al., 2011 tup1 mutant and wt for each subset of 90 locations, which were each plotted as an average trend over 1000 bp with standard deviation (SD). The SD for random regions (not shown for clarity) was very similar to the SD for non de-repressed genes.Expression array data generated by Chen et al. 3FLO1 gene promoter resides within a 5 kb chromatin region under the influence of long-range remodelling by the Cyc8–Tup1 and Swi–Snf complexes FLO1 gene was typical of a Cyc8–Tup1 repressed gene We previously reported that the subtelomeric 3.1tup1 deletion strain to examine the correlation between Tup1p promoter occupancy and gene repression attributable to the TUP1 gene TUP1 resulted in less than two-fold increase in transcription of 251 (79%) of these genes  retrieved a set of 92 genes that were distinguished by very low levels of transcription in the wild-type strain  and measured the respective Tup1p and Cyc8p abundance and occupancies.To investigate the molecular mechanism of sequence A. The recyc8 and tup1 deletion mutants confirmed the stability of each subunit was unaffected by the absence of the other (FLO1 in wt strains was completely absent in cyc8 strains (cyc8). This suggests Tup1p is recruited in the context of the Cyc8–Tup1 complex confirming observations at the RNR2 and STE6 genes tup1 strains. Thus, Cyc8p remains bound to the FLO1 promoter in the absence of Tup1p where it either increases in occupancy or is more amenable to ChIP analysis.Western blot analysis of Tup1p and Cyc8p levels in the respective he other . ChIP an strains B, cyc8. FLO1 repression by measuring FLO1 transcription in cyc8 and tup1 single and double deletion mutant strains. The data revealed that de-repression of FLO1 transcription was greatest in cyc8 strains compared to tup1 strains and wt , using the indirect end-labelling technique. FLO1 in repressed wild-type chromatin  (EGS) and formaldehyde as described in Materials and Methods. Occupancies at each site were expressed as the ratio of IP/input and normalised to the IP/input ratio from a strain containing untagged HDAC proteins. (B) Comparison of Tup1p ChIP analysis using either formaldehyde or EGS and formaldehyde as the cross-linking reagents. Tup1p ChIP analysis was performed in wt cells cross-linked with either formaldehyde (CH2O) or after sequential EGS and formaldehyde cross-linking. Tup1p occupancy at RNR2 and STE6 in the differently treated cells was compared. The results show that the use of the EGS did not alter the binding profile of Tup1p at RNR2 and STE6 promoters which were used as positive and negative Tup1p binding sites respectively.FLO1 gene promoter. (A) Diagram of the amplicons used in chromatin immunoprecipitation analysis covering a region up to 1800 base pairs upstream (− 1800), labelled by the distance (in bp) from their midpoints to the FLO1 translation start site (+ 1). Cross-linked chromatin fragments from wild-type (wt), CYC8 and RPD3 HDA1 deleted strains (cyc8 and rpd3 hda1 respectively) were immunoprecipitated with antibodies against Snf2p and the DNA content analysed by qPCR. Occupancies were normalised to the TEL-VI control region. The results represent the average from three to four independent experiments with bars representing SEM. The data from the amplicon centred around the − 585 bp FLO1 promoter region are shown in (B) Myc-Snf5p occupancy at the − 585 bp FLO1 promoter region was also measured by ChIP analysis in wt and cyc8 strains harbouring genomic copies of SNF5 tagged with a 9-myc epitope alongside relevant controls. Occupancies were normalised to the TEL-VI region as a control. The results represent the average from three independent experiments, with bars representing SEM and support the Snf2p ChIP data.Snf2 proteins are concentrated at a single location at the de-repressed The following are the Supplementary data related to this article.Conceived and designed the experiments: ABF YT SP. Performed the experiments: ABF SB MC. Contributed reagents/materials/analysis: ABF SB MC YT SP. Wrote the paper: ABF SP.10.13039/100004440Wellcome Trust [grant number 092533/Z/10/Z to ABF and grant number 045117 to SP]. SP acknowledges 10.13039/501100000268BBSRC funding. Funding for open access charge: [10.13039/100004440Wellcome Trust/092533/Z/10/Z].This work was funded by the


Tip60 is important for early mouse development and mouse embryonic stem cell (mESC) pluripotency, the function of Tip60 as reflected in a genome-wide context is not yet well understood.Tip60 (KAT5) is the histone acetyltransferase (HAT) of the mammalian Tip60/NuA4 complex. While Gel filtration of nuclear mESCs extracts indicate incorporation of Tip60 into large molecular complexes and exclude the existence of large quantities of “free” Tip60 within the nuclei of ESCs. Thus, monitoring of Tip60 binding to the genome should reflect the behaviour of Tip60-containing complexes. The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)-containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation. The comparison of genome-wide binding profiles of Tip60 and c-Myc, a somatic cell reprogramming factor that binds predominantly to active genes in mESCs, demonstrate that Tip60 and c-Myc co-bind at 50–60 % of their binding sites. We also show that the Tip60 complex binds to a subset of bivalent developmental genes and defines a set of mESC-specific enhancer as well as super-enhancer regions.Our study suggests that the Tip60 complex functions as a global transcriptional co-activator at most active Pol II promoters, co-regulates the ESC-specific c-Myc network, important for ESC self-renewal and cell metabolism and acts at a subset of active distal regulatory elements, or super enhancers, in mESCs.The online version of this article (doi:10.1186/s13072-015-0039-z) contains supplementary material, which is available to authorized users. The histone acetyl trasferase (HAT), Tip60  belongs to the MYST family of HATs that play key roles in acetylation of histones and other nuclear factors and thus influence chromatin structure and transcription regulation in the eukaryotic nucleus . The defTip60 complexes have three enzymatic functions: (1) a histone H2A/H4 acetlytransferase activity, (2) an ATP-dependent H2AZ.H2B dimer exchange activity and (3) a helicase activity , 8. SeveTip60 gene in mouse results in pre-implantation lethality at embryonic day 3.5 [Homozygous knockout of the  day 3.5 . Additio day 3.5 . Moreove day 3.5 . Interes day 3.5 . Chromat day 3.5 , though  day 3.5 , 15. Mor day 3.5 , 15.Interestingly, an additional HAT, Mof  was shown to be required for early mouse development and mESC pluripotency , 17. RecIn order to investigate whether Tip60 acts mainly in large molecular complexes in mESCs, nuclear extracts were prepared and subjected to gel filtration that allows separation of macromolecules of different sizes. The analysis of the gel filtration by western blot indicated that Tip60 is present mainly in fractions eluting around 2 MDa that may correspond to endogenous Tip60 complex about 1.3 MDa) , we compared the binding of Tip60 and Pol II, and the appearence of the H3K4me3 mark at all ENSEMBL TSSs  by k-meaTo better characterize the genome-wide Tip60 binding around promoters, we selected all Pol II positive genes and analysed the global distribution of Tip60 around annotated TSSs Fig. b. SurpriGenome-wide binding studies in differentiated human cells show a global co-localization of HATs and acetylated histones at transcriptional active promoters , 25. TheTo better dissect the function of these complexes, we compared the binding distribution of Tip60, Nsl1 and Msl1 at all Pol II positive promoters Fig. d. These Tip60 is known to interact with and regulate various transcription factors as a transcriptional co-factor . c-Myc iTo further characterize Tip60 function, we categorized the binding of Tip60 to different genomic regions. When the MACS identified 7693 Tip60 binding peaks were annotated to promoter-TSS, 5′- or 3′-untranslated regions (UTRs), exons, introns and intergenic regions, about 42 % of all high-confidence Tip60 binding peaks were found at promoter-TSS regions  or enriched at enhancer sites (H3K4me1 and H3K27ac) , 38. We The heatmap in Fig. Interestingly, two of the last Tip60 positive clusters within the heatmap Fig. a show loAs our above analyses suggested that Tip60 complexes could bind to enhancers; we wanted to examine the total enrichment of Tip60 at all known enhancer sites. To this end, we have taken annotated enhancer sites from mESCs . Since aAs two of the so-called ‘super enhancer’ regions of the Nanog gene and the enhancer of the Klf4 gene are bound by Tip60, but not by Nsl1 and Msl1 Fig. a, b, we In this study, we analysed the genome-wide binding of Tip60-containing complexes to understand their role in transcription regulation in mESCs. Our gel filtration analyses show that Tip60 incorporates into large molecular complexes in mESCs, as previously described in other systems, and that heterogeneous populations of Tip60-containing complexes exist, which might dynamically change their association with p400 and/or other subunits. However, our observations that there is very little free Tip60 within the nuclei of mESCs indicates that the ChIP binding profiles obtained with anti-Tip60 antibodies represent mostly the behaviour of the corresponding endogenous Tip60 complexes.Our study demonstrates that the majority of genome-wide Tip60 binding occurs at promoter regions, where it co-localizes with Pol II, H3K4me3 and DHS sites. Importantly, Tip60 bound genes are expressed and the Tip60 enrichment positively correlates with gene expression levels. Interestingly, using an unbiased clustering method of all mapped Tip60 reads against ENSEMBL TSS, and we observed that all transcriptionally active genes (Pol II positive) show Tip60 enrichment. Genome-wide binding and knock-down studies of Tip60-p400, NSL and MSL HAT complexes reported the binding of these complexes at Pol II positive genes and in mESCs , 19. WheThe frequent overlap of Tip60 and c-Myc binding strongly indicates a role of Tip60 complexes within the Myc-centred regulatory pluripotency network in mESC , 33. MorDrosophila, mESCs and human cells have revealed that c-Myc targets at least 10–15 % of all cellular promoters [Tip60 complexes preferentially acetylate histone H4 . Acetylaromoters . Neverthromoters  and (2) romoters . Our finromoters .Drosophila [Interestingly, the genome-wide Tip60 complex binding profile peaks at regions that are about 200 bps downstream of TSSs  are predicted to be involved in regulation of development, metabolism and proliferation of mESCs. Based on high Mediator occupancy, some of these enhancers can be clustered to 231 super-enhancer regions [Moreover, our results indicate that Tip60 complexes may be involved in gene expression regulation by occupying a set of previously identified ESC-specific enhancers . Tip60 e regions , which l regions . Thus, iHuman Tip60 is known to play a wide role in transcriptional regulation . The ideOur genome-wide binding analyses suggest that the Tip60 complex has a very broad role in regulating transcription in mESCs, which is in agreement with the observation that Tip60 and several other Tip60-complex subunits are required for mESC maintenance . In contOverall, we establish that Tip60-complexes are present at promoter regions of active RNA polymerase II genes and that half of Tip60 binding sites overlap with binding of the somatic cell reprograming factor, c-Myc, known to regulate an ESC specific transcriptional module. Importantly, Tip60, NSL and MSL coactivator HAT complexes have a genome-wide overlap at many active genes, but their specific functions might be reflected in their distinct binding profiles around the TSSs. Moreover, Tip60 complexes define a subset of bivalent developmental genes and a subset of ESC-specific enhancers. Thus, our study suggests that the Tip60 complex is important for mESC pluripotency by acting as a global transcriptional co-factor at active genes and distal regulatory elements.2) in DMEM  , 15 % foetal calf serum (PAA), 5uM LIF, 50 mM ß-mercaptoethanol , penicillin  and streptomycin (10 mg/ml) , 200 mM l-glutamine  and 1× non-essential amino acids . To work under feeder-free conditions cells were treated with 1 mg/ml Collagenase and 2 mg/mL Dispase  and cultivated for one passage without feeder cells on 0.1 % gelatine plates. Experiments were conducted with E14.wt cells at passages 26–29.The ES cell line, E14 was derived from 129P2/OlaHsd strain blastocysts  and cellChIP was carried out as described in . For 500http://www.ncbi.nlm.nih.gov/geo/): Input (GSM798320) [For further analyses the following ChIP-seq files were included from Gene Expression OmnibusM798320) , RNA polM798320) , H3K4me1M798320) , H3K9ac M798320) , H4K16acM798320) , p300 (GM798320) , H3K27meM798320) , Nsl1 (GM798320) . Tip60 dM798320) . DNAse I20 , RNA The Tip60 ChIP-seq was validated by ChIP coupled to quantitative PCR (ChIP-qPCR). Identified MACS14 Tip60 peaks were randomly taken based on different tag densitites (t). SYBR Green (Roche) was used according to the manufacturers protocol. Following primers were designed:The gel filtration experiment using a Superose 6 column was described in . Input nK-means linear clustering was conducted with seqMINER [K-means clustering was performed with normalized read densities, while resulting heatmaps show total number of reads. Obtained MACS14 peaks were annotated using the software HOMER [Density profile calculation around TSSs as well as seqMINER . K-meansre HOMER  based onTo determine the Tip60 or Pol II enrichment at genes, the peak tag density of the nearest peak to the TSS (in a region of +2 kb) was taken and correlated with gene expression levels. We considered a total of 26,460 ENSEMBL TSSs based on the ENSEMBL 67 database (mm9). For this, raw RNA-seq data of mESCs from Gene Expression Omnibus (GSE34473) were processed using the software tools TopHat . FPKM vaGene ontology (GO) term analyses of Pol II positive or bivalent peaks as well as enhancer sites were conducted with Manteia . Peaks wES-specific enhancers and super enhancer regions were taken from . ES-spec


The mechanism of transcription activation of mom gene by C protein is well understood. C activates transcription at Pmom by initial unwinding of the promoter DNA, thereby facilitating RNA polymerase (RNAP) recruitment. Subsequently, C interacts with the ß' subunit of RNAP to enhance promoter clearance. The mechanism by which C activates other late genes of the phage is not known. We carried out promoter-polymerase interaction studies with all the late gene promoters to determine the individual step of C mediated activation. Unlike at Pmom, at the other three promoters, RNAP recruitment and closed complex formation are not C dependent. Instead, the action of C at Plys, PI, and PP is during the isomerization from closed complex to open complex with no apparent effect at other steps of initiation pathway. The mechanism of transcription activation of mom and other late promoters by their common activator is different. This distinction in the mode of activation (promoter recruitment and escape versus isomerization) by the same activator at different promoters appears to be important for optimized expression of each of the late genes.Transactivator protein C is required for the expression of bacteriophage Mu late genes from  Mutants defective in lys make normal amounts of functional phage particles but do not release them from the cell . Amo. Amo1]. the cell ,3. I genassembly , while Page tail . The momfunction ,6 that cfunction . Althouge genome . Notablye genome ,9.mom promoter, alters the DNA conformation by unwinding, thereby, facilitating RNAP recruitment at the otherwise out-of phase promoter elements unmalleable for enzyme occupancy . Reactions were initiated by incubating 40 nM DNA, 80 nM RNAP in transcription buffer on ice for 10 min to allow the formation of closed complex. 300 nM C protein was used wherever required. The reactions were shifted to 37°C for 10 min to allow the formation of open complex. For single round transcriptions, 50μg/ml heparin was added and incubated at 37°C for 1 min. With the addition of 0.3 mM NTP mix and 3μCi [α32 P] UTP (6000 Ci/m Mol), the reactions were initiated and after 30 min at 37°C, terminated by the addition of urea loading dye , heat inactivated at 65°C for 3 min and quenched on ice. The samples were applied either to a 10% denaturing PAGE or 22% denaturing PAGE for analyzing run- off transcripts and abortive initiation respectively.Transcription reactions were carried out on the linear DNA templates of P


Oct4 distal enhancer in pluripotent cells. Here, we derive ES and iPS cells from a transgenic Oct4 distal enhancer reporter mouse. Using sonication-based Circularized Chromosome Conformation Capture (4C) coupled with next generation sequencing, we determined and compared the genome-wide interactome of the endogenous and transgenic Oct4 distal enhancers. Integrative genomic analysis indicated that the transgenic enhancer binds to a similar set of loci and shares similar key enrichment profiles with its endogenous counterpart. Both the endogenous and transgenic Oct4 enhancer interacting loci were enriched in the open nucleus compartment, which is associated with active histone marks , active cis-regulatory sequences (DNA hypersensitivity sites (DHS)), 5-hydroxymethylcytosine (5-hmc), and early DNA replication domains. In addition, binding of some pluripotency-related transcription factors was consistently enriched in our 4C sites, and genes in those sites were generally more highly expressed. Overall, our work reveals critical features that may function in gene expression regulation in mouse pluripotent cells.Genome structure or nuclear organization has fascinated researchers investigating genome function. Recently, much effort has gone into defining relationships between specific genome structures and gene expression in pluripotent cells. We previously analyzed chromosomal interactions of the endogenous  Pluripotent embryonic stem (ES) cells can self-renew indefinitely and generate all cell lineages in an organism, including the germ line12Recent studies have provided insight into chromatin folding patterns in 3D space as a mechanism governing stem cell pluripotencyOct4 locus, a region regulating ES cell pluripotency516A determinant of genome architecture is long-range chromatin-chromatin interactions occurring genome-wide. Since 2002, when chromosome conformation capture (3C) was first used to investigate interactions between two genomic segments1011Oct4 gene expression is governed by upstream distal and proximal enhancersOct4 expression in ES cells, while the latter controls expression in epiblastsOct4 regulation by the distal enhancer is achieved by spatial proximity via looping7Oct4 distal enhancer could regulate both nearby and distant genes on the same or even different chromosomes and that these interactions could be effectively detected by 4C-seq technology using the Oct4 distal enhancer as “bait”.Oct4 distal enhancer interacts with genomic loci that exhibit open chromosome features and contain active histone marks. Genes residing at these loci were expressed at levels higher than genes in other regions. We also demonstrate that long-range chromosomal interaction correlates with Oct4 gene transcription and show that somatic cells reprogrammed to iPS cells establish long-range chromosome interactions at the Oct4 locus before activating Oct4 transcription. When we compared the interactome of a transgenic Oct4 distal enhancer with its endogenous counterpart in iPS cells using sonication-based 4C-seq, we observed similar interacting loci. Overall, this analysis yields insight into high-fidelity interacting regions likely critical for gene expression in mouse pluripotent stem cells.Here, we report that the Oct4 distal enhancer “bait” in three pluripotent lines, including mouse ES cells, mouse transgenic ES cells and mouse transgenic iPS cells . 4C libraries were then constructed and subjected to next-generation sequencing.Details relevant to sonication-based 4C are found in Gao Based on our previously established protocol, we employed a paired-end tag (PET) mapping strategy15We initially determined reproducibility of inter-chromosomal interactions between biological replicates by counting interactions in every 2Mb genomic bin and correlating them between biological replicates. For inter-chromosomal interactions generated in replicates, Pearson’s correlation coefficient was >0.4 in all five 4C experiment groups . This fiet al.To evaluate consistency between biological replicates in a different manner, we employed a strategy devised by Favorov Application of our custom computational pipeline identified thousands of sites interacting with the bait region . To miniOct4 distal enhancer  compared with randomly shuffled genomic sites. By contrast, enrichment for the repressive mark H3K27me3 and the heterochromatin mark H3K9me3 was not significant , we established that Oct4 is located at an early DNA replication domain . Thus, we asked whether the interactomes identified were similarly correlated with replication timing. We found that Oct4 enhancer interacting sites overlapped primarily with early DNA replication domains in mouse ES and iPS cells  sites are universal features of active Oct4 enhancer interactomes than were randomly selected sites , and that pattern was comparable between endogenous and transgenic enhancer interactomes.DNA methylation marked by hydroxymethylcytosine (5-hmC) also regulates pluripotent cell activityOct4 enhancer interactomes with annotated gene locations, we examined distribution of genomic distances of 4C sites to nearby genomic elements, including to transcription start sites (TSSs) and CpG sites, which regulate gene expression and govern chromatin organization29Oct4 enhancers preferentially interact with gene-rich regions. Proximity of interactomes to defined TSSs supports the concept of “transcription factories”, in which multiple active gene loci are co-regulated31To correlate Oct4 enhancer interactomes are closely associated with genes, we identified all genes overlapping with or closest to 4C sites. Gene counts from 10 datasets are summarized in Since Oct4 enhancer interactome is enriched with transcription factor binding sites. To determine this, we analyzed ChIP-seq profiles of 13 sequence-specific transcription factors  and 2 transcription regulators (p300 and Suz12) reported previouslyTranscription factors can mediate chromatin-chromatin interactions by binding to two DNA segmentsOct4 gene locus in establishing and maintaining pluripotency, previous studies have mapped its interaction partners in mouseOct4 distal enhancer resides at a different locus, its interaction partners are comparable to those of the endogenous enhancer.To study the role of the Oct4 distal enhancer bait in transgenic mouse ES cells. We found that interacting partners tend to occupy active genomic regions labeled by active histone modifications and other epigenetic marks. This observation suggests that physical contact between the Oct4 enhancer and other loci may be crucial for activity of transcription of factors regulating pluripotency. Particularly, the Oct4 enhancer are closer to other enhancer regions in 3D space, thus may interact and synergize with enhancers of other genes functioning in pluripotent stem cells. In fact, synergy among enhancers has been previously shown in analysis of the mouse immunoglobulin kappa (Igκ) gene353637Oct4 enhancer interacts with a set of actively transcribed genes in pluripotent cells supports the association of genomic architecture with gene expression, as reported in a previous studyHere, we applied sonication-based 4C-seq to identify loci interacting with the We observed high reproducibility in our study, both between biological replicates and between endogenous and transgenic enhancer interactomes. However, we saw some variation in outcomes potentially due to the methodologies employed. The Pearson’s correlation coefficient of >0.4 is large enough in evaluating reproducibility between replicates, and is comparable to similar studies of this type. Chromatin-chromatin interactions are highly dynamic and transientOct4 enhancer. However, validation of the function of individual locus and gene is still required for unveiling the specific function. DNA fluorescence in situ hybridization (FISH) is a powerful approach to reveal nuclear positioning of a pair of loci42434C-seq provides genome-wide candidate of regions and genes that interact with Oct enhancer and conclude that interactions we observed are likely relevant to gene expression and pluripotency. It is important to keep in mind that we are relying on millions of cells and that our observations reflect behavior of cell populations. Such profiles should be interpreted with caution when predicting the function of a single cell.In summary, we report consistent interactome profiles for both the endogenous and transgenic Mouse E14 ES cells were grown in culture dishes coated with 0.1% gelatin in Glasgow Minimum Essential Medium (GMEM) supplemented with 15% fetal bovine serum (FBS), 100 nM nonessential amino acids, 1% sodium pyruvate, 200 mM glutamate, 1% penicillin streptomycin, 50 uM β-mercaptoethanol and 10 ng/mL LIF. Medium was replaced every 24 hours.Oct4 enhancer are as follows. 1st set, Forward 1: ACAGGCACTCTGAGGGCTAT, Reverse 1: TCGTTCAGAGCATGGTGTAGG; 2nd set, Forward 2: GTAATGGGATCCTCAGACTGGG, Reverse 2: AGGCTGTGTGATTCACCCTG. Primers targeting transgenic Oct4 enhancer are the same as those targeting endogenous enhancer, except for the Forward 2: GTAATGGGATCGTGACCCAAGG . Isolated chromatin pellets were resuspended in SDS lysis buffer  and sonicated to an average size of 500-bp. Fragments were diluted, blunt-end repaired, and ligated with T4 ligase for 24 hours at 4. Reverse crosslinking was carried out at 65 °C for 20 hours with proteinase K. Protein-free DNA was purified as template for nested PCR using two sets of primers. Primers targeting endogenous ACCCAAGG . Purifie4C libraries were sequenced using 90-bp paired-end reads. Using this method, we extracted 20-bp end tags from forward and reverse reads and aligned them to the mouse reference genome assembly (mm10) separately using Burrows-Wheeler Aligner BWA. We retai on chromosome W with length Lw was examined within window w with length lw. The number of interacting sites was defined as Ci,w, and a z score was assigned to the window based on the following calculation:We applied a statistical model to identify contact regions with significantly higher interaction frequency than that expected from backgroundμw is the expected number of interacting sites in window w on chromosome W.in which We then applied an FDR-based approach to determine statistical significance of each interacting site. We randomly permutated z-score data obtained using the above calculations 100 times for every chromosome, and selected sites with FDR ≤ 0.05 as significant interacting sites. An FDR for each site was calculated by counting randomly permutated z-scores that were larger than the experimentally determined z-score. All interacting sites within +/− 1 Mb range of significant interacting sites were merged into an enriched domain. Overlapping domains, if any, were further merged into a final candidate region.We retrieved ChIP-seq data for histone modifications, DHS data and RNA-seq data from ENCODE Project Portal. RNA-seq raw data were processed using Tophat and Cufflinks. ChIP-seq peak files of 15 DNA-binding proteins were retrieved from the GEO database (GSE11431). A file containing genome-wide 5-hmc peaks in mouse ES cells was provided by Dr. Hao Wuhttp://www.r-project.org/). Conversion of genomic coordinates between genome assemblies was executed using the liftOver tool from the UCSC Genome Bioinformatics Site.Statistical analysis was carried out using R .