Xvent homeobox genes encode transcription factors that repress organizer genes and are essential for dorsoventral specification during early embryogenesis in Xenopus. In contrast to the Xvent-2 gene subfamily, Xvent-1 subfamily members, including PV.1A, have been proposed as indirect targets of Bone Morphogenetic Protein-4 (BMP-4) signaling. Because PV.1A is a critical downstream mediator of, and tightly regulated by, BMP-4 signaling, we hypothesized that its promoter contains a direct BMP-4 response element to effect this transcriptional regulation. We demonstrate that direct regulation by BMP-4 is necessary for transcription of PV.1A: its proximal promoter contains cis-acting binding elements for Smads and Oaz crucial to induction in response to BMP-4 signaling. In addition to these direct cis-acting BMP-4 responsive elements, an indirect Xvent-2 response element and several repressive elements exist in the PV.1A promoter to regulate its transcription. In summary, PV.1A undergoes combinatorial regulation during early Xenopus development as both the direct target of BMP-4 signaling and as the direct and indirect target of positive and negative regulatory factors. Xenopus embryos is established in part by a gradient of Bone Morphogenetic Protein (BMP) signaling generated by the BMP antagonists Chordin, Noggin, and Follistatin in the extracellular space. Binding of BMPs, which effect ventralization, to antagonists prevents their interaction with their cognate receptors, leading to embryo dorsalization in overexpression studies Dorsoventral patterning in developing Xenopus, BMP-2, BMP-4, and BMP-7 ventralize the early mesoderm and negatively regulate neurogenesis Xvent-1PV.1Xvent-2Xmsx-1Xvents during early embryonic development In vertebrates, BMPs play critical roles in dorsoventral patterning of the early embryonic mesoderm and specification of the epidermis. In Xvent family members can suppress dorsal fates, and promote ventral, when ectopically expressed Xvent members that repress transcription of dorsal-specific genes Xvent family comprises two subfamilies subdivided on the basis of their amino acid sequence: Xvent-2 (Xvent-2Xbr-1bXomVoxXvent-2BXvent-1 (Xvent-1Xvent-1BPV.1PV.1A).Xvent-2 transcription is regulated by Smad1/4 and its co-activator Oaz in response to BMP-4 signaling Xvent families differs. According to previous reports, the Xvent-2, but not the Xvent-1Xvent-1B, a highly conserved Xvent-1 paralogue, is induced indirectly by BMP-4 signaling PV.1A, we isolated genomic DNA (gDNA) encompassing PV.1A and its 5′-flanking region (−2525 bp) and analyzed the regulatory elements in its promoter with regard to BMP-4 signaling during early embryonic development. The region 180-bp upstream of the major transcriptional initiation site promoted full reporter-gene activity and contained cis-acting elements responding directly to BMP-4 signaling. The proximal region of the promoter possessed putative binding sites for Smads and Oaz, both of which were necessary for response to BMP-4 signaling. Additionally, we determined that the promoter contains a BMP-4 response element (BRE) and an Xvent-2 response element (XRE). Furthermore, we identified negative cis-acting elements that responded to the dorsal-specific transcription factors AP-1 and Goosecoid in the proximal PV.1A promoter. These results suggest that the proximal PV.1A promoter contains multiple cis-acting elements, including direct, indirect, and negative response elements, as well as those binding transcriptional co-activators, allowing BMP-4 signals to converge and maintain tight transcriptional regulation during early embryonic development in Xenopus.In order to understand how BMP-4 signaling regulates the transcription of X. laevis were entrained in 12 hr light/dark (LD 12∶12 h) cycles at 18°C in containers from the Institutes of Laboratory Animal Resources built to specifications for laboratory animal maintenance.Approval from the Institutional Animal Care and Use Committee (IACUC) is not required for the experimental use of amphibians or reptiles in Korea. All members of our research group attended educational and training courses on appropriate care and usage of experimental animals. Adult Smad1 wild-type sequence and that of its 3SA and 4SA mutants was subcloned into pSP64TEN. Smad1(3.4SA) was generated from Smad1(3SA) by replacement of the linker region of Smad1(4SA). Smad2 and Smad2(EPSM) were subcloned into the pCS2+ vector in vitro transcription using a MEGAscript kit according to manufacturer's instructions . Synthetic RNAs were quantitated via ethidium bromide staining by comparison to a standard RNA control .cDNAs encoding BMP-4, DN-BR, Smad1(3.4SA), Smad1(3SA), Oaz, dominant-negative Xvent-2 (DN-Xvent-2) PV.1A genomic DNA (gDNA) was isolated by screening a Xenopus muscle gDNA library (Clontech) with a PCR-amplified cDNA probe corresponding to the segment from +48 to +215 bp of Xenopus PV.1A cDNA. PCR primers were (upstream) 5′-CCTTCAGCATGGTTCAACAG-3′ and (downstream) 5′-CATCCTTTCTCCTTGGCATCTCCT-3′. Approximately 7.0×106 plaque forming units (pfu) were screened using an ECL system in accordance with manufacturer's instructions  to identify putative positive clones, which were subsequently isolated and analyzed by restriction mapping and Southern blotting. A 3.8 kb DNA fragment in a positive clone was subcloned into the pBluescript SK(-) plasmid . Both strands of DNA were sequenced by the Sanger method to confirm the identity of the clone.PV.1A promoter mutants were made from this -2525 construct by PCR amplification (XhoI/HindIII and inserted into similarly digested pGL-2 basic plasmid. A triple-repeat BMP-4-response element (BRE) was generated by annealing two complementary oligos:A 3.8 kb fragment from a positive clone containing 2.5 kb of 5′-flanking region was subcloned into the pGL-2 basic plasmid ; the clone was designated the -2525 construct. Serially deleted fication . PCR con5′-ACTAACCTGACAGACTCACTAACCTGACAGACTCACTAACCTGACAGACTC-3′ and 5′-GAGTCTGTCAGGTTAGTGAGTCTGTCAGGTTAGTGAGTCTGTCAGGTTAGT-3′ before subcloning into the pGL-2 basic plasmid.Xenopus embryos were obtained by in vitro fertilization after induction of female frogs with 500 units of human chorionic gonadotropin . RNAs were injected into the animal pole of 2-cell stage embryos; animal caps were dissected from injected embryos at stage 8.5 and incubated to stage 11 in 0.5X modified Barth's saline  for RT-PCR, measurement of luciferase activity, and electrophoretic mobility shift assays (EMSAs).Bmp-4 mRNA (0.5 ng) was injected into the animal pole of 2-cell stage Xenopus embryos. At stage 7.5, embryos were treated with 25 µg/mL cycloheximide (CHX) in 0.5X MBS and maintained until control untreated embryos reached stage 8.5. Animal caps were then dissected from the injected embryos and incubated until stage 11 in 0.5X MBS containing 25 µg/mL CHX. Total RNA was isolated from whole embryos or animal caps using TRIzol reagent following the manufacturer's instructions  and treated with DNase I to remove gDNA contamination. RT-PCR was performed with Superscript II , as described by the manufacturer, with 2 µg total RNA per reaction. PCR was performed according to the following conditions: 1 minute at 94°C, 1 minute at each annealing temperature, 1 minute at 72°C; 20-28 cycles of amplification . Five different groups of animal caps  were harvested and homogenized in 10 µL lysis buffer per animal cap. 10 µL animal cap homogenate were assayed with 50 µL luciferase substrate and activity determined by luminometer . All experiments were repeated at least three times using independently derived sample sets.in vitro translation. In vitro translated proteins were prepared using the TNT Quick Coupled Transcription/translation System according to manufacturer's instructions . Promoter fragments or 3x-repeat oligonucleotides were amplified by annealing two complementary oligos, respectively, and labeled with α-32P-ATP using T4 polynucleotide kinase . Labeled DNA probes were incubated with 10 µg animal cap protein extract or 1 µg in vitro translated protein at room temperature for 20 minutes in 10 µL binding buffer . DNA/protein complexes were separated on 4% polyacrylamide gels. Competition experiments were performed using a 100-fold excess of unlabeled DNA probe.cDNAs encoding Smads 1, 3, and 4 were PCR-amplified and used as templates for Mutagenesis was performed by an overlap extension PCR method using the several oligonucleotides in accordance with instructions .PV.1A  cDNA sequence has been submitted to GenBank.The P<0.05.The data are presented as mean and standard deviation of measurements from at least three separate and independent experiments. Differences were considered significant at Xvent-1 on the ventral side of the embryo PV.1A transcription despite continuing transcription of Xvent-2 and GATA-2, indicating that transcription of Xvent-1 subfamily members relies to a greater degree on BMP-4 signaling than that of its direct targets Xvent-2 and GATA-2. Furthermore, the increases in Xvent-2 and GATA-2 transcription were less sensitive to BMP-4 overexpression than that of PV.1A, in contrast to our expectation (data not shown).Previous work posits that, unlike Xvent-2, Xvent-1 subfamily members are not under the direct transcriptional control of BMP-4 PV.1A transcription, finding that BMP-4 contributes both directly and indirectly to the process (PV.1A affected by dominant-negative Type I BMP receptor (DN-BR) in animal cap explants  was mitigated by co-injection of a constitutively active Smad1 mutant (Smad1(4SA), with four serine residues altered to alanine at the inhibitory phosphorylation sites in its linker region; We examined the necessity of BMP-4 signaling in the regulation of  process . We firsexplants ; co-injePV.1A expression in animal cap explants under CHX treatment. As shown in Xvent-2 and PV.1A transcription  induced by BMP signaling PV.1A promoter, 2 of 3 reporter activities were positively affected by BMP-4 in CHX-treated animal cap explants . The fragment contained the promoter and sequence upstream to -2525 bp, including a TATA-like element proximal to the transcriptional start-site (TATAA). The proximal sequence of our isolated PV.1A promoter was nearly identical to that of Xvent-1B, and differed from those of Xbr-1a/Xvent-2 and Xvent-2B; additionally, it contained putative BMP-4 and Oaz-response elements  at positions similar to the sites found in the Xbr-1a/Xvent-2 and Xvent-2B promoters  upstream of the major transcriptional initiation site did not alter promoter activity. However, the -262 construct had only half the capacity for transcriptional induction of the -300 construct . The 3BRE construct responded positively to BMP-4 and negatively to DN-BR  in response in BMP-4 stimulation, using the wild-type (-180 WT) and mutant (-180 MT) constructs. Expression of wild-type Smad1, Smad4, and Smad1(4SA) significantly increased PV.1A promoter activity in the -180 WT construct. In addition, co-injection of Smad1 and Smad4 further increased promoter activity of the -180 WT construct compared with either mRNA alone (Smad1 (Smad1(3SA), with the three serine residues in its C-terminal SSVS mutated to alanines); Smad1(3.4SA), generated from Smad1(3SA) by replacing the linker region with that of Smad1(4SA); and mutant Smad2 isoforms did not alter transcriptional activation of this minimal promoter  -136] into 2-cell stage embryos with Xvent-2 mRNA. In the XRE(M) -136 construct-injected embryos, promoter activity was not induced by Xvent-2, while the WT -136 construct retained activity  by BMP-4 signaling PV.1A expression (≥7-fold induction).We first investigated whether PV.1A promoter would elucidate the mechanics of its combinatorial regulation. We isolated the PV.1A genomic locus, including the 5′-flanking region (−2525 bp), and generated serial promoter deletions to investigate their several contributions to gene induction. The region 180 bp upstream of the major transcriptional initiation site had full reporter activity in response to BMP-4 signals; and within this region, putative binding sites for Smad family members and Oaz were necessary and sufficient to mediate the effects of BMP-4 signaling. Additionally, we detected a cooperative interaction between these factors in promoting PV.1A transcription. Notably, the BRE in the PV.1A promoter was insufficient for Smad specificity, which was achieved only in the context of an ORE proximal to a BRE.We reasoned that analysis of the PV.1A promoter contained a positive-regulatory cis-element and an XRE within 399 bp upstream of the major transcriptional initiation site: this latter element merely potentiated BMP-4 signaling for full transcriptional activation of PV.1A. We also investigated the negative regulation of the PV.1A promoter via co-injection of the dorsal-specific transcription factors AP-1 and Goosecoid, determining that each can repress PV.1A transcription. Our results suggest that Smads, Oaz, and Xvent-2 stimulate PV.1A expression and that the PV.1A promoter harbors the unique characteristic of possessing multiple cis-acting response elements for its regulation, including direct, indirect, co-activator, and negative response elements, to regulate the early embryonic development of Xenopus laevis.Using serial-deletion and point-mutation containing promoter fragments to drive reporter-gene expression, we determined that the PV.1A 5′ sequence to promoters of other Xvent family members, including Xbr-1a/Xvent-2 and Xvent-2B, found minor conservation of regulatory motifs, principally in the BRE and ORE, while PV.1A and Xvent-1 share considerable sequence identity upstream of their transcriptional start sites . We are currently biochemically investigating the mechanisms by which PV.1A is repressed within dorsal and organizer tissues using the PV.1A promoter to identify negative cis-acting elements that are bound and regulated by dorsal-specific proteins.Alternatively, ventral genes are repressed dorsally by dorsal-specific proteins such as Goosecoid and AP-1 through binding of dorsal-specific repressors to consensus sites in promoters of ventral-specific genes. PV.1A and identified cis-acting elements, including BRE, ORE, and XRE, in its promoter sequences. Additionally, we found that the dorsal-specific transcription factors Goosecoid and AP-1 exert negative effects on PV.1A reporter activity through BMP-4 signaling. Our results indicate that the complex transcriptional control exerted over downstream effectors of BMP-4 occurs through interplay between positive and negative regulators of transcription.In this study, we investigated the transcriptional regulation of Figure S1PV.1A. (A) Comparison among the 5′-flanking sequences of PV.1A, Xbr-1a/Xvent-2, and Xvent-2B indicates a high level of identity within BMP-4 response elements. BRE, ORE, and XRE indicate BMP-4, Oaz-, and Xvent-2 response elements, respectively. (B) The schematic diagram illustrates serial-deletion promoter constructs. Serially deleted and site-directed mutant constructs were subcloned into the pGL2-basic plasmid. Arrowheads indicate positions of site-directed mutagenesis.Bioinformatics analysis of the 5′-flanking region of (JPG)Click here for additional data file.Figure S2in vitro translated proteins  or uninjected animal caps . Unlabeled competitor probe was added at 100-fold excess (C) or the concentration indicated (D). Asterisks indicate specific bands.BRE binding assays. The BMP-4 response element was confirmed by EMSA using probes against triple-repeat BRE  or the TATA-box region of the promoter . The protein extracts indicated were obtained from (JPG)Click here for additional data file.Figure S3PV.1A.  The -180 WT construct (20 pg/embryo) were co-injected with the RNAs indicated (0.5 ng/embryo) into 2-cell stage embryos. Animal caps were dissected from injected embryos at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity as described in Smad binding within the 5′-flanking region of (JPG)Click here for additional data file.Figure S4PV.1A. The -2525 PV.1A promoter construct (20 pg/embryo) was co-injected with BMP-4 (0.5 ng/embryo) and Goosecoid (0.1 ng/embryo) or AP-1 (0.5 ng/embryo) into 2-cell stage embryos. Animal caps were dissected from injected embryos at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity as described in Repressive effects of dorsal-specific protein binding to the 5′-flanking region of (JPG)Click here for additional data file.


Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression.Product of the  Our understanding of the transcriptional regulation of gene expression has been considerably expanded as knowledge of the role of epigenetic modifications has become clearer. It is now apparent that the mechanisms that create and maintain a permissive or repressive epigenetic environment play a pivotal part in gene regulation controlling developmental and cellular differentiation programs. Studies focusing on the histone modifications underlying transcriptional regulation suggest that acetylation promotes transcriptional activity, perhaps by maintaining an open chromatin state Itga2b/CD41 expression is influenced by developmental mechanisms Identifying emerging definitive HPC and HSC in the embryo proper, Itga2b locus in these cellular models, mimicking different stages relative to hemangioblast emergence and commitment, and compare our findings to human data available from the ENCODE project Mpl gene regulation because, like Itga2b, it is; 1) a marker of HSC Itga2b and Mpl transcriptional control could exemplify such common sub-networks. To probe this hypothesis, the HPC7 line represents an ideal cellular system because of its unique capacity to recapitulating the differentiation process in vitro and produce normal mature megakaryocytes in response to thrombopoietin (TPO) Here, we characterise the epigenetic environment of the itga2b and Mpl expression in non-hematopoietic cells. Together with the switch in promoter usage, we highlight the profound restructuring of histone modification that needs to take place during development to enable the expression of the surface receptors in HSC. We show that Itga2b is then regulated by a HSC-specific transcriptional network that associates with a defined epigenetic landscape. Upon commitment to megakaryocyte differentiation, we find that the up-regulation of the H3K27me3 demethylase Jmjd3 plays a determining role in enabling the transition from HPC- to megakaryocyte-associated expression of both itga2b and Mpl.Our study points to a crucial role for internal alternative promoters in the silencing of Itga2b gene at the onset of haematopoiesis, we compared cell systems modelling different cellular contexts prior to and subsequent to haemangioblast commitment (Itga2b transcription start site (TSS). In contrast, regions within the core of the gene, between positions +2.5 to +3.5 kb and +12 to +14 kb from the Itga2b ATG, showed higher levels of modification in ES and MS1 lines. Finally, the heterogeneous foetal liver cells population recapitulates precisely the mix of both patterns, confirming the presence of the two types of profile in primary cells. The presence of transcription-associated histone modifications at the Itga2b locus suggests that in ES and MS1 lines, the gene is either transcriptionally active or bare marks reminiscent of a developmental priming of the gene.In order to approach the transcriptional regulation of the mmitment . We usedmmitment . This anmmitment . The disItga2b gene could indicate the existence of internal promoters. To substantiate this hypothesis, we searched for other promoter-like features. A search for clusters of transcription factor consensus binding sites highlighted the presence of six regions , suggesting that such positioning is dynamic and not fixed through developmental process.To cross-correlate our findings and further compare the regulatory features of ES cells . MoreoveItga2b gene regulation should dramatically differ in CD41+ HSC/HPC compared to CD41- non-hematopoietic cells, possible common regulatory features between the CD41+ HSC/HPC and megakaryocytes have never been assessed. To test this hypothesis, we generated megakaryocytes through TPO-dependent differentiation of the multipotential hematopoietic HPC7 line  common to HPC7 cells and megakaryocytes (HS1) and a megakaryocyte-specific site (HS2). Although displaying different sensitivities, the HS locations were confirmed in primary CD41+ fetal liver cells and fetal liver-derived megakaryocytes  and methylated histone H3  around the TSS for the full-length transcript which we will refer to as main TSS  and the megakaryocytic marker (GPIbá), we used shRNA-mediated silencing to impair jmjd3 up-regulation during megakaryocytic commitment and differentiation. HPC7 cells were transfected with jmjd3 shRNA and induced to differentiate towards the megakaryocytic lineage in the presence of TPO. Maturing cells were harvested at day 2 of the differentiation process to assess Jmjd3 expression as well as the levels of the Itga2b, Mpl and Gp1ba transcripts. Although Jmjd3 knockdown did not entirely overcome Jmjd3 up-regulation at this stage of differentiation, it correlated closely with a significant inhibition of Itga2b and Mpl transcription  and demethylases (Utx and Jmjd3) as well as co-effectors possessing acetyltransferase activity (CBP and p300) at different stages of maturation. As previously, HPC7-derived megakaryocytes were separated by density gradient fractionation into populations representing different levels of maturation. cDNA were generated from each cell population and the expression of by Q-PCR . The res2b locus  and an ipression . To testcription . As an ituration . We concItga2b and Mpl genes exhibit alternative promoters and that a switch in promoter usage needs to take place during development to permit CD41 and c-Mpl expression at the surface of the emerging HSC. Multiple promoter usage is an important gene regulatory mechanism, the global significance of which has been highlighted by comprehensive analysis of genomes showing that 30 to 50% of mouse and human genes utilise more than one promoter The regulation of gene transcription during development and differentiation is a coordinated process involving controls at many levels. Illustrated by a rapid acquisition of CD41 expression upon ES cell commitment to differentiation towards hematopoietic cells, this mechanism needs to be very dynamic. Our analysis points to a pivotal role for epigenetic modifications associated with promoter activity regulation in the mechanisms leading to cell-type specific gene expression. We find that both the Itga2b promoter is reduced through epigenetic means, whereas internal initiation of transcription may contribute in silencing CD41 expression. In CD41- ES and endothelial cells, the absence of the H3K27me3 Polycomb-associated modification indicate that the repression of CD41 expression is mediated through a Polycomb-independent mechanism. In effect, the observed patterns suggest that the presence of H3K9me3 over the main TSS region of the Itga2b gene, combined with the absence of marks associated with transcriptional activity, could be responsible for inhibiting the main promoter activity. In this setting, transcription initiation may occur from internal alternative promoters containing the active gene associated modifications H3K4me3 and H3K9ac. Further inquiries of the ENCODE/Broad Institute project data ITGA2b and MPL alternative promoters in human ES cells, while p300 and PolII positioning correlate with DNaseI hypersensitivity sites in regions we had previously defined in murine ES cells. As the first definitive HSC emerge, the acquisition of CD41 expression implies an epigenetic remodelling to produce the landscape that is permissive for the transcription of the full-length transcript, as observed in the CD41+ hematopoietic stem/progenitor HPC7 line. The relative importance of such a mechanism, compared to the well-described bivalent marks Mpl locus suggests that similar mechanisms could regulate both genes and possibly a class of HSC-related genes.Here we describe a mechanism in which, prior to the specification of the HSC, accessibility to the Itga2b and Mpl loci. Furthermore, the reinstatement of H3K4me3 and H3K9ac at the internal locations upon HSC commitment to the B cell lineage suggests that the alternative promoters serve a repressive rather than priming function on CD41 expression. In contrast, CD41 and Mpl expression are up regulated during megakaryocyte differentiation.The dissociation of transcriptional activity and protein expression resulting from RNA transcription could serve diverse purposes such as priming genes for expression by maintaining them within transcription factories or silencing genes that are retained in the transcription factories as a result of adjacent actively transcribed loci. In fact, ubiquitously expressed genes flank both Itga2b regulation is a high degree of acetylation on H4K8. Changes in histone acetylation, and the association with specific HAT and HDAC, are well documented in relation to transcriptional regulation during differentiation Itga2b promoter is restricted to the uncommitted cells. Interestingly, the potential of Pu.1 to inhibit CBP-mediated acetylation of histones suggests that cross-regulation between HAT and Pu.1 possibly participates in the maintenance of the HPC-related epigenetic profile and level of transcription of the Itga2b gene. Moreover, Gata-1, which is specifically up regulated during megakaryopoiesis, can induce cell-type specific histone acetylation following its own acetylation Itga2b cis-regulatory domain occupancy, would ultimately facilitate histone acetylation and the implementation of the H4K8ac lineage-specific pattern on the Itga2b promoter.Previous studies have identified Ets and Gata factors as key regulators for both genes in the maturation process Itga2b implying a pivotal role for H3K27me3. Although H3K27me3 is a defined mark of polycomb-mediated repression of transcription, there is no strict correlation between H3K27me3 and transcriptional silence as it is also found in ES cells associated with transcribed loci such as wnt, fgf and hedgehog Itga2b locus provides another example of a low level of H3K27me3 being compatible with transcriptional activity. Here we find that the up-regulation of the H3K27-specific demethylase Jmjd3 during megakaryopoiesis participates in implementing a permissive context for Itga2b and Mpl lineage-specific expression. The role of Jmjd3 in the transcriptional program associated with early stage commitment Itga2b locus following megakaryocyte lineage commitment; however, the lower levels of H3K27me3 across the Itga2b promoter in mature megakaryocytes suggest that Itga2b is a likely target for Jmjd3 demethylase activity.More surprisingly, we have defined an HPC-related epigenetic pattern on Itga2b expression involves a broad range of mechanisms. We show that these are closely linked to epigenetic modulation and are regulated in a developmental stage and differentiation-associated manner.Overall, we have demonstrated that the regulation of HPC7 cells were kindly provided by Dr Lief Carlsson, and were cultured in Stem Pro 34 medium (Invitrogen) and recombinant SCF 100 ng/ml. To induce megakaryocytic differentiation, the SCF concentration was lowered to 20 ng/ml and recombinant TPO (Peprotech) was added to a concentration of 100 ng/ml for two days, than the SCF was removed whilst the TPO remained for an additional 5 days. Mature megakaryocytes were purified by density gradient as previously described Cells were cytospun and stained using Diff-Quik reagents as specified by the manufacturer . DNA content was determined by staining with 50 µg/mL propidium iodide  as previously described Cross-linking and X-ChIP were performed as previously described Itga2b locus and compared against the mouse genome using BlastN to avoid repeated or cross-reacting sequences. The oligonucleotides were arrayed in triplicate onto Codelink slides  using a Microgrid II arrayer  and stored at room temperature until hybridised. Samples were hybridized as previously described A series of 60 base-long oligonucleotides were designed to span the http://zlab.bu.edu/~mfrith/cister.shtml) using the default parameters: 35 bp average distance between motifs within a cluster, average number of 50 motifs in a cluster, average distance between clusters of 2 kb. The motif probability threshold was set to 0.01.Clustering of transcription factor consensus binding sites was assessed using the web-based Cister algorithm nd generation (Roche). The different primers used for first strand synthesis and nested PCR are listed in Rapid amplifications of cDNA were achieved using the 5′/3′RACE kit 22 5 mM, glucose 300 mM, EGTA 0.5 mM, NP40 0.1%). For digestion of nuclei, 0 to 100units of DNaseI were added to each aliquot and incubated 10 minutes at 37°C. The reaction was terminated by adding 330 µl of stop solution . RNA and proteins were sequentially digested by addition of 100 µg of RNase A and 100 µg of proteinase K, incubated respectively for 1 hour and overnight at 37°C. Following phenol/chloroform extractions, DNA was ethanol precipitated and resuspended in water. Note that when treating small amounts of nuclei, all volumes were halved. Quantitative PCR was performed on 30 ng of undigested and DNaseI-treated DNA. The different sets of Itga2b oligonucleotides used are listed in Cells were washed with PBS and nuclei prepared by resuspension in 1 ml aliquots of digestion buffer  and the Jmjd3 shRNA or control vectors (5 µg) , using the Amaxa transfection kit  according to the manufacturer’s instructions. Puromycin (Invitrogen) was added to the medium to a final concentration of 0.5 µg/ml, 24 hours post transfection.20×10Figure S1Multi-species sequence alignment of the Itga2b nuclease hypersensitive regions. Boxes indicate the locations of Ets and Gata conserved consensus binding sites.(TIFF)Click here for additional data file.Figure S2ITGA2b and MPL loci in human ES cells. (B) DNaseI hypersensitivity, p300 and pol II binding on Itga2b and Mpl in murine ES cells (ES-Bruce4) and bone marrow cells.Encode project ChIP-seq data from CD41- cells. (A) Transcription factors binding on (TIFF)Click here for additional data file.Figure S3RACE PCR sequences.(DOC)Click here for additional data file.Table S15′ RACE PCR primers sequences.(DOC)Click here for additional data file.Table S2Itga2b locus.Sequences and positions of the Q-PCR primers spanning the (DOC)Click here for additional data file.Table S3Sequences of Q-PCR primers.(DOC)Click here for additional data file.


Nucleosomes are present throughout the genome and must be dynamically regulated to accommodate binding of transcription factors and RNA polymerase machineries by various mechanisms. Despite the development of protocols and techniques that have enabled us to map nucleosome occupancy genome-wide, the dynamic properties of nucleosomes remain poorly understood, particularly in mammalian cells. The histone variant H3.3 is incorporated into chromatin independently of DNA replication and requires displacement of existing nucleosomes for its deposition. Here, we measure H3.3 turnover at high resolution in the mammalian genome in order to present a genome-wide characterization of replication-independent H3.3-nucleosome dynamics.in vivo: rapid turnover, intermediate turnover and, specifically at telomeres, slow turnover. Our data indicate that H3.3-containing nucleosomes at enhancers and promoters undergo rapid turnover that is associated with active histone modification marks including H3K4me1, H3K4me3, H3K9ac, H3K27ac and the histone variant H2A.Z. The rate of turnover is negatively correlated with H3K27me3 at regulatory regions and with H3K36me3 at gene bodies.We developed a system to study the DNA replication-independent turnover of nucleosomes containing the histone variant H3.3 in mammalian cells. By measuring the genome-wide incorporation of H3.3 at different time points following epitope-tagged H3.3 expression, we find three categories of H3.3-nucleosome turnover We have established a reliable approach to measure turnover rates of H3.3-containing nucleosomes on a genome-wide level in mammalian cells. Our results suggest that distinct mechanisms control the dynamics of H3.3 incorporation at functionally different genomic regions. Drosophila, H3.3 replaces H3 during gene activation and becomes deposited in active chromatin and especially very highly expressed ribosomal DNA [Incorporation of histone variants into chromatin critically influences the properties of nucleosomes that play important roles in regulating transcription and epigenetic memories -6. The homal DNA ,15. In momal DNA , althougomal DNA . In spitomal DNA . Nucleosomal DNA ,19. In momal DNA -23.Drosophila. Transgenic epitope-tagged histones can be inducibly expressed to estimate nucleosome turnover and allow detection of specific histone incorporation [Intrinsic nucleosome characteristics such as inclusion of histone variants as well as extrinsic factors such as ATP-dependent nucleosome remodeling, the transcriptional machinery and various other factors shape the dynamic profile of nucleosomes ,25. Despporation -28. Alteporation . Using tporation ,30.Apart from the measurement of turnover, inducible expression systems with tagged histones have also contributed to our understanding of mechanistic aspects that pertain to histone deposition. For instance, studies from yeast have shown that Asf1 is required for the deposition of H3 and that the amino termini of both H2B and H3 are not required for their incorporation into nucleosomes ,28.While these techniques measure average histone deposition rates across cell populations, alternative techniques such as FRAP and SNAP-tag have allowed the deposition of histones in individual cells to be visualized -33.The genome-wide turnover of the histone variant H3.3 in mammals has not been studied up until now. Global FRAP studies in HeLa cells with green fluorescent protein (GFP)-tagged histones revealed only cycling of H2B. Canonical H3 and H4, in contrast, exhibited very slow cycling, and the majority of H3 remained permanently bound outside S phase . Slow tuIn this study, we developed a versatile technique to map the dynamics of histone variant incorporation into chromatin in mammalian cells. Using this technique, we mapped the replication-independent incorporation of the histone variant H3.3 in mouse embryonic fibroblasts (MEFs). We were able to track H3.3 incorporation across a relatively short time window of several hours after induction of H3.3 as well as over a longer time frame of up to 72 hours. By combining our chromatin immunoprecipitation (ChIP)-based technique with high-throughput sequencing, we measured the H3.3-nucleosome turnover kinetics at the genome-wide level. Our results reveal three major categories of H3.3 nucleosome turnover: (1) rapid turnover at enhancers and promoters; (2) intermediate turnover at gene bodies; and (3) slow turnover at heterochromatic regions. We find faster H3.3 turnover at enhancers and promoters is positively correlated with active histone modifications, including H3K4me1, H3K4me3, H3K9ac, H3K27ac and the histone variant H2A.Z, whereas slower turnover is negatively correlated with H3K27me3 and H3K36me3 modifications. These results show that distinct mechanisms of histone deposition and eviction pertain to the dynamics of nucleosomes at different functional chromatin regions. We also show that turnover is related to the presence of specific histone marks, strongly suggesting that histone modifications are important determinants of nucleosome stability.In order to track histone incorporation and thereby assay the genome-wide dynamics of the histone variant H3.3, we generated MEFs that carry a cytomegalovirus-controlled tetracycline (TET) transactivator and hemagglutinin (HA)/FLAG-tagged H3.3 expression cassette controlled by tetracycline response elements. This TET-ON expression system allowed us to induce the expression of a HA/FLAG-tagged version of H3.3 by addition of the tetracycline analog doxycycline (DOX) Figure A. In ourIn order to minimize the effect of replication-coupled histone disassembly, we arrested the cell cycle of confluent NIH/3 T3 MEF cells by treatment with the DNA polymerase (Pol) inhibitor aphidicolin. After 18 hours of aphidicolin treatment (time point 0) and across the time course of HA/FLAG-H3.3 induction, the MEF cell population was essentially devoid of cells in S phase and arrested at the G1/S phase boundary, as indicated by bromodeoxyuridine and propidium iodide staining  and negatively correlated with repressive marks (H3K9me2 and H3K27me3). While the steady-state level of H3.3 is positively correlated with H3K36me3, its turnover rate is negatively correlated with H3K36me3.We found that HA-H3.3 is enriched in tRNAs and rRNA genes Figure C. The inCompared to promoters, enhancers, gene body regions, tRNAs and rRNAs, the incorporation of HA-H3.3 at telomeres exhibited the slowest rate. It was detected at 48 hours and reached a maximum at 72 hours after induction Figure C, suggesNucleosome dynamics and histone turnover are not well understood in mammals. In this report, we have developed a robust, TET-inducible system to study the kinetics of genome-wide deposition of the histone variant H3.3 in MEF cells. Using this system, we were able to measure turnover rates of genome-wide H3.3 target sites and were able to infer important differences between turnover rates of various genomic regions. Several categories of H3.3 deposition kinetics were observed: invariably high turnover at promoters, a broad range of turnover rates at enhancers, and slow turnover at gene bodies. In addition, repeat elements displayed dramatically different turnover rates with relatively high turnover at tRNA and rRNA elements and very slow H3.3 exchange at telomeres. These results provide novel insights into the genome-wide turnover of H3.3-containing nucleosomes and suggest distinct mechanisms of nucleosome assembly, stability and eviction in order to fulfill their function in regulating transcription and maintaining chromatin stability and integrity.Unlike canonical histones, the histone variant H3.3 can be deposited onto chromatin in a replication-independent manner ,8. It isOur analysis of H3.3 distribution at the steady state revealed its presence at intergenic regions that overlap with histone modifications that typically mark enhancers, such has H3K4me1, H3K27ac and H2A.Z, in addition to the enrichment at TSS regions. In coding regions, we find that H3.3 is broadly distributed within the gene body of actively transcribed genes as well as TESs and regions immediately after. This association is well correlated with gene expression levels as has been reported from other cell lines . At promOur results on the dynamics of H3.3 incorporation revealed three basic modes of H3.3 deposition kinetics. The most dynamic exchange of H3.3 was observed at promoters and enhancers. Robust signals of induced H3.3 were detected within 2 to 3 hours of induction at these regulatory regions, which is consistent with the rapid nucleosome turnover at chromatin boundaries observed in yeast . The secThe fast turnover at enhancers and promoters may involve histone chaperones such as HIRA and Atrx-Daxx and other chromatin remodeling enzymes as well as sequence-specific transcription factors. High turnover at promoters suggests that high nucleosome turnover is a pre-requisite for binding of the transcriptional machinery as well as transcription factors.The concept of nucleosome versus transcription factor competition has been well established from the Pho5 promoter in yeast ,23,41. WAlthough simple nucleosome depletion could accomplish accessibility, ‘transient’ nucleosome deposition may stabilize DNA integrity, which may be necessary for accurate transcription factor binding. Several families of transcription factors, so-called ‘pioneer factors’ , requireThe modest rate of H3.3 incorporation in gene bodies and TES regions is closely related to active transcription, and the accumulative signals of H3.3 in these regions may require multiple rounds of transcription of a gene unit. It is generally thought that RNA polymerase displaces nucleosomes during elongation, as it cannot pass by nucleosomes ahead of it ,45. NuclOur analyses suggest that heterochromatic regions turn over more slowly than euchromatic regions. We observed very slow turnover at telomeres and no turnover at all at pericentromeric regions, both of which are known to be enriched in H3.3. This suggests that pericentromeric nucleosomes are replaced only during replication, whereas telomeres undergo continuous exchange of their nucleosomes. Continuous exchange of nucleosomes at telomeres may be necessary for telomeric maintenance, although bulk telomeric nucleosomes may be exchanged during replication.In summary, we have provided the basis for our understanding of nucleosome dynamics in mammals by providing a detailed picture of genome-wide replacement of the histone variant H3.3. This study will provide the platform for further studies into the determinants and mechanisms of nucleosome exchange.In this study, we mapped the genome-wide H3.3-specific nucleosome occupancy and the dynamic turnover of this histone variant in mammalian cells. We found that H3.3 turnover rates vary dramatically across functionally distinct genomic regions: turnover was highest at promoters and enhancers, intermediate at gene bodies and slowest at telomeres. Furthermore, we delineated striking correlations between turnover rates, histone modifications and H2A.Z, suggesting that intrinsic nucleosome properties such as histone modifications and histone variant inclusions are important properties of nucleosome stability.cDNA of human H3.3B or mouse H3.1 (Hist1h3h) was subcloned in-frame with a HA and FLAG tag at the carboxyl terminus and cloned into the lentiviral plvx-Tight-Puro Vector . Lentiviral particles were packaged in 293 T cells with the psPAX2 packaging plasmid. Subsequently, we transduced NIH/3 T3 Tet-On® 3G cells (Clontech) and drug-selected with puromycin for stable integration.Cells were lysed with RIPA buffer, and whole-cell lysates were resolved on SDS-PAGE, transferred onto nitrocellulose membranes, and blotted with anti-HA , anti-FLAG  antibodies at 1:1,000. For anti-H3.3 western blotting, histones were isolated by acid extraction as described in  prior toCells were examined for BrdU incorporation with the BrdU Labeling and Detection kit (Roche). For further cell cycle and DNA content analysis, cells were fixed with 70% ethanol, and subsequently stained with 50 μg/ml propidium iodide in 0.1% PBS-Triton-X with 0.2 mg/ml RNase A before analysis by flow cytometry.NIH/3 T3 MEFs were cultured in standard conditions with medium containing TET-compatible fetal bovine serum. MEFs were grown to 100% confluence and treated with 1 μg/ml aphidicolin for 18 hours. Adding 3 μg/ml doxycycline hyclate before crosslinking with formaldehyde at various time points induced HA/FLAG-H3.3 expression. ChIP-Seq experiments were performed as described previously  with an ChIP-Seq reads were mapped to the mouse genome  using Bowtie , only alThe RefSeq gene annotation (mm9 or NCBI37) was downloaded from the UCSC browser. The mouse genome was classified into 5’ UTR, exon, intron, 3’ UTR and intergenic based on the UCSC annotation. Then we defined another two categories: promoter (3 kb upstream of the TSS) and TES (from TES to 5 kb downstream). The total mapped reads in each category were normalized to its total length. All mouse consensus repetitive element sequences, including pericentromeric element, L1, SINE1 and telomeric element, were obtained from Repbase Update (RU) . The relWe used the position of the center of each peak for peak annotation. Two peaks from different libraries were considered as overlapping if the overlapped region accounts for 10% of the length of one peak. We classified genes with RPKM (reads per kilobase of exon model per million reads) <1 as lowest (inactive) genes and the other genes as active genes. We grouped genes into five categories with equal numbers of genes  based on their RPKM values. For each gene, reads falling in peaks were counted according to their shifted positions in 50 bp windows for the regions from 3 kb upstream of the TSS to the TSS and from the TES to 5 kb downstream of the TES. Within gene bodies, reads falling in peaks were counted according to their shifted positions in windows equal to 1% length of each gene. The number of reads in each window was normalized by the total number of bases within the window, and the total number of peak-filtered reads in the corresponding sample to obtain a normalized read-tag density.The reads from RNA-Seq libraries were mapped to the mouse genome  using TopHat , a fast TI) for each individual peak. Assuming that enrichment of H3.3 at 0 hour is E0, then Et = TI × t + E0, where Et equals H3.3 enrichment at each time point , t = time point . For peaks that reached their maximum enrichment before the end time point of analysis (t = 72 h), multiple linear regression coefficients were calculated by fitting the end time points from time point of maximum enrichment to 72 h and t was adjusted correspondingly. We adopted the regression coefficient with the best fit  as the turnover index of the peaks. The turnover index was scaled from 0 and 1 in order to compare the reproducibility between duplicate experiments.The total number of H3.3 peaks was identified by peak-calling at the 72-hour time point when both early appearing and late appearing peaks were readily detected. The number of reads in each of these peak regions was recorded and normalized over the total mapped reads for each ChIP-Seq library. The relative H3.3 enrichment of each peak was calculated by normalizing the normalized reads in the peak over the normalized reads in input. A linear regression model was used to calculate turnover indices  based on the distribution of turnover indices in Figure Click here for file


Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast  Schizosaccharomyces pombe as a model system, we have identified two Cdk9-dependent pathways that modulate RNAPII elongation. One pathway involves the direct interaction of phosphorylated Spt5 with Prf1/Rtf1, a multi-functional transcriptional regulator. A second pathway, involving a different Cdk9 target, directs the Polymerase Associated Factor (PAF) complex to transcribed chromatin. The Prf1 and PAF pathways are functionally distinct and have opposing effects on the RNAPII elongation complex. Our data provide novel insights into Cdk9 functions and suggest that Cdk9 auto-regulatory mechanisms are an important feature of RNAPII elongation.The expression of many eukaryotic genes is regulated during the elongation phase of transcription. Cyclin-dependent kinase 9 (CDK9) enzymes are key positive regulators of RNA polymerase II (RNAPII) elongation, and are required for co-transcriptional events such as mRNA processing and chromatin modification. Cdk9 phosphorylates RNAPII and the elongation factor Spt5, but the molecular pathways that underlie Cdk9 functions in transcription remain poorly defined. Using fission yeast  Saccharomyces cerevisiae (Bur1) and Schizosaccharomyces pombe (Cdk9), where they have imputed positive roles in RNAPII elongation Regulation of RNA polymerase II (RNAPII) transcription elongation affects the expression of many eukaryotic genes 1SPTSPS7 and is involved in coordinating co-transcriptional events Cdk9 phosphorylates both Rpb1 and Spt5 within repeated amino acid motifs at their C-termini. The Rpb1 C-terminal domain (CTD) repeat has consensus sequence YThe Polymerase-Associated Factor (PAF) complex is a key mediator of Cdk9 function during elongation. PAF is minimally composed of conserved subunits Paf1, Leo1, Cdc73, and Ctr9 PAF1 and RTF1 revealed shared functions in telomeric gene silencing in S. cerevisiae, somite development and hematopoiesis in zebrafish, regulation of flowering time in A. thaliana, and mouse embryonic stem cell pluripotency S. cerevisiae revealed pleiotropic deficiencies for paf1Δ and ctr9Δ mutants but few phenotypes overall in an rtf1Δ strain rtf1Δ mutation was found to suppress the phenotypes of paf1Δ, suggesting that the relationship between Rtf1 and PAF could, in some circumstances, be antagonistic bur1Δ in budding yeast is also suppressed by rtf1Δ, as are phenotypes caused by mutation of the associated cyclin Bur2 Rtf1 is a stable component of PAF in budding yeast, but is biochemically separate from PAF in metazoans S. cerevisiae indicate a particularly close functional connection between Rtf1 and these histone modifications S. pombe, phenotypes associated with loss of H2Bub1 are suppressed by reduction of activity of Cdk9. These include a general 3′ shift of RNAPII density within genes, a pattern that could reflect enhanced elongation through coding regions caused by aberrant Cdk9 function Although both Rtf1 and PAF are required for formation of H2Bub1 and H3K4me, genetic analyses in S. pombe as a model system, we report that PAF and Rtf1 (which we designate Prf1 in S. pombe) are involved in two distinct and opposing regulatory pathways, both of which depend on Cdk9 activity. We further show that these divergent functions involve different Cdk9 targets, and mirror the opposing functions of Cdk9 and H2Bub1 in elongation.Using the fission yeast S. pombe, we assessed the biochemical and functional properties of orthologs of known PAF complex subunits. We first tagged the genes encoding homologs of Paf1 (systematic ID # SPAC664.03), Ctr9 , and Rtf1 , 200 µl of sterile glass beads were added, and cells were lysed at 4°C using a mini-beat beater (4×30 s with 1 min rests on ice). The lysates were transferred to 1.5 mL tubes and centrifuged at top speed for 15 min to collect the chromatin. Chromatin pellets were resuspended in 1 mL lysis buffer, sonicated for 20 minutes using a Bioruptor waterbath sonicator (Diagenode) (30 s ON/OFF with 2 min rest on ice after 10 min) and centrifuged at top speed for 5 minutes. The supernatant (700–800 µl) was incubated for 2–3 h with either 50 µl of IgG sepharose beads (for TAP immunoprecipitation) or with 15 µl of protein G Dynabeads (Invitrogen) coupled to 8WG16 antibody (for RNAPII immunoprecipitation) at 4°C with rocking. The beads were washed successively with 0.5 mL of each of the following buffers: lysis buffer +0.1% SDS, lysis buffer +0.1% SDS+500 mM NaCl, LiCl buffer  and TE . Chromatin was eluted with 100 µl of elution buffer  at 65°C for 30 min. Reversal of crosslinks, DNA purification, and quantitative PCR were as described +act1, +adh1, +nup189, and +SPBC354.10 genes have been described previously ChIP was carried out as previously described 15 µl of streptavidin Dynabeads (Invitrogen) were washed three times with 400 µl of 1xPBS/0.1% Triton-X100. Beads were coupled to 50 µg biotinylated peptide in 380 µl of 1xPBS. Incubation was at room temperature for 3 h with rocking. Spt5 CTD peptides were synthesized at the Keck Foundation Research Laboratory  and had the sequence [biotin]-GSKTPAWNSGSKTPAWNS. In the phospho-peptide both Thr residues were phosphorylated. Rpb1 CTD peptides were a generous gift from Francois Robert  and had the following sequences: [biotin]-YSPTSPSYSPTSPSYSPTSPS, [biotin]-YSPTSPSY(pSer)PTSPSYSPTSPS, [biotin]-SPTSPSYSPT(pSer)PSYSPTSPSY, [biotin]-TSPSYSPTSP(pSer)YSPTSPSYSP, [biotin]-PTSPSYSPT(pSer)P(pSer)YSPTSPSYS. Beads were washed twice with washing buffer  and resuspended with 150 µl of the binding buffer . Binding reactions contained 20 mM Hepes pH 7.6, 0.075% Triton X-100, 0.75 mM PMSF, 7.5 mM β-glycero-3-phosphate, 5% glycerol, 37.5 mM KOAc, 2.5 mM MgOAc, 0.25 mM EDTA, 0.25 mM DTT and 50–200 ng of purified factors (∼50 ng for Prf1-TAP or 200 ng for Paf1-TAP and Tpr1-TAP) in a volume of 200 µl. The mixtures were incubated at 4°C for 1 h with shaking. Beads were washed 4 times with 1 mL of washing buffer, resuspended with 20 µl of SDS-PAGE sample buffer, boiled for 5 minutes at 95°C, and subjected to SDS-PAGE and immunoblotting with a TAP antibody. Signal intensities were quantified using ImageJ software and used to calculate percent bound.Figure S1TAP-tagged Paf1, Tpr1, and Prf1 proteins are functional. Whole-cell extracts from the indicated TAP-tagged strains were analyzed by SDS-PAGE and western blotting with the indicated antibodies.(TIF)Click here for additional data file.Figure S2PAF and Prf1 have shared functions in promoting co-transcriptional methylation of histone H3 lysine 4. Whole-cell extracts from strains of the indicated genotypes were analyzed by SDS-PAGE and western blotting with the indicated antibodies.(TIF)Click here for additional data file.Figure S3act1+ and adh1+ genes. (A) The indicated TAP-tagged strains were treated with DMSO (−) or 20 µM 3-MB-PP1 (+) for 2 hours prior to whole-cell extract preparation and analysis by SDS-PAGE and western blotting. Levels of TAP-tagged proteins were assessed with the TAP antibody. (B) ChIP of RNAPII was carried out in the ascdk9 strain and quantified by qPCR using primers specific to the nup189+ gene. Error bars denote standard deviations from three independent experiments.Inhibition of Cdk9 does not affect total levels of PAF or Prf1 and does not dramatically alter RNAPII occupancy at the constitutive (TIF)Click here for additional data file.Figure S4A) and (B). Strains used for ChIP with IgG resin (recognizing the TAP tag) are indicated at the bottom. All strains also harbored the ascdk9 allele and were treated with either DMSO (−) or 20 µM 3-MB-PP1 (+) for two hours before crosslinking for ChIP. Assays were quantified by qPCR using primers specific for the adh1+ gene. Length of the gene coding region (in base pairs) and positions of PCR amplicons are as indicated at the top. Error bars denote standard deviations from 2–3 independent experiments.Prf1 and PAF are recruited to chromatin via alternate mechanisms. ((TIF)Click here for additional data file.Figure S5A) Whole-cell extracts from strains of the indicated genotypes were analyzed by SDS-PAGE and western blotting with either the 8WG16 antibody  or the H5 antibody (to detect RNAPII phosphorylated at Rbp1-Ser2). Relative band intensities were quantified using ImageJ software and the ratios of Spt5-P signal to total Spt5 signal for each strain are plotted. Error bars denote standard deviation from 3 independent experiments and asterisks denote significant differences from the wild-type intensity . (B) As in (A).Prf1 and PAF differentially regulate phosphorylation of Spt5 CTD and Rpb1-Ser2. ((TIF)Click here for additional data file.Figure S6act1+ (left) or adh1+ (right) genes. Lengths of the gene coding regions (in base pairs) and positions of PCR amplicons are indicated at the top. Error bars denote standard deviations from 2–3 independent experiments.Rpb1-Ser5 is not required for recruitment of Prf1 or PAF to chromatin. ChIP was performed on the indicated strains with IgG resin (recognizing the TAP tag). Assays were quantified by qPCR using primers specific for the (TIF)Click here for additional data file.Figure S7Purified Prf1 and PAF complex interact differently with the Spt5 CTD. Material purified from the TAP-tagged strains indicated at the top was incubated with magnetic steptavidin beads alone (“no peptide”) or beads coupled to the indicated biotinylated peptides. Input and bound fractions (50%) were analyzed by SDS-PAGE and western blotting with the TAP tag antibody. Molecular weight markers (in kD) are shown on the left.(TIF)Click here for additional data file.Figure S8cdk9 and PAF mutants do not require Prf1, Brl2, or H2Bub1. Five-fold serial dilutions of the indicated strains were spotted on agar plates containing rich media (YES), minimal media (EMM), rich media with dimethyl sulfoxide (YES+DMSO), or rich media with mycophenolic acid (YES+MPA).Phenotypes of (TIF)Click here for additional data file.Figure S9A) ChIP of RNAPII was carried out in the indicated strains and quantified by qPCR using primers specific to the SPBC354.10+ gene. Values were normalized to that for primer pair 1. Error bars denote standard deviations from three independent experiments. Significant differences from wild-type values (unpaired t-test) are indicated. (B) Levels of mRNA from the indicated genes were quantified by qRT-PCR using primer pair 3 in either nup189+ or SPBC354.10+ and normalized to the signal for primer pair 3 in act1+. The resulting value in the wild-type strain was set to 1. Error bars denote standard deviations from two independent experiments. Significant differences from wild-type values  are indicated by asterisks. (C) ChIP of RNAPII was carried out in the indicated strains and quantified by qPCR using primer pair 1 in either nup189+ or SPBC354.10+. Values were normalized to that for primer pair 3 in act1+. Error bars denote standard deviations from three independent experiments. Significant differences from wild-type values  are indicated by asterisks.Differential effects of Prf1 and PAF pathway mutations on intragenic RNAPII distribution and gene expression. ((TIF)Click here for additional data file.Table S1Mass spectrometry analysis of Tpr1-TAP and Prf1-TAP purifications. Shown are all proteins identified with a confidence level of 95% or greater in at least one of the samples analyzed. PAF complex components and Prf1 are highlighted in yellow.(PDF)Click here for additional data file.Table S2S. pombe strains used in this study.(PDF)Click here for additional data file.Text S1Supplemental materials and methods.(PDF)Click here for additional data file.


Plants have a remarkable ability to react to seasonal changes by synchronizing life-cycle transitions with environmental conditions. We addressed the question of how transcriptional re-programming occurs in response to an environmental cue that triggers the major life cycle transition from seed dormancy to germination and seedling growth. We elucidated an important mechanistic aspect of this process by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3. Histone methylation patterns of major dormancy regulators changed during the transition to germination and seedling growth. We observed a switch from H3K4me3 and high transcription levels to silencing by the repressive H3K27me3 mark when dormancy was broken through exposure to moist chilling, underscoring that a functional PRC2 complex is necessary for this transition. Moreover, this reciprocal regulation by H3K4me3 and H3K27me3 is evolutionarily conserved from gymnosperms to angiosperms. Our current knowledge about histone methylation depicts a rather static picture. Moreover, with the exception of a few developmental regulators involved in flowering induction DNA in the eukaryotic nucleus is organized to form chromatin. The minimal unit, the nucleosome, is composed of the DNA wrapped around complexes of eight histone cores. Histones can be modified at their N-terminus at a number of residues and with different modifications Physcomitrella patensHistone modifications are important in regulating various life cycle transitions in plants, including the transition from vegetative to reproductive growth in flowering plants pickle (pkl) mutant, which is affected in H3K27me3 deposition Mutants in the polycomb repressive complex 2 (PRC2) display a significant delay in germination and express markers of seed development and dormancy in seedlings In this work, we elucidated an important mechanistic aspect underlying the global transcriptional network re-programming during seed dormancy breakage and the transition to germination. This was achieved by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3.Arabidopsis thaliana ecotype Cvi plants were grown in a growth chamber at 22°C in soil under 16-h photoperiod. Mature dry seeds were harvested and surface-sterilized with 70% and 100% ethanol. Seeds were either monitored for their germination capacity immediately (no moist chilling), or were first moist-chilled at 4°C in the dark on half-strength Murashige and Skoog (MS) media (pH 6.5) solidified with 1% agar for 7 and 14 d prior to germination testing. For germination tests, 3×50 seeds were sown on plates containing solid half-strength MS media (pH 6.5) and the plates were transferred to a Conviron CMP3244 growth chamber . Seeds were scored as germinated based on radicle emergence with the aid of a dissection microscope. A control treatment for the moist chilling was conducted in which seeds were sown on plates containing solid half-strength MS media and kept at 22°C under long day conditions for 14 d.Yellow-cedar seeds (seedlot 48827) were obtained from the BC Ministry of Forests and Range Tree Seed Centre . The conditions for dormancy termination and germination were as described ABI3 (At3g24650), LEC2 (At1g28300), DOG1 (At5g45830), SOM (At1g03790), RAB18 (At5g66400), 2S1 (At4g27140), FLC (At5g10140), MBK20.1 (At5g07580), COR 47 (At1g20440), EXP10 (At1g26770), MDAR6 (At1g63940), PBC2 (At1g77440), HB1 (At3g01470).The target genes for this study are noted in Table 1. The TAIR numbers for these genes were as follows: For ChIP and RNA extractions, three replicates of 100 mg of Arabidopsis seeds were used for each treatment. After the described treatment, each seed batch was aliquoted equally into two tubes, flash frozen in liquid nitrogen and stored at −80°C until use. One tube was used for RNA extraction and the other for ChIP. For yellow-cedar samples, megagametophytes and embryos were excised with a scalpel, flash frozen in liquid nitrogen and stored at −80°C until use.Whole seeds of Arabidopsis, yellow-cedar embryos and yellow-cedar megagametophytes were ground in liquid nitrogen and total RNA was extracted as described qRT-PCRs were run in 15 µl reaction volumes on an ABI7900HT machine (Applied Biosystems) using the PerfeCTa Sybr Green Supermix with ROX (Qanta Biosciences). Primers  were des∧ (−CT). No-template-controls (NTCs) for each primer pair were included to check for contamination of the reagents. Only samples whose corresponding NTC showed no amplification signal were used in the analysis.The efficiency E of the primer pairs was calculated as the average of the Es of the individual reactions by using raw fluorescence data with the publicly available PCR Miner tool 2, 5 mM beta-mercaptoethanol, 20% glycerol, 1×protease inhibitor complex)  on ice. The suspension was filtered through a double layer of miracloth and Triton X-100 was added to a final concentration of 1%. After rotation at 4°C for 10 min and centrifugation at 4000 g for 10 min at4°C, the pellet was resuspended in 300 µl nuclease buffer  for micrococcal nuclease digestion. For nChIP of isolated Arabidopsis embryos, 300 embryos were carefully dissected from enclosing seed tissues, ground in liquid nitrogen and transferred directly to nuclease buffer. Micrococcal nuclease (2.5 µl)  was added and the sample incubated for 8 min at 37°C. The reaction was stopped with EDTA  and the nuclei lysed by adding 900 µl lysis buffer , vortexing for 1 min, incubating the tubes on ice for 10 min, followed by vortexing for 1 min. Samples were centrifuged for 2 min at 4000 rpm at 4°C and the supernatant transferred to new tubes. The sample were pre-cleared with 30 µl salmon-sperm DNA- and BSA-blocked A/G beads at 4°C for 2 h on a rotating wheel. After centrifugation at 4°C, 4000 rpm, the supernatant was collected. This is the pre-cleared chromatin sample.Samples were ground in liquid nitrogen, and added to 15 ml nuclei enrichment buffer  was added to all samples including the untreated input sample. All samples were incubated at 65°C for 10 min, centrifuged at 4000 rpm for 1 min at room temperature and the supernatants were collected. The elution step was repeated and the eluates pooled.Pre-cleared chromatin (250 µl) was used per immunoprecipitation (IP). One hundred µl of the sample was used as input and left at 4°C overnight. Chromatin was immunoprecipitated with antibodies against unmodified histone H3 , H3K4me3  and H3K27me3 , respectively. As a negative control, a mock IP with IgG (Sigma Aldrich) was performed. Thirty µl blocked A/G beads were added to each reaction and the samples rotated at 4°C overnight. The beads were then spun down and washed twice with 400 µl wash buffer , containing 150 mM NaCl for 5 min at 4°C, and once with 400 µl wash buffer containing 500 mM NaCl. After the last centrifugation, the supernatant was discarded and 100 µl elution buffer (100 mM NaHCOTen µg RNAase A (Fermentas) was added to the eluates, and the mixture was incubated for 30 min at room temperature. DNA was purified with a nucleic acid purification kit (Epoch Lifescience) and eluted with 20 µl elution buffer. The resulting DNA solution was diluted five times and 2 µl was used as a template for qPCR.For yellow-cedar embryos and megagametophytes as well as seedling nChIP, 60 seed parts or 20 seedlings underwent the same protocol as described for Arabidopsis in http://epigenomics.mcdb.ucla.edu, 21) indicated the presence of the marks in seedlings. The reaction mixture consisted of 2 µl diluted ChIP-DNA (4 µl for yellow-cedar), 7.5 µl mastermix, and 280 nM of each primer, and the temperature regime was 10 min at 95°C and 40 cycles of 15 s at 95°C/1 min at 60°C followed by a dissociation curve. Template abundance for individual samples was calculated as described above. For the comparison of different samples, the IPs of modified histones were normalized to the IPs of unmodified H3 to account for possible variability in histone density.qPCRs using purified nChIP-DNA as a template were run in 15 µl reactions on an ABI7900HT machine with EvaGreen qPCR mastermix with ROX (abmgood). Primers  were plaCape Verde Island (Cvi) is a model for seed dormancy, and its transcriptomic changes during dormancy maintenance and termination have been characterized The Arabidopsis ecotype prc2 mutants of Arabidopsis display enhanced dormancy Given the evidence for chromatin changes during other plant lifecycle transitions see , and theFor our analyses we focused on two key histone marks: H3K4me3, which is associated with transcriptionally active genes, and H3K27me3, an inhibitory mark. The deposition of both marks is mediated by trxG- and PcG SET domain proteins respectively COR47, which carried H3K27me3 in the dormant state. This repressive mark on COR47 was gradually lost and exchanged for increasing amounts of H3K4me3 following transfer of seeds to germination conditions . For markers and regulators of germination, we selected six genes that exhibit the strongest up-regulation during dormancy termination of Cvi seeds based on published transcriptomes (Table 1) nditions  ThereforABI3 (24) and LEC2Delay of Germination1 (DOG1) is a major dormancy QTL in Arabidopsis SOM) positively influences signaling of the dormancy-inducing and germination-inhibiting plant hormone abscisic acid (ABA) and negatively influences signaling of its antagonist gibberellin (GA) FLC has been implicated in the regulation of temperature dependent seed germination RAB18. RAB18 likely plays an indirect role in dormancy, as this protein is connected more to the survival of the seed in the dispersed, dormant state  (Table 1). The RAB18 gene is expressed during late seed maturation, and exhibits reduced expression during germination. Therefore it is most accurately considered a “dormancy marker”, and not a dormancy regulator.Our new nChIP protocol allowed us to focus on the dynamics of major dormancy regulators in seeds at key physiological stages . We deciper se (e.g. DOG1 and FLC), or once germination had been induced (e.g. ABI3 and LEC2)  . SOM expmination . The “mamination .The switch from activating H3K4me3- to repressive H3K27me3-deposition was associated with a change in transcript level of the dormancy regulators . We are FLC deviates from the general pattern of a maintenance of repressive marks throughout the rest of the life cycle. Although this gene also showed a replacement of H3K4me3 by H3K27me3 during seed dormancy release by moist chilling and germination, FLC must be reset to an active state very soon after germination to fulfill its role as a negative regulator of flowering. However FLC has been tested both positive and negative for H3K27me3 in Arabidopsis plants, depending on natural variation, developmental state, and possibly growth conditions, respectively FLC is caused by exposure to low temperatures, and not by the physiological process of dormancy breakage per se. The exposure of seeds to moist chilling might thereby lead to FLC repression on the chromatin level such that earlier flowering is promoted in the adult plants.FLC-locus during induction of flowering competence through vernalization: H3K27me3 accumulates slowly over weeks of cold exposure in one segment of the FLC gene in the sampling population. When plants are returned to warm conditions, the mark spreads over the whole gene depending on the length of period of cold exposure, and the presence of the mark is quantitatively correlated with FLC expression competence is reached at a certain threshold ratio of active to repressive marks on dormancy regulators, and that germination proceeds only when genes specifying positive germination regulators are activated. This would provide a quantitative means to measure the extent of cold exposure as a result of the output of the ratio of activating and repressive marks.A. Angel et al. Interestingly, we detected both histone marks at the corresponding loci for maturation/dormancy-related genes in our sampling population in non-dormant seeds before their transfer to germination conditions  and S2. prc2 mutants ABI3, DOG1, and FLC, still show H3K4me3 in seedlings upon loss of H3K27me3 Very little is known about the mechanistic aspects of the interactions between different histone modifications in plants. By combining our findings with H3K4me3 profiling in PRC2-defective endosperm but heterozygous embryos exhibit germination behavior that is indistinguishable from that of wild-type seeds PRC2-mediated dormancy control appears to take place at the level of the embryo, as seeds with homozygous Callitropsis nootkatensis). The seeds of this conifer species are deeply dormant at maturity and upon dispersal they typically require a minimum of 6 months of moist chilling in natural stands to terminate dormancy Having found that major dormancy regulators such as ABI3 are transcriptionally regulated at the chromatin level in Arabidopsis, we asked whether the same is true for an evolutionarily distant species, the gymnosperm yellow-cedar  and expression analyses (right column); averages of three biological replicates are shown +/− SE. Refer to Table 1. ER = endosperm rupture and radicle emergence (completion of germination). Note that the Y-axis for the RNA data is in log-scale.(JPG)Click here for additional data file.Figure S2Expression analyses and histone H3 methylation pattern changes of markers of seed maturation/dormancy (2S1 and RAB18) in Arabidopsis Cvi. Supplementary results to support data of . nChIP/qPCR (left column) and expression analyses (right column); averages of three biological replicates are shown +/− SE. Refer to Table 1. ER = endosperm rupture and radicle emergence (completion of germination). Note that the Y-axis for the RNA data is in log-scale.(JPG)Click here for additional data file.Figure S3Comparison of H3K4me3 and H3K27me3 marks on dormancy regulators in WT seedlings and fie-seedlings based on microarray data from Bouyer et al., 2011. Supplementary results to support data of . Upon loss of PRC2 activity in fie-mutants, the H3K4me3 mark stays on dormancy regulators through to the seedling stage.(JPG)Click here for additional data file.Figure S4Histone H3 methylation pattern changes of regulators and markers of seed maturation/dormancy in Arabidopsis Cvi embryos of non-dormant seeds. Supplementary results to support data of . Embryos were cleanly excised from seeds that had been subjected to 14 d of moist chilling. Data are based on the average of two biological replicates +/− S.D.(JPG)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.


Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes , revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth , while upregulating both yeast form-associated genes  and repressors of morphogenesis . On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth , while downregulating yeast form-associated genes and repressors of morphogenesis . Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. We show, indeed, that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis.Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in  Candida albicans can switch from a harmless colonizer of body organs to a life-threatening invasive pathogen. This switch is linked to the ability of C. albicans to undergo a yeast-to-filament shift induced by various cues, including temperature. Sfl1p and Sfl2p are two transcription factors required for C. albicans virulence, but antagonistically regulate morphogenesis: Sfl1p represses it, whereas Sfl2p activates it in response to temperature. We show here that Sfl1p and Sfl2p bind in vivo, via divergent motifs, to the regulatory region of a common set of targets encoding key determinants of morphogenesis and virulence and exert both activating and repressing effects on gene expression. Additionally, Sfl2p binds to specific targets, including genes essential for hyphal development. Bioinformatic analyses suggest that Sfl1p and Sfl2p control C. albicans morphogenesis by cooperating with two important regulators of filamentous growth, Efg1p and Ndt80p, a premise that was confirmed by the observation of concomitant binding of Sfl1p, Sfl2p and Efg1p to the promoter of target genes and the demonstration of direct or indirect physical association of Sfl1p and Sfl2p with Efg1p, in vivo. Our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis. Candida albicans is the most frequent causative agent of superficial as well as disseminated, life-threatening fungal infections C. albicans as a major fungal pathogen of humans relies on a number of pathogenic traits, among which its capacity to grow and switch between at least three distinctive morphological forms: budding yeast, pseudohyphae and hyphae C. albicans knock-out mutants suggests that pathogenesis can be dissociated to some extent from morphological switching 2 and serum The yeast-to-hyphae transition is triggered by a variety of environmental stimuli including nutrient availability, temperature, pH, COSaccharomyces cerevisiae, which has been used as a model for studying the transcriptional control of the morphological transition suppressor gene for flocculation 1) is a target of the cAMP-PKA pathway SFL1 encodes a negative regulator of pseudohyphal growth and invasion in vitro to a GAA triplet motif C. albicans genome encodes two structural homologs of ScSfl1p, namely Sfl1p and Sfl2p SFL1 or SFL2 functionally complement an S. cerevisiae sfl1 mutation C. albicansC. albicans Sfl1p showed that deletion of SFL1 promoted filamentous growth and cell flocculation and correlated with induction of HSGs  and genes involved in cell adhesion , whereas its overexpression inhibited hyphal formation lacZ reporter analyses in C. albicans correlated with a repressor function SFL1 attenuated C. albicans virulence in a mouse model of systemic infection SFL2 impaired filamentation in response to different cues, whereas SFL2 overexpression promoted hyphal growth, even under non hyphae-stimulating conditions sfl2Δ/sfl2Δ strain exhibited reduced damage in a reconstituted human oral epithelium model and displayed attenuated virulence in a mouse model of gastrointestinal colonization and dissemination model C. albicans pathogenesis. Similar to Sfl1p, an Sfl2p-GFP fusion localized to the nucleus, in line with a role in transcriptional regulation In the yeast SFL1 and SFL2 were shown to genetically interact with at least transcription factor FLO8. Hyphal development in sfl1Δ/sfl1Δ was abolished upon deletion of FLO8 but enhanced upon FLO8 overexpression SFL2 triggered filamentation in a FLO8- and EFG1-dependent manner SFL2 is required for hyphal maintenance at high temperature and that a temperature increase from 25°C to 37°C leads to upregulation of both the RNA and protein levels of Sfl2p, indicating that Sfl2p is a temperature-responsive regulator et al. showed that the putative HSF domains of Sfl1p and Sfl2p were required for their functional divergence by testing HSF domain-swapped hybrids for their ability to retain their effect on filamentation C. albicans morphogenesis and, potentially, pathogenesis and virulence.It is still unknown how Sfl1p and Sfl2p exert their antagonistic functions. Both in vivo, by chromatin immunoprecipitation. To this end, we generated triple-hemagglutinin epitope (HA3)-tagged versions of SFL1 and SFL2 and used the pCaEXP system MET3 promoter-dependent expression of the tagged alleles in sfl1Δ/sfl1Δ (sfl1-CaEXP-3SFL1-HA) and sfl2Δ/sfl2Δ (sfl2-CaEXP-3SFL2-HA) mutant strains, respectively  lacking methionine (PMET3-inducing condition) at 37°C and allowed to resume growth during 4 h prior to microscopic examination  software We performed genome-wide location of Sfl1p or Sfl2p under hyphae-inducing conditions by chromatin immunoprecipitation coupled to massively parallel high-throughput sequencing  and Sfl2p (28 peaks) binding peaks were not detected by the peak-finding algorithm and were added to our target lists  was also among the most overrepresented categories of the identified GO terms and included the above-mentioned transcription factors in addition to HMS1, encoding a transcription factor that controls C. albicans morphogenesis mediated by HSP90 compromise or high temperature MSB2, CHT2, GAP1, ALS1, RAS2, others (P = 2.40×10−4) was also among the most significantly enriched functional categories among Sfl1p and Sfl2p common targets and is consistent with the known roles of Sfl1p and Sfl2p in C. albicans virulence WOR2, FLO8, EFG1 and AHR1 , as well as genes involved in biofilm formation , suggesting wider functions for these two regulators in C. albicans. These functions may include the ability to respond to a variety of stimuli, such as drug treatment , nutrient availability  and pH levels .We used the GO Term Finder tool from the CGD s genome . Strikin, others . In line, others . As expeP = 2.06×10−5), “Symbiosis, encompassing mutualism through parasitism” , “Adhesion to host”  and “Fungal-type cell wall” . Sfl2p also bound specifically to 11 genes encoding transcription factors such as CPH2, ECM22, CZF1, FCR3, RFX2 and ROB1  on selected targets . The URAand TEC1 . The profl1p-HA3 .C. albicans morphogenesis and potentially confer virulence through direct binding to the promoter of genes encoding key regulators of these processes. They also revealed that, while both transcription factors bind to common targets, Sfl2p specifically binds to additional target genes that appear to be involved in processes pertaining to interaction with the host.Taken together, our results suggest that Sfl1p and Sfl2p regulate sfl1-CaEXP-3SFL1-HA versus sfl1-CaEXP and sfl2-CaEXP-3SFL2-HA versus sfl2-CaEXP grown 3 times independently under the same conditions than those in the ChIP-Seq experiments (see P≤0.05) in the sfl1-CaEXP-3SFL1-HA strain as compared to strain sfl1-CaEXP (Table S7 in P≤0.05) in strain sfl2-CaEXP-3SFL2-HA relative to sfl2-CaEXP  functional TAP (tandem affinity purification)-tagged SFL1 or SFL2 alleles PCK1 promoter (PCK1-inducing) conditions during 0, 2 and 4 hours and total RNA was isolated followed by qRT-PCR  and increased expression of UME6 and ALS3 (UME6 and ALS3 panels), consistent with our microarray data  as well as genes encoding or predicted to encode permeases or transporters .Sfl2p binding to its 75 specific target genes correlated with increased and decreased expression of 24 and 25 genes, respectively . StrikinC. albicans morphogenesis, iii) show that Sfl2p additionally specifically controls the expression of HSGs and iv) reveal a direct SFL1-SFL2 cross-factor negative control.Taken together, combination of the ChIP-Seq and the transcriptomics data i) indicate that Sfl1p and Sfl2p have dual transcriptional regulatory functions, acting as both activators and repressors, ii) suggest that Sfl1p and Sfl2p antagonistic functions in regulating hyphal morphogenesis is mediated through direct transcriptional modulation of genes encoding key regulators of C. albicans morphogenesis and virulence fostered us to assess the genetic interactions between SFL1, SFL2 and these target genes. Data mining of our ChIP-Seq and transcriptomics results showed that Sfl1p directly negatively regulates SFL2 expression  and the Efg1p (5′-taTGCAta-3′) binding motifs 5′-TtCtaGaA-3′ and 5′-TCGAACCC-3′, carrying GAA triplets that are characteristic of HSEs  >10 for RSAT analyses and >25 for SCOPE analyses). Ndt80p is a transcription factor that controls the expression of genes involved in many cellular processes, including drug resistance, cell separation, morphogenesis and virulence through the recognition of mid-sporulation elements on the promoter of its targets 5′-TCGAACCC-3′ or 5′-TtCtaGaA-3′. We searched the YeTFaSCo and the JASPAR databases for similarity with known transcription factor binding sites 5′-TtCtaGaA-3′ sequence was strongly similar to the S. cerevisiae Hsf1p motif , while database searches did not identify any known motif that closely resembled the 5′-TCGAACCC-3′ sequence (data not shown). On the other hand, we found 3 high-scoring motifs in Sfl2p-enriched sequences, including the Efg1p and Ndt80p binding motifs as well as the GAA-containing sequence, 5′-aaNAATAGAA-3′  . All these identified motifs were distributed preferentially around the center of the sequences corresponding to peak locations  . To confTAGAA-3′ . The 5′-ocations , suggestet al. under yeast-form growth conditions at 30°C et al. during both yeast-form growth (30°C) and hyphal induction (YP serum at 37°C) C. albicans, with rare or no overlap under hyphal induction and increased overlap under yeast-form growth   Both Sfl1p and Sfl2p proteins physically associate with Efg1p, in vivo.Taken together, our results show that i) the Efg1p protein binds to many Sfl1p and Sfl2p targets, in vivo approaches that, when combined, allow to provide mechanistic insights into the function of transcriptional regulators. When associated with both genetic and physical interaction analyses, the overall generated data are cross-validated and provide a comprehensive view of the regulatory interactions within transcriptional networks. They also shed more light into the epistatic relationships to explain the phenotypes associated with transcription factor function. In the present report, we used such approaches to decipher the regulatory network of two HSF-type transcription factors, Sfl1p and Sfl2p, both required for C. albicans virulence but with antagonistic functions in regulating C. albicans morphogenesis. One limitation of our ChIP-Seq design was the use of ectopic promoter-driven expression of the 3SFL1-HA and 3SFL2-HA alleles , transcriptional activators of hyphal growth  and yeast-form associated genes . While Sfl1p exerts direct negative and positive regulation on the expression of activators  and repressors  of hyphal growth, respectively, Sfl2p directly upregulates and downregulates the expression of positive  and negative  regulators of hyphal growth, respectively  whereas Sfl2p directly downregulates their expression , SFL2  and their target genes  and yeast-form specific genes   and multiple input motifs in Sfl1p or Sfl2p transcriptional network dictates the commitment of the C. albicans cells to form hyphae or yeast-form cells. This model is consistent with Sfl1p and Sfl2p acting as “switch on/off” proteins, with Sfl1p directly turning off the expression of positive regulators of hyphal growth while turning on the expression of both yeast-form associated genes and genes encoding repressors of hyphal development, whereas Sfl2p directly turns on the expression of HSGs and positive regulators of hyphal growth while turning off the expression of yeast-form associated genes as well as negative regulators of hyphal development  . We prop1, RHD3)  and 10, nditions  could benditions , 8 and 9et genes . On the et genes , provideelopment .SFL2 RNA levels were undetectable by Northern blotting at either 25°C or 30°C, but were greatly enhanced upon temperature increase vice versa for Sfl1p  as well as interaction with multiple protein partners, followed by recruitment of the co-activating mediator complex and initiation of the transcriptional process or Sfl1p . Based oor Sfl1p . A tempeor Sfl1p . As we sor Sfl1p  and 10, or Sfl1p and 10 isC. albicans genes under yeast-form growth conditions, suggesting wide functions for Ndt80p C. albicans ndt80Δ/ndt80Δ mutant is unable to form true hyphae under different filamentation-inducing conditions and, in the presence of serum at 37°C, it fails to activate the expression of HSGs, including HWP1, ECE1, RBT4, ALS3, HYR1 and SAP4TEC1 and UME6 which are both directly modulated by Sfl1p and Sfl2p  transcription factor, has been recently isolated in a screen aimed at identifying transcription factors whose function is required for the HSP90- or high temperature-mediated filamentous growth HMS1 gene, while Sfl2p downregulates its expression (HSP90-dependent filamentation response (at least under the conditions used in the present study). Similar to Sfl2p, Hsf1p is an HSF-type transcription factor that induces transcription following a temperature increase, but, unlike SFL1 and SFL2, HSF1 is essential for viability HSP104, HSP90, HSP70 as well as other classical heat-shock protein (HSP)-encoding genes such as HSP60, HSP78, others C. albicans to efficiently act as an opportunistic yeast of warm-blooded animals through converting temperature-sensing inputs into a morphogenesis programming output using HSF-type regulators like Sfl1p and Sfl2p. Nevertheless, we detected Sfl1p and Sfl2p binding at the promoter of the HSP104, HSP70 and SIS1 genes , suggesting that a reminiscent classical heat-shock response may have been retained in Sfl1p and Sfl2p. It is intriguing that one of the two potential binding motifs of Sfl1p  mep1Δ/mep1Δ mep2Δ/mep2Δ (encoding ammonium permeases) filamentous defect S. cerevisiae. Interestingly, Mga1p was shown to act as a master regulator of S. cerevisiae pseudohyphal development through direct transcriptional control of key genes involved in morphogenesis S. cerevisiae Mga1p, although either SFL1 or SFL2 could complement an sfl1Δ mutation and SFL2 could not complement the pseudohyphal growth defect of an mga1Δ mutant 5′-AtAGAACA-3′ for Mga1p 5′-ANATAGAA-3′ for Sfl2p . Third, the regulatory networks to which they belong are intriguingly similar: Mga1p establishes cross talks with major regulators of S. cerevisiae pseudohyphal growth including Phd1p, Sok2p (Efg1p orthologs), Flo8p and Tec1p, as in the case of Sfl2p . Secondof Sfl2p  [39],  [pH 8.0], 80% ethanol) and elution (4 mM Phosphate buffer [K2HPO4/KH2PO4O4] [pH 8.5]) buffers. The purified first-strand cDNAs were subsequently labelled with the monoreactive Cy dye N-hydroxysuccinimide esters Cy3  and Cy5 (cDNA from strains sfl1-CaEXP-3SFL1-HA or sfl2-CaEXP-3SFL2-HA)  and the uncoupled dye was removed using the standard Qiagen PCR purification kit protocol. The Cy3- and Cy5-labeled cDNA lyophilized pellets were resuspended in 10 µl of DNase-free water then 2.5 µl and 12.5 µl of 10X blocking agent and 2X hybridization buffer (Agilent Technologies), respectively, were added. The resulting samples were mixed, incubated at 95°C during 3 min and snap cooled on ice during 1 min then hybridized to a Candida albicans expression array (Agilent Technologies) designed such that two nonoverlapping probe sets are targeting each of 6,105 C. albicans ORFs for a total of 15,744 probes, thereby allowing two independent measurements of the mRNA level for a given gene .Prior to first-strand cDNA synthesis, the purity and concentration of RNA samples were determined from A260/A280 readings  and RNA integrity was determined by a Bioanalyzer 2100 instrument (Agilent Technologies) per the manufacturer's instructions (RNA concentration was ranging between 7.92 and 10.48 µg/µl). First-strand cDNA was synthesized from 20 µg total RNA, using the Superscript III indirect cDNA labeling system (Invitrogen) with the following minor modifications to the manufacturers' instructions. Briefly, the Qiagen PCR Purification kit was used to remove unincorporated aminoallyl-dUTP and free amines with substitution of the Qiagen-supplied buffers with phosphate wash . Images were subsequently analyzed with the GenePix Pro 6.1.0.2 software . GenePix Results (GPR) files were imported into the Arraypipe 2.0 sfl1Δ/sfl1Δ) and CEC1997 (sfl1Δ/sfl1Δ PPCK1-SFL1-TAP) or CEC1535 (sfl2Δ/sfl2Δ) and CEC1509 (sfl2Δ/sfl2Δ PPCK1-SFL2-TAP) (PCK1-inducing conditions). Cells from 100 mL cultures were mechanically disrupted with glass beads using a Fastprep  and total RNA was extracted using RNAeasy (QIAGEN) according to the manufacturer's instructions. The quality and quantity of the isolated RNA were determined using an Agilent 2100 Bioanalyzer. Before cDNA synthesis, total RNA samples were DNase-treated using the Turbo DNA-free kit (Ambion). 2 µg of total RNA were used to perform cDNA synthesis using Superscript II Reverse Transcriptase according to the manufacturer's instructions (Invitrogen). Quantitative PCR was carried out on a Mastercycler ep realplex (Eppendorf) with a 2X SYBR Green master mix . The oligonucleotide primers used are listed in Table S9 in C. albicans translation elongation factor CEF3 transcript as a calibrator. The relative expression was calculated as 2CEF3 CEC1509 or CEC1997) – (Ct target– Ct CEF3 CEC1535 or CEC2001)  during aStrains co-expressing Sfl1p-TAP and Efg1p-HA or Sfl2p-TAP and Efg1p-HA . The orf19 list of the Sfl1p and Sfl2p common targets or the orf19 list of the Sfl2p-specific targets was used as input for functional grouping. To decide which of the two ORFs sharing the same bound promoter are included among the GO-term finder input list, we selected those ORFs showing differential expression in Sfl1p and Sfl2p transcriptomics data . This led to a list of 110 (Sfl1p and Sfl2p common targets) and 73 (Sfl2p specific targets) genes for GO term enrichment analyses  http://rsat.ulb.ac.be/rsat/) peak-motifs algorithm C. albicans (genome sequence available at www.broad.mit.edu/annotation/genome/);“fixed” was selected for the upstream sequence control set and both strands of the DNA sequence inputs were searched for motif discovery.Gene Ontology functional enrichment analyses were conducted using the CGD Gene Ontology (GO) Term Finder tool  or ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) databases under series numbers GSE42886 or E-MEXP-3779, respectively.ChIP-Seq and microarray data can be found at the Gene Expression Omnibus  Strains SFL1-TAP (CEC1922), SFL2-TAP (CEC1918) and EFG1-HA (HLCEEFG1), carrying chromosomally tagged SFL1 , SFL2  and EFG1  alleles were grown in SC medium at 30°C or Lee's medium at 37°C during 4 h together with the SC5314 strain as a control (CTRL) prior to microscopic examination (40× magnification). (B) Western blot (WB) analyses of strains SFL1-TAP, SFL2-TAP (upper panel) and EFG1-HA (lower panel) together with the SC5314 control strain (CTRL). Strains were grown in SC medium at 30°C (30°C) or in Lee's medium at 37°C (37°C) during 4 h and total protein extracts were prepared then subjected to SDS-PAGE. Western blotting was performed using an anti-TAP antibody  or an anti HA antibody , Roche). Positions of the molecular mass standards are indicated on the left (kDa). Antibody cross-reacting signals were used as a loading control (Loading Control).(TIF)Click here for additional data file.Text S1Includes Tables S1–S9 and full description of Tables S1–S9.(XLSX)Click here for additional data file.