Arabidopsis thaliana, regulators of floral determinacy include several transcription factor genes, such as APETALA2 (AP2), AGAMOUS (AG), SUPERMAN (SUP), and CRABSCLAW (CRC), as well as a microRNA (miRNA), miR172, which targets AP2. How the transcription factor and miRNA genes are coordinately regulated to achieve floral determinacy is unknown. A mutation in POWERDRESS (PWR), a previously uncharacterized gene encoding a SANT-domain-containing protein, was isolated in this study as an enhancer of the weakly indeterminate ag-10 allele. PWR was found to promote the transcription of CRC, MIR172a, b, and c and/or enhance Pol II occupancy at their promoters, without affecting MIR172d or e. A mutation in mature miR172d was additionally found to enhance the determinacy defects of ag-10 in an AP2-dependent manner, providing direct evidence that miR172d is functional in repressing AP2 and thereby contributes to floral determinacy. Thus, while PWR promotes floral determinacy by enhancing the expression of three of the five MIR172 members as well as CRC, MIR172d, whose expression is PWR-independent, also functions in floral stem cell termination. Taken together, these findings demonstrate how transcriptional diversification and functional redundancy of a miRNA family along with PWR-mediated co-regulation of miRNA and transcription factor genes contribute to the robustness of the floral determinacy network.Termination of the stem cells in the floral meristem  is critical for the reproductive success of plants, and the molecular activities regulating floral determinacy are precisely orchestrated during the course of floral development. In  APETALA2 (AP2) and thereby promotes the determinate growth of flowers , we show that the five MIR172 genes undergo differential transcriptional regulation. POWERDRESS (PWR), a previously uncharacterized SANT-domain-containing protein, promotes floral determinacy by enhancing the expression of MIR172a-c. MIR172d, whose expression is PWR-independent, was found to be functional in floral determinacy by repressing AP2. PWR also promotes floral determinacy through a transcription factor previously implicated in this process. Thus, transcriptional diversification of a miRNA family and PWR-mediated co-regulation of miRNA and transcription factor genes involved in floral determinacy contribute to the robustness of this developmental network.microRNAs (miRNAs) are 20–24 nucleotide RNAs that play regulatory roles in many developmental processes in plants and animals. Some miRNAs are encoded by multi-member gene families, and the members may exhibit differential expression patterns. However, the basis of this expression diversification and its developmental impact are poorly understood. By studying miR172, which represses its target  Arabidopsis thaliana, the floral homeotic gene AGAMOUS (AG) is required for both reproductive organ specification and floral determinacy. AG's role in floral determinacy involves the negative regulation of the stem-cell-promoting gene WUSCHEL (WUS) in the floral meristem, via direct and indirect mechanisms Flowers are a key adaptation of the angiosperm lineage that helps ensure reproductive success. At a particular point in floral development, stem cells in the floral meristem are terminated, thereby preventing the continual growth that typifies the shoot and root apical meristems of plants. In CRC's dual functions in determinacy and carpel development are notably similar to those of AG, and the subtly indeterminate crc-1 mutant background has helped uncover the involvement of a number of other genes in floral determinacy AP2 encodes an AP2-domain-containing transcription factor and is regulated by miR172. A role for AP2 in maintaining stem cell fate was first proposed in the shoot apical meristem AP2 was expressed and found to confer indeterminate floral organ production WUS to confer floral determinacy.Other known regulators of floral determinacy represent a wide range of functions and include signaling proteins, transcription factors, and microRNAs. Of particular importance for the present study are the YABBY transcription factor CRABSCLAW (CRC) and the floral homeotic transcription factor APETALA2 (AP2). MIR genes) typically reside between protein-coding genes and are transcribed by RNA polymerase II (Pol II) MIR genes typically belong to families with multiple members MIR gene families in plants indicates that their evolution has features in common with the evolution of protein-coding gene families. For example, analysis of the MIR156, MIR159, and MIR166 families revealed differences in the spatial and temporal expression of genes within these families, which suggests that expression diversification occurred after gene duplication miRNAs, regulatory molecules 20–24 nucleotide in length, play critical roles in various aspects of plant development through sequence-specific regulation of their targets MIR172 genes to these processes are unknown. Three unique mature miR172 sequences are produced from these five loci , and publicly available datasets indicate that the three mature miR172 species differentially accumulate in inflorescences, rosette leaves, seedlings, and siliques MIR172c and MIR172e in the outermost floral whorl by a transcriptional corepressor indicates that the MIR172 family members are differentially regulated in the flower miR172 regulates AP2 transcription factor genes involved in the distinct processes of flowering time and floral development MIR172 and transcription factor genes in the termination of the floral stem cells makes this developmental paradigm well suited for the investigation of how individual MIR family members contribute to the functions broadly assigned to the mature miRNA species. In the present study, the POWERDRESS (PWR) gene was found to promote floral determinacy through CRC and miR172. PWR promoted the transcription and/or enhanced Pol II occupancy at the promoters of MIR172a, b, and c but did not affect MIR172d or e. Coincidentally, a mutation in MIR172d was also found to enhance the determinacy defects of a weak ag allele. Taken together, these findings indicate that differential regulation of MIR172 genes with overlapping functions enhances the robustness of the genetic network underlying floral stem cell termination.The complex interplay of both ag-10 background, as previously reported ag-1 mutant, which produces sterile flowers that indeterminately produce sepals and petals ag-10 mutant has normal floral organ specification and slightly bulged gynoecia that very rarely, if at all, contain additional floral organs inside  were observed along with the presence of ectopic floral organs in all of the dissected gynoecia, indicating a consistent enhancement of the floral determinacy defect of ag-10 (http://arabidopsis.org). SANT domains have high structural similarity to Myb DNA-binding domains but have been characterized as histone-binding domains important for chromatin remodeling activity in both plants and animals POWERDRESS (PWR) , and the ag-10 enhancer mutation was designated pwr-1. The floral determinacy defects of ag-10 pwr-1 were rescued by transformation with an 8.1-kb genomic PWR fragment. As with ag-10 plants, the flowers from five independent pPWR:PWR-GFP lines in the ag-10 pwr-1 background rarely produced gynoecia containing ectopic floral organs, and elongated gynophores were not observed , was obtained from the Arabidopsis Biological Resource Center PWR  background, Colag-10 siliques are nearly indistinguishable from wild-type siliques, indicating the presence of a genetic suppressor of determinacy defects in Col Col pwr-2ag-10 double-mutant flowers were found to exhibit weak determinacy defects, namely, the infrequent production of visibly bulged gynoecia containing additional floral organs inside  background, SALK_071811C .Both 1 petals  versus a2 petals ), pleiotPWR in Ler wild-type roots, rosette leaves, cauline leaves, and inflorescence tissues revealed high PWR transcript abundance in inflorescences , SUPPRESSOR OF CONSTANS 1 (SOC1), CAULIFLOWER (CAL), and SEPALLATA4 (SEP4), were observed in pwr-2   . FT acts mutants .pwr-2 were three direct targets of the floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI): BANQUO1 (BNQ1) and BNQ2 and NAC-LIKE, ACTIVATED BY AP3/PI (NAP) AP3 and/or PI function, neither of them was identified as having a significant change in expression in the microarray analysis. Real-time RT-PCR analysis for PI and AP3 in pwr-2 revealed no significant change in PI transcript levels and a small reduction for AP3 , these transcript level changes may provide a starting point for investigating the petal defects observed in pwr mutants.Another related group of genes with altered transcript levels in  for AP3 , which mPWR acts in any of the known pathways regulating floral determinacy, genetic analyses were carried out by combining pwr-1 and pwr-1 ag-10 with loss-of-function mutations in CLAVATA3 (CLV3), SUPERMAN (SUP), WUS, AG, CRC, and AP2.To address whether clv3-1 was combined with ag-10 pwr-1. clv3-1 single mutant flowers produce slightly larger numbers of each floral organ type clv3-1 ag-10 flowers resembled clv3-1 flowers . In addition to this analysis, crosses to a pWUS:GUS reporter line (for β-glucuronidase) containing a 3.2-kb WUS promoter ag-10 background, GUS expression was only observed in approximately 10% of flowers beyond stage 6  showed prolonged GUS expression in late stage floral meristems  of the examined miRNA species in both pwr alleles  and pwr-1 plants harboring a p35S:MIR172a transgene  (CRC was examined by chromatin immunoprecipitation (ChIP). Pol II occupancy was clearly reduced at the MIR172b, MIR172c, and CRC loci in pwr-1  . The occin pwr-1 , while o67a loci . These fpwr mutants, the mutation in another enhancer isolated from the ag-10 genetic screen was mapped to the MIR172d locus. A G-to-A mutation was identified at the ninth position of the 21-nucleotide sequence that corresponds to the mature miRNA , suggesting that other MIR172 genes are sufficient to confer this developmental function.miR172 is also known to promote flowering by targeting several members of the PWR, confers floral determinacy through two distinct players: miR172 and CRC  or MIR172d each has little effect on proper floral determinacy in the wild-type background.The termination of the floral meristem requires various regulatory factors whose functions are orchestrated during the different stages of floral development. The data presented here indicate that a previously uncharacterized gene,  and CRC , which at of PWR , also afpwr mutants, the other pleiotropic defects initially observed in pwr-1 ag-10, including early flowering, petal defects, and small plant size, were observed in pwr-1. Some preliminary hypotheses can be drawn from the microarray and transcript level analysis, including the possible role of increased FT expression and decreased AP3 expression in disrupted flowering time and petal development, respectively. Although further investigation of the pleiotropic defects of pwr is required, the data clearly indicate that the defects are not attributable to widespread changes in miRNA abundance, as is the case in miRNA biogenesis mutants. First, the early flowering phenotype of pwr is in contrast to the late-flowering phenotype expected for a reduction of miR172 and is instead consistent with the increased FT transcript levels observed. Second, the widespread reduction observed for pri-miRNA levels in pwr were not accompanied by widespread decreases in mature miRNA abundance. One possible explanation for this discrepancy is that decreases in precursor levels do not always correspond to decreases in mature miRNAs, due in part to differences in the post-transcriptional maturation mechanisms for individual miRNAs Although determinacy defects were not observed in single Arabidopsis and other organisms identified PWR orthologs in other plant species but no homologs in Arabidopsis. Furthermore, PWR was found to have higher amino acid sequence similarity to histone deacetylase (HDAC) complex proteins in animals than to any other proteins in Arabidopsis. Studies in animals and yeast have uncovered the involvement of SANT domain-containing enzyme subunits in chromatin remodeling activity, including histone acetylation, histone deacetylation, and ATP-dependent chromatin remodeling PWR and the spacing between them (173 amino acids) are similar to the domain structures of the HDAC subunits SMRT and N-CoR (nuclear receptor co-repressors), whose first and second SANT domains are necessary for HDAC activation and binding to unacetylated histone tails, respectively PWR function yields pleiotropic defects and changes in the transcript levels of otherwise unrelated genes, including MIR172B, CRC, and FT.The predominant feature of the predicted PWR protein is a pair of SANT domains encoded in the fourth and largest exon of the gene. A BLAST search for proteins similar to PWR in ap2-like phenotypes) or lines in which the miR172 target AP2 was made resistant to regulation by the miRNA AP2, it does not distinguish among the mature miR172 produced from the five MIR172 loci. Previous reports of differences in the accumulation of the individual pri-miRNAs have suggested variations in their individual functions throughout the plant. For example, the pri-miR172b precursor is particularly abundant in the shoot apex at the induction of flowering miR172d-1 mutation provides direct genetic evidence for the role of miR172 as a positive regulator of floral determinacy, and the absence of flowering time defects in this mutant suggests that the developmental functions  assigned to miR172 can be uncoupled. Considered together with the effects of PWR on the transcription of some, but not all, of the MIR172 genes, these findings also demonstrate that the contributions of individual members of a miRNA family to one particular developmental process can be distinguished.In addition to its role in floral development, miR172 regulates flowering time as a negative regulator of floral repressors that belong to the AP2 transcription factor family erecta (Ler) ecotypic background, except for pwr-2, which is in the Columbia (Col) background, and Colag-10, which is the ag-10 mutation introgressed into Col through six consecutive backcrosses. All plants were grown at 23°C under long-day conditions . ag-1clv3-1sup-1ap2-2wus-1crc-1ag-10The mutants and transgenic lines used in this study are in the Landsberg pwr-1 and miR172d-1 were backcrossed into the ag-10 background and Ler two times prior to further analysis to reduce the number of extraneous background mutations. pwr-2 is a T-DNA insertion line, SALK_0713811C, obtained from the Arabidopsis Biological Resource Center ag-10 pwr-1 and ag-10 mir172d-1 mutants were isolated from the ag-10 screen and crossed to Colag-10 to generate the respective mapping populations. For ag-10 pwr-1, 20 F2 plants exhibiting the enhanced phenotype were used for rough mapping, and linkage was observed for the ciw4 marker on the lower arm of chromosome 3. The mapping population was expanded to 888 plants for fine mapping. SSLP and CAPS markers were designed based on identified polymorphisms between the Ler and Col ecotypes (http://arabidopsis.org/browse/Cereon/index.jsp). The mapping region was narrowed to a 154-kb window encompassing portions of two BAC clones, F4F15 and T25B15, and spanning 55 open reading frames. A G-to-A mutation that produces a premature stop codon in At3g52250/PWR was identified by sequencing analysis. For ag-10 mir172d-1, 24 F2 plants exhibiting the enhanced phenotype were used for rough mapping, and linkage was again observed for the ciw4 marker on chromosome 3. Using an expanded mapping population of approximately 200 plants, the mapping region was narrowed to an 1,100-kb window spanning portions of the BAC clones T26I12 and T16L24. The sequencing analysis revealed a G-to-A mutation in At3g55512/MIR172d.The pwr-1, the dCAPS primers PWRgeno-F and PWRgeno-R produce an HphI  site exclusively in the mutant. The pwr-2 mutation was genotyped using primers EN1_XcmIF and 3G52250R1, which fail to amplify a fragment in the homozygous mutant. The ag-10 mutation eliminates an HpyAV restriction site and was genotyped using primers AGp1 and ag10_genoR followed by HpyAV digestion . For miR172d-1, the primers R194geno-F and R194geno-R were used to amplify the genomic fragment. The fragment amplified from miR172d-1 fails to be cut by Hpy188III .The primer sequences used for genotyping are listed in pPWR:PWR-GFP, the PWR genomic region was amplified using primers EN1_full_CACC and EN1cDNA_NS  and introduced into the pMDC107 Gateway-compatible vector ag-10 pwr-1 and pwr-1 plants were transformed with the plasmid by agroinfiltration for the phenotypic rescue analysis.To generate 1cDNA_NS . The 8.1http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and are listed in The harvesting of inflorescence tissue was consistently performed at the same time of day. RNA was extracted using TRI reagent , and DNA was removed using DNase I . Oligo-dT primer  and reverse transcriptase  were used to synthesize cDNA. All procedures used were according to the manufacturers' instructions. Quantitative PCR was performed in triplicate on a Bio-Rad IQ cycler apparatus using iQ SYBR Green Supermix . The primers used for real-time RT-PCR were previously described or designed using the Primer3 online tool (32P antisense DNA oligonucleotides were used to detect mature miRNA species. U6 was used as the loading control. The oligonucleotide probes used were previously described http://rsbweb.nih.gov/ij/).The hybridization and detection of miRNAs were performed as previously described Total RNA extracted from inflorescences was purified using the RNeasy Plant Mini Kit . Affymetrix Gene Chip probe preparation, hybridization, and quality control were performed according to the manufacturer's instructions. The SimpleAffy package available for the R language was used to analyze the microarray data, and expression values were determined using the MAS 5.0 method as previously described 4, pH 7.2; 0.5 mM K3Fe(CN)6; and 0.5 mM K4Fe(CN)6]. After the infiltration solution  was added, the inflorescences were vacuum infiltrated for 10 min then incubated at 37°C overnight.GUS staining was performed as previously described 2 and 0.5% Triton X-100) and one time with M3 buffer . The chromatin was resuspended in nuclei lysis buffer and sonicated to generate DNA fragments approximately 500 bp in length. The lysate was pre-cleared by incubation with 50 µL protein-A agarose beads/salmon sperm DNA (Millipore) for 1 h then incubated with anti-RBP2 antibody (Abcam 10338) overnight. The bound DNA fragments were recovered and purified using columns from the Qiagen Plasmid Extraction Kit according to the manufacturer's instructions. Quantitative real-time PCR was performed on bound and input DNAs. The primers used are listed in ChIP was performed as previously described AP2 3′ cleavage products were amplified and sequenced. For amplification, a primer specific to the 5′ adaptor and an AP2-specific primer were used.Total RNA was extracted from dissected inflorescences, and mRNA was isolated using the Sera-Mag Magnetic Oligo(dT) kit (Thermo Scientific) according to the manufacturer's instructions. 5′ RACE was performed using the GeneRacer Kit (Invitrogen) according to the manufacturer's instructions. Briefly, an RNA oligonucleotide adaptor was ligated to the isolated mRNAs, and reverse transcription was conducted using oligo-dT. Figure S1pwr-1 and mir172d-1 reduce the accumulation of the 3′ cleavage products from AP2 mRNA. The 3′ products resulting from cleavage of AP2 mRNA by miR172 were detected using 5′ RACE RT-PCR for Ler, ag-10, pwr-1, ag-10 pwr-1, and ag-10 mir172d-1. UBIQUITIN 5 was used as the loading control. ACTIN2 (ACT2) RT-PCR was conducted using an intron primer and an exon primer and served as a control for DNA contamination. The serial dilutions for Ler cDNA show that the PCR results are semi-quantitative (“1” stands for undiluted samples).(TIF)Click here for additional data file.Table S1pwr-2.Genes down-regulated in (PDF)Click here for additional data file.Table S2pwr-2.Genes up-regulated in (PDF)Click here for additional data file.Table S3Sequences of oligonucleotide used in this study.(PDF)Click here for additional data file.


Cells and unicellular organisms are similar to their progenitors because information is transmitted from one generation to the next. The information is mainly transmitted in the primary sequence of the genome (genetic information), but there are heritable traits that are transmitted by other mechanisms. Epigenetics studies these alternative modes of inheritance. According to classic definitions, epigenetics refers to heritable differences between cells or organisms that occur without changes in DNA sequence, and do not depend on different external conditions Epigenetic information can be transmitted by several different molecular mechanisms, which include but are not limited to DNA methylation and histone post-translational modifications (PTMs). However, the term “epigenetics” is often used to refer to any transcriptional regulation mechanism that involves histone PTMs or other chromatin-based processes, and the study of chromatin modifications on a genome-wide level is commonly termed “epigenomics”. Several voices have questioned this use of the word epigenetics, because many histone PTMs do not carry heritable information Plasmodium falciparum  P. falciparum also occurs in many other gene families linked to different processes such as erythrocyte invasion, nutrient transport, protein folding, and lipid metabolism Clonally variant gene expression (CVGE) lies at the base of a bet-hedging adaptive strategy consisting of the stochastic generation of phenotypic diversity followed by natural selection upon environmental changes  can maintain a variant gene  in a different transcriptional state, active or silenced, and this state will be transmitted to the next generations (with infrequent switches). This has been clearly demonstrated by subcloning experiments , and the histone variant H2A.Z all contribute to specify the active or silenced state of a var gene P. falciparumP. falciparum, and conceivably they may play a role in the control of CVGE cis-acting DNA elements are bound by the ApiAP2 factor PfSIP2, and this interaction plays a likely role in var gene regulation var genes, ncRNAs initiated from var introns are involved in the control of a mutually exclusive expression program Plasmodium ncRNAs still awaits experimental characterization. Another important layer of CVGE regulation is subnuclear localization. Activation of a var gene is associated with spatial repositioning within the nucleus to a specific perinuclear active site, possibly involving dissociation from telomeric clusters var intron and actin Plasmodium parasites undergo closed mitosis and that their chromosomes do not condensate during nuclear division The molecular players involved in regulating histone PTM patterns in CVGE have been partially characterized only for Plasmodium biology are regulated at the transcriptional level and are associated with changes in chromatin structure. A key process that is largely controlled at the transcriptional level is the progression along the life cycle, which involves multiple stages in two different hosts, the Anopheles mosquito and humans P. falciparum asexual cycle progression comes from studies that demonstrated massive alterations in temporal transcription profiles upon treatment of parasites with histone deacetylase or histone acetyltransferase inhibitors Histone PTMs and other chromatin modifications are key regulators of transcription in eukaryotes Plasmodium lies within the DNA sequence, both in regulatory cis-acting modules and in the genes encoding transcription factor networks. This hypothesis is supported by the observation that promoters on transfected plasmids retain correct stage-specific patterns of activity P. falciparum relative to its genome size Plasmodium parasites. However, the identification of the ApiAP2 family of transcription factors, which conceivably has sufficient members to coordinate cascades of gene expression trans-activator binding How these chromatin modifications are regulated remains unclear, but it is unlikely that they transmit information for cycle progression from one generation of asexual parasites to the next . Progresvar genes are silenced by default in the majority of parasites, regardless of the state of the endogenous promoter, at least when coupled to a second transcriptional unit such as the var intron trans-acting factors, dictate temporal regulation, whereas chromatin conformation, which does not necessarily coincide between episomal and endogenous promoters, regulates CVGE.In transfection experiments, naked plasmid DNA produced in bacteria is introduced into parasite nuclei, and chromatin is assembled de novo on the episome during S phase. In transient transfection experiments, promoter activity is analyzed in the first or second cycle after transfection, whereas in stable transfection experiments episomes are maintained for many generations. In both types of experiments, the episomes do not carry epigenetic information from before transfection. However, temporal regulation of promoter activity is maintained in plasmid promoters, recapitulating the correct stage-specificity of the endogenous promoter  [36], [7P. falciparum the H3K4me3 and H3K9ac marks as well as the histone variant H2A.Z define euchromatic intergenic regions P. falciparum these modifications typically occupy the full intergenic region Plasmodium promoters recapitulates the function of the endogenous elements P. falciparum chromosome ends are demarcated by H3K9me3/HP1, similar to other eukaryotes, but the centromeres of this parasite are unusual because they are characterized by a specific sequence signature and contain CenH3 but not pericentric heterochromatin P. falciparum.The demarcation of different functional elements within the genome is another process in which chromatin modifications are involved, including histone PTMs, histone variants, and specific nucleosome positioning. In model eukaryotes, elements of the genome with particular functions in regulating transcription  show specific chromatin features P. falciparum is able to sense the environment and produce adaptive transcriptional responses remains controversial P. falciparumYet another process where chromatin modifications play an important role in many eukaryotes is adaptation via directed transcriptional responses. When an external signal or condition is sensed, resulting in an adaptive transcriptional response, it often involves chromatin alterations P. falciparum asexual blood cycle because chromatin modifications and CVGE have only been characterized in some detail during this part of the life cycle. Likewise, little is known about epigenetics and chromatin modifications in other Plasmodium species. Chromatin changes undoubtedly play a key role in multiple aspects of parasite biology. However, to date CVGE has emerged as the only regulatory process where there is actual flow of epigenetic information from one generation to the next through heritable chromatin modifications. Importantly, CVGE is also the only known chromatin-based process for which alternative options for the parasite exist, i.e., to express or not to express a clonally variant gene. Chromatin modifications confer the ability to “remember” the option chosen in the previous generation. In contrast, in other processes such as asexual blood cycle progression, the recruitment of chromatin modifying enzymes and the resulting nucleosome modifications are downstream regulatory events that do not transmit differential information between genetically identical parasites. There are no alternative options for the parasite, and the single possible option is dictated by the genetic program, which makes epigenetic transmission of information unnecessary. This situation is different from development in multicellular eukaryotes, where in different cell types the same genome will translate into different stable patterns of gene expression that must be “remembered” over multiple generations.Most of this opinion article focused on the An important consideration is that some of the same chromatin modifications that we consider unlikely to carry epigenetic information in processes such as stage-specific transcription or genome indexing  are also used in the epigenetic inheritance of CVGE and are thus involved in both types of processes Plasmodium epigenetics has gained momentum in recent years, our knowledge in this field is still very limited and much remains to be discovered. We still do not understand the actual mechanisms underlying the establishment and maintenance of alternative states of transcription in CVGE. Furthermore, epigenetic inheritance and/or CVGE are likely to play an important role in other life cycle stages where alternative decisions are made, such as gametocyte conversion or P. vivax hypnozoite formation.Although research on


Steatohepatitis occurs in alcoholic liver disease and may progress to liver cirrhosis and hepatocellular carcinoma. Its molecular pathogenesis is to a large degree unknown. Histone modifications play a key role in transcriptional regulations as marks for silencing and activation of gene expression and as marks for functional elements. Many transcription factors (TFs) are crucial for the control of the genes involved in metabolism, and abnormality in their function may lead to disease.We performed ChIP-seq of the histone modifications H3K4me1, H3K4me3 and H3K27ac and a candidate transcription factor (USF1) in liver tissue from patients with steatohepatitis and normal livers and correlated results to mRNA-expression and genotypes.We found several regions that are differentially enriched for histone modifications between disease and normal tissue, and qRT-PCR results indicated that the expression of the tested genes strongly correlated with differential enrichment of histone modifications but is independent of USF1 enrichment. By gene ontology analysis of differentially modified genes we found many disease associated genes, some of which had previously been implicated in the etiology of steatohepatitis. Importantly, the genes associated to the strongest histone peaks in the patient were over-represented in cancer specific pathways suggesting that the tissue was on a path to develop to cancer, a common complication to the disease. We also found several novel SNPs and GWAS catalogue SNPs that are candidates to be functional and therefore needs further study.In summary we find that analysis of chromatin features in tissue samples provides insight into disease mechanisms. CYP2E1) and aldehyde dehydrogenase have an increased level of transcription in NASH patients compared to controls [PNPLA3 gene was strongly associated with increased hepatic fat levels and with hepatic inflammation [NCAN, GCKR and LYPLAL1[Alcoholic steatohepatitsis (ASH) is a chronic liver disease that develops in approximately 20% of heavy drinkers. ASH leads to characteristic morphological alterations in the liver, such as accumulation of fat (steatosis), ballooning of hepatocytes, appearance of hepatocytic protein aggregates , necrosis and apoptosis, fibrosis, inflammation, and cholestasis. These alterations are similar to steatohepatitis that develops in patients without alcoholic liver diseases  in the context of obesity, diabetes and the metabolic syndrome . The simcontrols . Genome-ammation . Anotherd LYPLAL1.USF1 have been associated to the disease familial combined hyperlipidemia [Chromatin Immunoprecipitation coupled with high throughput sequencing (ChIP-seq) allows the study of binding sites for TFs or sites of modified histones to be identified in a single experiment on a genome-wide scale. In this study, we used bio-bank samples from patients with steatohepatitis and compared it to samples from healthy controls. N-terminal modifications of histones may change the chromatin accessibility to transcription and they are associated with activation and silencing of the genes . We compipidemia  and furtipidemia . We therWe find that the pattern of histone modifications was similar when comparing normal liver tissue and alcoholic steatohepatitis. However, in differentially enriched regions we find genes known to be involved in the disease as well as new genes that are candidates to contribute to the pathology. In particular, in the patient we find genes in cancer pathways suggesting that the tissue is en route to a malignancy, a known complication to the disease.Frozen liver tissue samples from three patients with ASH and three controls without chronic liver diseases (patients who underwent liver surgery because of liver metastasis from colon cancer) were obtained from Graz bio-bank, Medical university of Graz, Austria. The first control and patient set was used to map USF1, the second set was used to study the histone modifications and a third set along with the above two sets was used for RNA expression analysis by qRT-PCR.3 and 1% SDS) with vigorous shaking of beads at room temperature and crosslinks were reversed at 65°C with 0.3 M Sodium chloride and RNase A for 6 hours followed by 45°C overnight incubation with Proteinase K. The DNA was extracted by phenol/chloroform and ethanol precipitation and the pellet was dissolved in water. The ChIP DNA enrichment was verified by semi-quantitative PCR using two known positive and two negative targets.Frozen liver tissues were chopped into small pieces and crosslinked with a final concentration of 0.37% formaldehyde for 10 minutes and the crosslinking was stopped by adding glycine to a final concentration of 0.125 M. Crosslinked tissue was washed by 1X PBS and cell lysis buffer with protease inhibitors (PIs) was added and nuclei were prepared by using a dounce homogenizer. RIPA buffer  with PIs was added to the nuclei and chromatin was sonicated to 100-300 bp fragments using a BioRuptor (Diagenode). After preclearing with Protein-G-Agarose beads (Roche), chromatin was incubated with antibody overnight. Antibodies were from Santa Cruz Biotechnology for USF1 (sc-229) and from Abcam for histone modifications . Protein-G agarose beads were added and incubated for two hours and then the chromatin-antibody-bead complex was washed four times with RIPA buffer, once with ChIP wash buffer 2 (0.01 M Tris–HCl (pH 8), 0.25 M LiCl, 0.001 M EDTA, 1% NP-40, 1% Sodium deoxycholate) and once with TE buffer. The protein-DNA complex was eluted in IP elution buffer (0.1 M NaHCOSOLiD fragment libraries were prepared according to the manufacturer’s protocol. ChIP DNA fragments were end repaired and ligated with adapters. Ligated fragments were amplified using adapter specific primers and size selected on Invitrogen’s flash gel. Approximately 200-300 bp fragments (including adapters) were collected. These libraries were sequenced on the ABI SOLiDV3 platform.Pfu ultra high sensitivity master mix (Agilent) and adapter specific primers. The PCR product was size-selected on 2% TAE agarose gel and purified with Qiagen gel extraction columns. Qiagen MinElute columns were used for purification after end repair and A-tailing and AMPure XP beads were used to purify and size-select the ligation mix before amplification. Sequencing was done on Illumina Hiseq platform using paired-end technology. Quality and size range of libraries for both the platforms were checked on Bioanalyzer (Agilent technologies) before sequencing.ChIP enriched DNA was end repaired using end repair kit (Epicentre) at room temperature for 45 minutes, ‘A’ base was added to the 3′ ends of the fragments using Klenow (3′-5′ exo-) (NEB) by incubating at 37°C for 30 minutes. These fragments were ligated with Illumina adapters using quick ligase kit (NEB). Adapter ligated fragments were enriched by PCR with Primers were designed for the SNPs located near a motif for USF1 in peaks with potential differential enrichment. USF1 enriched ChIP DNA and genomic DNA of patient and control was amplified and purified and PCR products were sequenced using BigDye terminator v3.1 (Applied Bio system) and capillary electrophoresis on a ABI 3730XL DNA analyzer. Obtained sequences were analyzed by using Sequencher software.New replicate of ChIP was performed with the tissues from the same individuals using anti-USF1 antibody and primers for qPCR were designed around the USF1 enriched regions and the regions without enrichment. qPCR in triplicates was used to measure enrichment by comparing to a standard curve obtained for each primer by serial dilution of input DNA and to a background level calculated as the average + 2*standard deviation of the values for negative regions. Enrichment of more than two-fold over this background level was considered as positive.Total RNA was extracted from tissue samples using mammalian total RNA extraction kit (Sigma Aldrich). First Strand cDNA was synthesized from 5 μg of total RNA using the Maxima First Strand cDNA synthesis kit (Thermo Scientific). The reaction was performed at 50°C for 30 minutes and terminated by incubating at 85°C for 5 minutes. qPCR was performed with cDNA in triplicates and normalized to the house keeping genes GAPDH, β-Actin and RSP18.in silico to the average fragment length (170 bp) and the overlaps of the extended fragments were calculated throughout the genome. Thresholds for significance (more than 5 and 10 reads for ASH and control respectively) were set based on the number of reads used for peak calling and the enrichment compared to background to get comparable datasets for patient and control, and peaks of enrichment higher than the thresholds were identified. The center position with highest overlap count was identified in each peak and used as basis for motif discovery and location analysis. De novo motif analysis was done using the online version of MEME-ChIP (http://meme.nbcr.net/meme/cgi-bin/meme-chip.cgi) based on the 500 highest and lowest peaks for each dataset.The SOLiD 50 bp reads for USF1 were aligned to the hg18 reference genome using BFAST (v. 6.4) with the -A 3 setting which excludes reads with more than one best scoring alignment. We further removed all alignments with more than five mismatches in the first 40 colors and a mapping quality below 20. Only one read per start position was retained. Reads were extended http://picard.sourceforge.net). MACS was used to call peaks, and for differential enrichment analysis SICTIN [The Illumina reads for histone modifications were aligned to hg18 using bwa . Duplicas SICTIN  was usedbuild binary was used to transform the bed files into a binary format and make footprint to count the average number of reads for a region of +/− 1000 bp from the TSS. The gene annotations were collected from the Ensembl [H.sapiens 54_36p). Only the 19 950 genes annotated as protein coding were used. Each histone modification was normalized to have the same total sum over all 2001 positions, and then scaled so that the maximal observed enrichment had a value of 1.SICTIN  Ensembl  system  and SNPs in either sample that had been reported in GWASs related to certain keywords  were identified. In the GWAS database, only the strongest associations are reported when there are multiple hits in the same region. In order to also identify the weaker hits in the samples, we searched for SNPs in high LD with GWAS SNPs using the SNAP proxy search (http://www.broadinstitute.org/mpg/snap/ldsearch.php) with data from the CEU panel in the 1000 Genomes Pilot project, using an r2 threshold of 0.8 and a distance limit of 500 kb. The SNPs were annotated with whether they had overlap to a HM peak only in control, only in patient, in both, or in none.The annotated SNPs in patient and control were compared to “A Catalog of published Genome-Wide Association Studies”  between the ChIP-seq and qPCR enrichment indicating good quality in the data  for the three histone modifications Figure C. As exphttp://david.abcc.ncifcrf.gov/summary.jsp) [http://liverome.kobic.re.kr/index.php) [We used the web based functional annotation tool (DAVID) (ary.jsp)  for genedex.php)  for livehttp://cbl-gorilla.cs.technion.ac.il) [We have used another web based tool GORilla (n.ac.il)  to furthTPI1) catalyses the isomerisation of Glyceraldehyde 3 phosphate (G3P) and Dihydroxy acetone phosphate (DHAP) which is involved in glycolysis and in gluconeogenesis  had lower signal for activating histone modifications in the patient than in the control suggesting that the gene is inactivated in the patient  and the Toll like receptor (TLR4) , which is in line with previous studies, which have shown that there is an increased expression in patients for this gene. It should also be noted the involvement of TNF4a in cancer which is in line with our findings on cancer described above  and for transforming growth factor (TGFβ1), which are both known to mediate hepatic fibrosis, a well-known component of ASH [We have investigated the histone modification pattern of the genes involved in alcohol metabolism and alcohol-related liver disease in the patient and control. There were stronger peaks in the patient than the control for the histone modifications at the promoters for Monocyte differentiation antigen (t of ASH  (AdditioSingle Nucleotide Polymorphisms (SNPs) are associated with many common diseases such as diabetes and high blood pressure . SNPs anhttp://www.genome.gov/gwastudies)  , which had a significant USF1 peak only in ASH, and Oxysterol binding protein like 6 (OSBPL6) and Peroxisome proliferator-activator receptor γ (PPARγ) [PPARγ is a TF involved in lipid metabolism and treatment with ligands of PPARγ can improve NASH [ADK and OSBPL6 but for PPARγ higher USF1 signal was associated with lower expression  ,34, whicove NASH . We anal; PPARGC1a, HNF4a, ARL6IP4, ATAD2B, IGFBP1, IL6R, ELP3, TCEB3, ALAS1, PRKAR1B and IL15RA) and 1 gene had a significant change in the opposite direction . Four genes did not show any significant difference between ASH and control . Most of the tested genes are involved in lipid metabolism and cholesterol metabolism, directly or in the presence of other TFs. We have tested Peroxisome proliferator-activated receptor γ co-activator 1α (PPARGC1α) which is a TF previously known to be involved in developing of NAFLD [PPARGC1a gene polymorphisms and lower expression of PPARGC1a are important contributors of NAFLD [Gene expression levels can be predicted using histone modifications . To see 5of NAFLD ,38. PPARof NAFLD  and alsoBy studying key TFs and histone modifications in tissue samples from cases with ASH and from controls we may learn more about the etiology of the disease. In a previous study, we mapped USF1 binding regions along with USF2 and H3Ac in HepG2 cell line using ChIP-chip  and founKEGG pathways identified many genes involved in liver metabolism in normal tissue and many cancer related genes in liver tissue from a ASH patient. This indicates that the mapping of histone modifications may provide further knowledge about disease progression. Potentially they can serve as biomarkers that could be mapped in liver biopsies from disease subjects and results like those in this study could provide support for more intense intervention to lower alcohol consumption and other types of therapy. The genes found in this study may be involved in the disease but more functional studies are needed to verify this. We have also observed the change of gene expression and function of these genes using the GORilla and Liverome databases. Based on these studies, our data proved that using ChIP-seq of histone modifications and TFs we can identify genes involved in the disease as well as new disease candidates. More functional studies of these genes are needed to determine their relation to ASH. Due to the limitation of tissue material availability from the same individual, we have used tissue material from different individuals for the ChIP-seq experiments but used the same tissues along with a third set of tissues for RNA expression and found good correlation between histone peaks and gene expression, which suggests that similar mechanisms are present in multiple patients. There was a partial correlation between USF1 ChIP-seq signal and RNA expression. The study thus gives input to the design of biobanks so that enough material is collected to allow a series of assays to be performed on a tissue sample from the same individual, or preferentially from larger cohorts.MLXIPL gene  with the accession number SRA066400.All ChIP-seq data is freely available at NCBI SRA webpage (ChIP: Chromatin Immunoprecipitation; ChIP-seq: Chromatin Immunoprecipitation coupled with sequencing; GO: Gene ontology; GWAS: Genome wide association studies; H3K27ac: Histone 3 lysine 27 acetylation; H3K4me1: Histone 3 lysine 4 mono-methylation; H3K4me3: Histone 3 lysine 4 tri-methylation; HCC: Hepatocellular carcinoma; NAFLD: Non-alcoholic fatty liver disease; ASH: Alcoholic steatohepatitis; NASH: Non-alcoholic steatohepatitis; qRT-PCR: Quantitative real time PCR; SNP: Single nucleotide polymorphism; TF: Transcription factor; TSS: Transcription start site; USF1: Upstream stimulatory factor 1.The authors declare that they have no competing interests.CW and KZ conceived the study and CW coordinated experiments and analysis. MSB performed most of the experiments. MSB, OW and SB analyzed data after input from CW and JK. MSB wrote a draft of the manuscript and major editing was done by CW, OW and SB. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/6/50/prepubShows the USF1 peaks and read counts for control and ASH.Click here for fileFigure S2. Comparison of the ChIP-seq signal over the peak regions between disease and control for the histone modifications. Figure S3. The number of genes that contain histone modification peaks both in ASH and in control, only in ASH and only in control. Figure S4. Different biological processes identified using the genes associated with histone modifications in ASH. Figure S5. Different biological processes identified using the genes associated with histone modifications in control. Figure S6. Histone modification pattern for the genes associated with alcoholic liver disease and ASH. Figure S7. Fraction of peaks with USF1 motif ranked on peak height, with comparison to peaks called with MACS for the same dataset. Figure S8. Correlation between ASH and control signals for USF1 for peaks close to TSS. Table S1. Sanger sequencing results of SNPs identified at USF1 peaks and alleles identified for Genomic DNA and ChIP DNA of USF1. Allele frequencies obtained from dbSNP129 and AA, AB and BB indicate the frequencies calculated by using Hardy-Weinberg equation. Table S2. GWAS catalogue dbSNPs identified using ChIP-seq data of histone modifications in control. Table S3. GWAS catalogue dbSNPs identified using ChIP-seq data of histone modifications in ASH. Table S4. Novel SNPs identified using ChIP-seq data of histone modifications in control. Table S5. Novel SNPs identified using ChIP-seq data of histone modifications in ASH. Table S6. Primers used for USF1 qPCR validations, mRNA primers for USF1 and histone modifications.Comparison of ChIP-seq signals with ChIP-qPCR represents the good correlation between qPCR and ChIP-seq signal. Click here for filePeak regions with the highest differences in histone modification levels between ASH and control.Click here for fileList of genes within 2 kb from the histone modification peaks with highest difference between ASH and control. Peaks for all three histone modifications are combined. This is the list we have used for Gene Ontology.Click here for file


Maintaining the proper expression of the transcriptome during development or in response to a changing environment requires a delicate balance between transcriptional regulators with activating and repressing functions. The budding yeast transcriptional co-repressor Tup1-Ssn6 is a model for studying similar repressor complexes in multicellular eukaryotes. Tup1-Ssn6 does not bind DNA directly, but is directed to individual promoters by one or more DNA-binding proteins, referred to as Tup1 recruiters. This functional architecture allows the Tup1-Ssn6 to modulate the expression of genes required for the response to a variety of cellular stresses. To understand the targeting or the Tup1-Ssn6 complex, we determined the genomic distribution of Tup1 and Ssn6 by ChIP-chip. We found that most loci bound by Tup1-Ssn6 could not be explained by co-occupancy with a known recruiting cofactor and that deletion of individual known Tup1 recruiters did not significantly alter the Tup1 binding profile. These observations suggest that new Tup1 recruiting proteins remain to be discovered and that Tup1 recruitment typically depends on multiple recruiting cofactors. To identify new recruiting proteins, we computationally screened for factors with binding patterns similar to the observed Tup1-Ssn6 genomic distribution. Four top candidates, Cin5, Skn7, Phd1, and Yap6, all known to be associated with stress response gene regulation, were experimentally confirmed to physically interact with Tup1 and/or Ssn6. Incorporating these new recruitment cofactors with previously characterized cofactors now explains the majority of Tup1 targeting across the genome, and expands our understanding of the mechanism by which Tup1-Ssn6 is directed to its targets. Eukaryotic enhancers often consist of adjacent binding sites for multiple proteins that work together cooperatively to recruit co-activator proteins S. cerevisiae genes In budding yeast, Tup1 shares structural and functional properties with Groucho, and is considered its homolog The proteins that bring Tup1-Ssn6 to DNA vary in both their DNA-binding and protein-protein interaction domains. Tup1-Ssn6 recruitment and corresponding complex formation occurs by relatively weak protein-protein interactions with either Tup1 or Ssn6 To provide a more comprehensive model for genome-wide Tup1 recruitment, we used ChIP-chip assays to identify the genome-wide distributions of Tup1 and Ssn6. We then compared the binding pattern to published ChIP-chip data and found that the majority of loci bound by Tup1 were not co-occupied by a known Tup1 recruiter. In addition, individual deletions for seven known Tup1 recruiters did not significantly alter the Tup1 binding profile. These observations suggest that novel Tup1-Ssn6 recruiting proteins remain to be discovered and that Tup1 recruitment usually depends on multiple cofactors. To identify unknown Tup1 cofactors, we utilized an unbiased approach where we compared the genomic distribution of Tup1-Ssn6 to the distribution of more than 200 transcription factors. Using this method we identified several novel candidate cofactors along with the majority of the known Tup1-Ssn6 cofactors. Subsequently, we experimentally validated that the candidate cofactors Phd1, Cin5, Yap6, and Skn7 by showing that they physically interact with Tup1 and/or Ssn6. The newly identified cofactors are involved in regulating processes in which Tup1 has been previously implicated, including pseudohyphal growth (Phd1)Dataset S1). For each peak of Tup1 binding, the highest-scored array element was used for all further analysis. We also determined the binding pattern of Ssn6 using two biological replicates. As expected, most Ssn6 targets (73%) were also bound by Tup1, and the Tup1 binding profile was highly correlated to Ssn6 occupancy (R2 = 0.48). Also as expected, the majority of genes downstream of Tup1 binding events were derepressed in a tup1Δ strain  and included known targets such as the hexose transporters (GO term enrichment p = 7.05×10−8). The ChIP-chip results provided a high-confidence set of 282 Tup1 binding sites to use in down-stream analysis of Tup1 targeting.To better understand how Tup1-Ssn6 is directed to its targets on a genome-wide scale, we first determined the genomic binding pattern of Tup1 and Ssn6 in yeast by ChIP-chip during growth in rich media Figure 1To identify which cofactors occupied each of the 282 Tup1 target sites, we used published ChIP-chip data for the known Tup1 DNA-binding cofactors Aft1, Rfx1, Mig1, Nrg1, Rox1, Mcm1, Sko1, Cup9, and Sut1 . Next, all of the DNA sequences near sites of Tup1 binding were screened for the presence of DNA sequence motifs that corresponded to the specificities of each of the recruiters To further explore the mechanism of Tup1 recruitment to specific targets, we examined Tup1 binding in seven strains, each carrying a deletion of a different known recruiter of the Tup1-Ssn6 complex. Then, for each Tup1 target, we compared the wild-type ChIP values to the values obtained in each of the seven deletion strains Figure 1R2 = 0.500 (P<1×10−10) . To ensure that this correlation was specific to recruiters of Tup1, we calculated the regression coefficient between Tup1 occupancy at all intergenic regions to the total number of proteins bound using all the ChIP data, and found the relationship to be greatly reduced (R2 = 0.097). The strong association between the number of recruiting proteins present and Tup1 binding signal suggests that Tup1 is simultaneously recruited by multiple cofactors to its targets.To test the hypothesis that multiple recruiters direct Tup1 to its targets, we once again divided Tup1 targets into groups, this time based on the number of different recruiters bound to each target. We then calculated the average Tup1 binding score for each group of targets. We observed a positive relationship between the magnitude of either Tup1 or Ssn6 binding signal and the number of recruiters bound to the target, which was not seen for mock ChIP experiments  across all five tests for each transcription factor.Despite evidence for multiple recruiters, over 40% of Tup1 genomic targets were not bound by a known recruiter, even when using a relatively liberal cutoff (P<0.01). Thus, either Tup1 binds directly to some of its targets without assistance, possibly through association with histone tails There are three readily apparent biological explanations for why a transcription factor might score highly in our prediction tests. First, Tup1 may modulate the binding of the transcription factor to some of the transcription factor’s targets. Second, Tup1 may independently co-regulate some of the transcription factor’s targets. Third, the transcription factor may recruit Tup1 to a subset of its targets or be a member of the Tup1-Ssn6 complex. We are interested in this third class.Multiple lines of evidence suggest that some of the predicted recruiters do in fact function to direct Tup1 to its target genes. First, six of the eight  previously characterized Tup1 recruiters for which we have data rank in the top twenty of the 204 tested transcription factors. Second, we have observed that Tup1 itself binds to the regulatory regions upstream of nine of the top 20 genes, including all six of the known recruiters on the list   experiments in strains harboring Myc-tagged versions of the potential Tup1 recruiters. In the first experiment, we immunoprecipitated with anti-Ssn6 antibodies and then immunoblotted with anti-Myc to determine whether Cin5, Phd1, Yap6, and Skn7 were associated with Ssn6. As a positive control, we confirmed the ability of Ssn6 to immunoprecipitate Tup1-MYC in an Ssn6-dependent manner . The signal produced by the Cin5 and Phd1 is comparable to that of the known recruiters.To confirm the strong interaction of Skn7 and Yap6 with Tup1-Ssn6, we repeated our co-IP experiments. For these experiments we added a HA-tag to Tup1 in all of the strains harboring a MYC-tagged recruiter protein to confirm that the Tup1-Ssn6 complex was intact throughout our IP experiments , treatment with DNAse I did not prevent the ability of Ssn6 to pull down Yap6, Skn7, Phd1, or Cin5  . Additionally, the regression between the number of binding recruiters and Tup1 occupancy improves from an R2 of 0.50 to 0.58 when these four proteins are included  from a function of regressor variables (Tup1-Ssn6 recruiting proteins) and parameters. The parameters are adjusted so that a measure of fit is optimized. There is a single parameter for each recruiter protein in the model that defines the weight given to a recruiter protein in the regression model. A value close to zero has low weight while a large positive or negative parameter value is weighted highly for its ability to predict Tup1 occupancy.We first tested each recruiter independently to determine if each single recruiter was significantly positively correlated to Tup1 occupancy. At this step, we removed from the model Rfx1, which did not have a significant relationship with Tup1 binding, and Aft1, which was negatively correlated See . Using oR2 of 0.577 to 0.648  (Dataset S3). Overall, this means that our model can explain 65% of Tup1 binding variance at Tup1 bound sites and 43% of variance in Tup1 binding for all yeast intergenic regions  to an ODChIP and input DNA were amplified by ligation-mediated (LM) PCR as described previously ChIP samples were competitively hybridized with input DNA to low-resolution spotted-DNA yeast whole genome spotted microarrays containing coding and non-coding regions at approximately 800 bp resolution. The arrays were scanned with an Axon 4000 scanner, and data was extracted using GenePix 6.0 software. Only spots of high quality by visual inspection, with less than 10% saturated input pixels, with a background corrected sum of medians for both channels greater than 500 were used for the analysis.http://buffalo.edu/mjbuck/Tup1-recruiters.html.The DNA microarrays were normalized using block-by-block loess with the limma R package as part of Bioconductor The median standardized value was determined across all biological replicates. The standardized log ratio was used as input for ChIPOTle tup1Δ expression data tup1Δ and the top quartile of binding for each transcription factor. The last approach is similar to the fourth but instead of using in vivo Tup1 binding data we used the change in expression for downstream genes in a tup1ΔStarting with our Tup1 ChIP-chip data we utilized published ChIP-chip data for 204 transcription factors 2 ratios from ChIP-chip experiments R2. The yeast intergenic dataset was then randomly split 4 additional times and the regression model estimation was repeated. The final parameter estimates were determined as the average across all five regression models. The final regression model was determine as outlined above but included the additional Tup1-Ssn6 recruiting proteins . Mcm1 and Mig1 were removed from the final model because they were both non-significant predictors (p>0.01). All regression analysis was performed using SAS v 9.1 .Regression analysis examines the relationship between one variable (Tup1 occupancy) and another set of variables (Tup1-Ssn6 recruiting proteins). The data for each recruiter was the log600 of 0.5–1.0 and for each strain, 25–30 OD600 units were collected at 4°C . Pellets were washed with Co-IP buffer  plus protease inhibitors , transferred to ependorf tubes, and lysed with glass beads. The lysates were removed from the glass beads and cleared by spinning at 15,000 RPM for five minutes. For the DNAse I experiments, DNAse I was added to a final concentration of 100 U/µl, samples were incubated for one hour at 4°C, and EDTA was added to a final concentration of 10 mM to stop reactions. For inputs, 25 µl of cleared lysates was added to 25 µl of 2X sample buffer . For IPs, antibody (either 1 µl of anti-Ssn6 antibody or 5 µl of anti-HA antibody) was added to 500 µl of cleared lysates and incubated for three hours at 4°C. Antibody-protein complexes were recovered by incubation with Protein G Sepharose beads in Co-IP Buffer, beads were washed two times with Co-IP Buffer and once with high-salt Co-IP Buffer . Following the final wash, all buffer was removed from the beads and 20 µl of 2X sample buffer was added to the beads. For detection of proteins by Western blot, 5 µl of each input was loaded for each blot . For anti-Ssn6 co-IP blots, 10 µl was loaded for anti-Myc blots , 4 µl was loaded for anti-HA blots  and 2 µl was loaded for anti-Ssn6 blots . For anti-HA co-IP blots, 10 µl was loaded for anti-Myc blots , 4 µl was loaded for anti-Ssn6 blots  and 2 µl was loaded for anti-HA blots .Appropriate strains were inoculated and grown in YPD to an ODLysates were electrophoresed on 4–12% NuPAGE Bis-Tris gels with MOPS running buffer according to manufacturer’s instruction (Invitrogen). Separated proteins were transferred to a nitrocellulose membrane according to standard methods. Membranes were blocked with 5% NFDM (nonfat dry milk) in 1X TBS /0.1% Tween. Following blocking, the membranes were incubated overnight in either a 1∶6000 dilution (in 1X TBS/0.1% Tween/5% NFDM) of rabbit anti-Ssn6 (Sharon Dent), a 1∶500 dilution (in 1X TBS/0.1% Tween/1% NFDM) of mouse anti-HA (Santa Cruz) or a 1∶2500 dilution (in 1X TBS/0.1% Tween/1% NFDM) of mouse anti-Myc (Upstate). The membranes were then washed three times in 1X TBS/0.1% Tween/1% NFDM and incubated in a 1∶15000 dilution (in 1X TBS/0.1% Tween/1% NFDM) of HRP conjugated donkey anti-rabbit IgG (anti-Ssn6 blots) or HRP conjugated donkey anti-mouse IgG (anti-HA and anti-Myc blots) (Amersham). Following washing, blots were developed by enhanced chemiluminescence (ECL) using an Amersham ECL Plus Detection Kit.Figure S1Genes derepressed in a tup1Δ strain are bound by Tup1. (A) Tup1 ChIP-chip data at single promoters are plotted versus derepression of the downstream genes in a tup1Δ strain B) All genes were sorted into 10 bins depending on the degree to which they were derepressed in a tup1Δ strain (TIF)Click here for additional data file.Figure S2Tup1 interacts with the known Tup1 recruiters Sut1, Nrg1, or Sko1. This figure is a longer exposure for the same blot shown in (TIF)Click here for additional data file.Figure S3Characterization of DNAse I-treated Co-IP experiments. Top, Genomic DNA isolated from the supernatant of Co-IP experiments in the presence or absence of DNAse I. Middle and bottom, To show digestion of the DNA, PCR was performed using genomic DNA prepared from the TOP panel as a template. The ability to amplify through small regions (∼400 bp) in the RPS1A gene (middle) and Tup1-HA tagged region (bottom) were examined.(TIF)Click here for additional data file.Table S1Strains used in this study.(DOCX)Click here for additional data file.Dataset S1Tup1 bound sites with data shown in .(XLSX)Click here for additional data file.Dataset S2Regulatory regions bound by each recruiter with Tup1 and Ssn6 occupancy.(XLSX)Click here for additional data file.Dataset S3All regulatory regions with regression model results compared to experimental results (data from ).(XLSX)Click here for additional data file.


The strict temporal and spatial regulation on Myog expression in the embryo makes it an ideal gene to study the developmental regulation of tissue-specific expression. Over the last 20 years, our knowledge of the regulation of Myog expression has evolved from the identification of the minimal promoter elements necessary for the gene to be transcribed in muscle, to a mechanistic understanding of how the proteins that bind these DNA elements work together to establish transcriptional competence. Here we present our current understanding of the developmental regulation of gene expression gained from studies of the Myog gene.Expression of the  Drosophila have identified Trithorax (TrxG) and Polycomb (PcG) group proteins as the mediators of this epigenetic cellular memory . Inand Myog . In contthe same . AlternaMyog promoter region has been extensively studied, and multiple conserved binding elements have been characterized  region of the ized see . Indeed,Myog promoter [Myog promoter driving expression of a reporter gene in chick myoblasts show that deletion of this element within the context of the proximal promoter completely blocks its expression [The TATA box is required for the binding of TFIID that directs the assembly of the general transcriptional machinery at the promoter region. It has also been shown to bind the TAF3/TRF3 transcriptional complex to the promoter . Studiespression .The E-box (E1) present between the TATA box and the transcription start site is the binding site for myogenic bHLH protein complexes, including MyoD/E-protein and Myog/E-protein heterodimers. Mutation of the E1 E-box in the context of the proximal promoter led to a block of Myog expression in the mouse myotome during development . It is iMyog promoter is bound by members of the Mef2 family of transcription factors—including Mef2a, Mef2c, and Mef2d. Mutation of the Mef2 binding element in the context of the proximal promoter driving expression of the reporter gene blocks the activation in both chick myoblasts and fibroblasts undergoing myogenic conversion [In vivo, the Mef2 binding element is required for the activation of the LacZ reporter construct in the somites posterior to somite 7  of day E10.5 mouse embryos in the context of a −1565 to +18 bp promoter construct [Myog in some developmental contexts, but that activation of the Myog gene can also be achieved through alternative binding elements.The Mef2 binding element in the nversion . In vivoMyog promoter has also been shown to be crucial to the proper expression of the reporter gene in the developing embryo [Six1 in mice leads to an impaired primary myogenesis, muscle hypoplasia, and decreased endogenous Myog in the limb buds [The Mef3 binding element serves as a binding site for the Six family of transcription factors—including Six1 and Six4. Mutation of the Mef3 site in the context of the −184 to +18 bp g embryo . Consistimb buds .myogHCE) has been shown to serve as a binding site for at least two different proteins—Pbx-Meis heterodimers [Myog promoter through a tethering mechanism [The Pbx binding element  has shown that the repressed Myog locus is localized to the nuclear lumen in myoblasts [Myog locus shows a relatively low density of CpG residues [Myog promoter appears to play a role in the negative regulation of transcription [Myog proximal promoter (−192 to +58 bp) is more extensively methylated in anterior somites that have not yet expressed Myog compared to posterior somites that do express Myog [Myog in growing myoblasts [de novo methyltransferase activity (DMNT3a/DNMT3b) targeted to the Myog promoter or maintenance methyltransferase activity (DNMT1) during DNA replication.In proliferating myoblasts, the yoblasts . Howeveryoblasts , 23. Exaresidues  suggestiresidues . Neverthcription , 23, 25.yoblasts . What reMyog proximal promoter in growing myoblasts suggesting that the presence of DNA methylation is not inhibitory to the targeting of these important factors to the loci. Instead, the methylation of DNA within the proximal promoter appears to be essential to the recruitment of the transcriptional repressor CIBZ which directly binds isolated methylated CpG sequences [Myog gene remains unclear but it is interesting to note that knockdown of the methyl-binding protein leads to transcriptional activation of the promoter in the absence of CpG demethylation [Myog promoter region helps repress expression through the recruitment of the CIBZ protein  and lysine 27 (H3K27). Indeed, the repressed Myog promoter has been shown to be marked by both dimethyl-H3K9 (H3K9me2) and trimethyl-H3K9 (H3K9me3) in proliferating myoblasts [Myog promoter has been attributed to the H3K9 methyltransferase KMT1A/Suv39h1 [Myog promoter is modulated by the phosphorylation of MyoD by p38γ MAPK [Myog promoter in repressive conditions [Myog promoter to mediate a methylation of the muscle regulatory factor [Myog promoter remains to be investigated.In addition to DNA methylation the posttranslational modifications of histones play an important role in maintaining a transcriptionally repressive environment at the yoblasts . The pre/Suv39h1  which is/Suv39h1   is a very well-characterized histone modification that has been shown to mark developmentally regulated genes to maintain them in a transcriptionally silent state . Consistyoblasts , 35, andnditions , 35. Nev the TSS  as well  the TSS   enzymes that are responsible for removing acetyl groups from lysines within the Histone H3 and H4 of the nucleosome. Targeting of the HDAC enzymes to the Myog promoter occurs through a direct interaction with MyoD [Myog promoter to mediate repression in proliferating myoblasts. Interestingly, studies using innervated muscle show that Mef2 forms a complex with Dach2, MITR (HDAC9) and class I HDACs to mediate the downregulation of Myog expression [ The repression of Myog expression is also associated with the removal of transcriptionally permissive histone modifications. In particular, the ith MyoD , 37, 38.ith MyoD . This supression , 41. It Myog promoter [Myog promoter in proliferating myoblasts. However, this association occurs as a subcomplex, as BRG1/BRM and other core SWI/SNF subunits are not associated with the repressed Myog promoter [Myog promoter. Lastly, recent studies have shown that two subunits of the ATP-dependent chromatin remodeling complex SWI/SNF are associated with the repressed promoter , 43. Indpromoter . While iMyog gene is activated relatively early in the developmental program. In differentiating C2C12 myoblasts, a change in DNA methylation status can be observed as early as 2 hrs after induction of differentiation, though 24 hrs is required to see complete demethylation [Myog promoter element suggest that Six1 and Mef2 binding is required for the demethylation of DNA at this locus [Myog promoter in proliferating myoblasts [ Under conditions permissive to terminal myogenesis, expression of the hylation . Studiesis locus . Howeveryoblasts —where thα MAPK is responsible for this activation of transcription, where it phosphorylates several proteins involved in regulating Myog expression. These p38α MAPK targets include the E-proteins E12/E47 , the SWith MyoD—), and Mecineurin  and AKT cineurin .Myog promoter, several key repressors of transcription are downregulated early in terminal muscle differentiation. Indeed, both H3K27 methyltransferase Ezh2 and the H3K9 methyltransferase G9a undergo decreased expression at the onset of terminal myogenesis [Myog promoter [Myog promoter, p300 then mediates the acetylation of nucleosomes through the modification of specific lysines within histone H3 and H4 to create a transcriptionally permissive environment  by the Msk1 kinase that occurs upon differentiation [Myog promoter to mediate this exchange of PRC2 complexes. Finally, the decreased levels of H3K27me3 are also established through the recruitment of the histone demethylase UTX/KDM6A [Myog promoter through an association with the Six4 transcriptional activator [Myog promoter after expression has been initiated. Thus, multiple mechanisms appear to be working together to mediate efficient removal of the transcriptionally repressive H3K27me3 mark.The final step in the activation of the view see ). The efone H3.3   that lie far outside their proximal promoter, we expect that many of the principles that modulate their transcription to be common to those elucidated on the Myog promoter. In particular, we highlight the fact that tissue-specific factors such as MyoD must cooperate with more ubiquitously expressed proteins  to establish a transcriptionally permissive environment within the gene. Though the MyoD-Six1/4-Mef2-Pbx1 axis of transcription factors is important to the proper developmental expression of Myog, it is well established that additional combinations of transcription factors work synergistically in the activation of other muscle-specific genes. With the recent advance in high-throughput analysis of transcription factor binding and chromatin structure analysis, we expect that evolving transcriptional network models will provide us with important new insight into the many other axes of transcription factors that modulate expression of the specific genes that make up the muscle-specific gene expression program.The control of


Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. One critical virulence attribute is its morphogenetic plasticity. Hyphal development requires two temporally linked changes in promoter chromatin, which is sequentially regulated by temporarily clearing the transcription inhibitor Nrg1 upon activation of the cAMP/PKA pathway and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling. Molecular mechanisms for the temporal connection and the link to Tor1 signaling are not clear. Here, through a forward genetic screen, we report the identification of the GATA family transcription factor Brg1 as the factor that recruits Hda1 to promoters of hypha-specific genes during hyphal elongation. BRG1 expression requires both the removal of Nrg1 and a sub-growth inhibitory level of rapamycin; therefore, it is a sensitive readout of Tor1 signaling. Interestingly, promoters of hypha-specific genes are not accessible to Brg1 in yeast cells. Furthermore, ectopic expression of Brg1 cannot induce hyphae, but can sustain hyphal development. Nucleosome mapping of a hypha-specific promoter shows that Nrg1 binding sites are in nucleosome free regions in yeast cells, whereas Brg1 binding sites are occupied by nucleosomes. Nucleosome disassembly during hyphal initiation exposes the binding sites for both regulators. During hyphal elongation, Brg1-mediated Hda1 recruitment causes nucleosome repositioning and occlusion of Nrg1 binding sites. We suggest that nucleosome repositioning is the underlying mechanism for the yeast-hyphal transition. The hypha-specific regulator Ume6 is a key downstream target of Brg1 and functions after Brg1 as a built-in positive feedback regulator of the hyphal transcriptional program to sustain hyphal development. With the levels of Nrg1 and Brg1 dynamically and sensitively controlled by the two major cellular growth pathways, temporal changes in nucleosome positioning during the yeast-to-hypha transition provide a mechanism for signal integration and cell fate specification. This mechanism is likely used broadly in development. Candida is part of the gut microflora in healthy individuals, but can disseminate and cause systemic disease when the host's immune system is suppressed. Its ability to grow as yeast and hyphae in response to environmental cues is a major virulence attribute. Hyphal development requires temporary clearing of the transcription inhibitor Nrg1 upon activation of cAMP/PKA for initiation and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling for maintenance. Here, we show that, during hyphal initiation when Nrg1 is gone, expression of the GATA family transcription factor Brg1 is activated under reduced Tor1 signaling. Accumulated Brg1 recruits Hda1 to hyphal promoters to reposition nucleosomes, leading to obstruction of Nrg1 binding sites and sustained hyphal development. The nucleosome repositioning during the yeast-hyphal transition provides a mechanism for temporal integration of extracellular signals and cell-fate specification. The hypha-specific transcription factor Ume6 functions after Brg1 in this succession of feed-forward regulation of hyphal development. Since misregulation of either Nrg1 or Ume6 causes altered virulence, and Brg1 regulates both Nrg1 accessibility and Ume6 transcription, our findings should provide a better understanding of how Candida controls its morphological program in different host niches to exist as a commensal and a pathogen. Candida albicans is a major opportunistic fungal pathogen of humans C. albicans exists as a harmless commensal in the oral cavity and the gastrointestinal and urogenital tracts. But the fungus can cause mucosal infections and systemic disease in immunocompromised people. One critical virulence attribute of C. albicans is its ability to undergo hyphal development in response to environmental cues. Mutants that are defective in hyphal formation display much reduced virulence in animal models of systemic candidiasis HWP1, ALS3, and RBT5 encode cell wall proteins that are important for adhesion to host cells and iron acquisition from the host 1 cyclin-related protein Hgc1 Hyphal development is regulated by multiple signal transduction pathways. Among them, the cAMP-dependent protein kinase A (PKA) pathway is essential for morphogenesis and virulence C. albicans contains a conserved Tor1 protein kinase Saccharomyces cerevisiaeC. albicansbiofilm regulator Brg1 BRG1 expression is a sensitive readout of Tor1 signalling level in C. albicans, and Brg1 expression during hyphal elongation leads to nucleosome repositioning. Different nucleosome positions at the promoters of hypha-specific genes lead to different accessibility of Nrg1 and Brg1 to their DNA-binding sites at the promoters. This provides molecular insights to the sequential and integrative nature of promoter chromatin regulation. We further show that Brg1 functions before Ume6 in a feed-forward loop that regulates the yeast-to-hypha transition in C. albicans.hda1 mutant, we expected the mutants of interest to be capable of germ-tube formation, but defective in sustained hyphal growth. Furthermore, they were expected to block rapamycin-mediated hyphal elongation. Mutants defective in hyphal elongation, but with defects which could be suppressed by rapamycin, were not studied further as their gene products were expected to act upstream or parallel to Tor1 signaling. We screened a knockout library of 165 transcription factor genes in C. albicansbrg1, rob1, and ahr1 mutants compared to the wild-type strain by chromatin immunoprecipitation (ChIP)  , and Hda1 mutant . This isg1 cells . The brgto serum . The Brgalbicans Brg1 is most homologous in protein sequence to S. cerevisiae Gat2 by BLAST analysis. Both proteins contain a GATA family zinc finger motif most similar to Gat3, Gat4, Gat1 and Gln3 of S. cerevisiae. Functions and regulations of Gat2, Gat3, and Gat4 in S. cerevisiae are not well studied. Gln3 and Gat1 are phosphorylated by the TOR kinases MAL2 promoter. Unlike Gln3 and Gat1, Myc-Brg1 displayed nuclear localization under all conditions with or without rapamycin  and found 3 regions of ∼250 bp long that contain binding motifs for both Nrg1 and Brg1 . Both binding sites are accessible during the hyphal initiation phase . In contrast to Gln3, Brg1 nuclear localization is not regulated by rapamycin. Instead, BRG1 expression requires the presence of 5–10 nM rapamycin, making it a sensitive readout of Tor1 signaling. In addition to reduced Tor1 signaling, activation of BRG1 expression also requires the removal of Nrg1 inhibition. This is different from hypha-specific genes where lack of Nrg1 is sufficient for their expression. Therefore, BRG1 expression uses an AND gate logic gating for signal integration. This type of logic gating is also seen in other GATA factor regulated gene expression. For example, the expression of nitrate and nitrite reductase in Aspergillus nidulans requires both the presence of nitrate and the absence of ammonium and glutamine BRG1 promoter. Similar to hyphal specific promoters, Nrg1 dissociates from the BRG1 promoter during hyphal initiation and subsequently, Brg1 binds to the promoters during hyphal elongation in a rapamycin-dependent manner. Brg1 protein is not a direct target of the Tor1 pathway because Brg1 association with hypha-specific promoters is independent of rapamycin and ectopic expression of Brg1 can sustain hyphal elongation in the absence of rapamycin. Therefore, Tor1 function in hyphal development is through the transcriptional regulation of BRG1. Experiments are underway to identify the additional regulators of BRG1 expression.HWP1 promoter, we find dynamic changes in nucleosome positioning at the UAS region. The observed change in nucleosome positioning provides a mechanism for differential accessibility to Nrg1 and Brg1 in yeast and hyphal cells. Other hypha-specific promoters, including UME6 and HGC1 as well as BRG1, likely have different nucleosome positioning in yeast and hyphae similar to HWP1 as differential Brg1 and Nrg1 binding is observed for all hypha-specific promoters examined. Furthermore, Brg1 bound regions on the examined promoters all have both Brg1 and Nrg1 binding sites within the distance of one nucleosome. We propose that hyphal development undergoes sequential changes in nucleosome positioning from the yeast state, to a transition state, and then to the hyphal state  containing the C-terminal BRG1 coding region was inserted into the BamHI-MluI sites of pPR671 and pPR673 SpeI for target integration into the BRG1 loci to express Brg1-13Myc. The pMAL2-MYC-BRG1 plasmid was constructed by amplifying BRG1 (primers 3 and 4) to replace HGC1 from the pMAL2-MYC-HGC1 plasmid AscI to target integration into the ADE2 locus to express Brg1-13Myc. A 1.3-kb PCR product (primers 5 and 6) containing the C-terminal HDA1 coding region was digested with BglII and MluI to replace CPH1 from the pACT1-CPH1-TAP plasmid (our unpublished data). The resulting plasmid was digested with BamHI to target the integration of the plasmid into the HDA1 locus in HLY3636 to express Hda1-TAP. The pMAL2-UME6-13MYC plasmid was constructed by amplifying UME6 (primers 7 and 8) to replace NRG1 from the pMAL2-NRG1-MYC plasmid. The resulting plasmids were digested with AscI for target integration into the ADE2 locus in HLY4032 to express Ume6-13Myc. To construct a mutant HWP1 promoter with a site-specific mutation at the predicted Brg1 binding site in the UAS region, the AGGTAA sequence at −1130 to −1125 upstream of HWP1 was replaced by CTAGCC. Two pairs of primers  were used to PCR amplify overlapping HWP1 promoter fragments with the mutation in the overlapping region. The resulting PCR products were purified and mixed as templates for another round of PCR amplification using the primers 17 and 20, which produced the full-length mutated HWP1 promoter sequence. The resulting PCR product was cloned into the KpnI-ClaI site of the plasmid pHL471 BglII within the HWP1 promoter region (from −1465 to −1460) for integration into the endogenous HWP1 promoter locus. Primers 21 and 22 were used to quantitate the binding of Brg1 to the wild type HWP1 promoter, and primers 21 and 23 were used to quantitate the binding of Brg1 to the mutated HWP1 promoter.The Chromatin immunoprecipitation was performed as described with modifications et al., 2006) with modifications. Protein extract containing 10 mg protein were subjected to immunoprecipitation using 60 µl of immunoglobulin G (IgG) agarose bead slurry (Sigma), which was preincubated once with 0.2 mg/ml sheared salmon sperm DNA, 0.5 mg/ml bovine serum albumin in phosphate-buffered saline. Proteins were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane . After blocking in 3% skim-milk powder in Tris-buffered saline/0.05% Tween 20, a peroxidase-conjugated anti-c-Myc antibody (Roche) was used to probe for Myc-tagged proteins, which were then detected using the enhanced chemiluminescence system .Protein extraction and immunoprecipitation were performed as described previously , and incubated at room temperature for 5 min with gentle stirring. We harvested the cells, washed it in 1 ml of 1 M sorbitol, then resuspended the pellet in 500 µl of digestion buffer  for 8 min at 37°C. After terminating the MNase digestion by adding 50 µl quench buffer , DNA was extracted with phenol/chloroform, and then we proceeded with the qPCR analysis. The PCR product was designed to be ∼100 bp, and the neighboring PCR primers are ∼40 bp apart. In the nucleosome map, the x-axis represents the midpoint of the PCR product. The signal of each primer pair obtained from genomic DNA from each sample was used for normalization. The values of nucleosome occupancy at −5188 on the WOR1 promoter, which is not regulated by the yeast-hypha transition, were set to be 1.00.For the MNase assay, we used the protocol described as previously described Figure S1Brg1 is required for hyphal elongation and promoter recruitment of Hda1 in serum-containing media. (A). Wild type and brg1 mutant cells were inoculated into YPD+10% serum medium at 37°C and grown for 4 h. (B). Wild type and brg1 mutant cells carrying Hda1-Myc were inoculated into YPD+10% serum at 37°C and grown for 3 h. ChIP DNA was quantitated with primers at the UAS region of HWP1.(TIF)Click here for additional data file.Figure S2Recruitment of Hda1 to promoters of ALS3 and ECE1 in the presence rapamycin is Brg1 dependent. Wild type and brg1 mutant cells carrying Hda1-Myc were inoculated into YPD+10 nM rapamycin medium at 37°C and grown for 6 h, as described in ALS3 and ECE1 using primers described in ALS3 promoter that contains a GATA factor binding site.(TIF)Click here for additional data file.Figure S3Brg1 is constitutively localized in the nucleus. Wild-type strain expressing Myc-Brg1 under the MAL2 promoter (HLY3636) was grown in YPD medium at 37°C in the presence or absence of 10 nM rapamycin. Cells were fixed at 3 h after inoculation and processed for indirect immunofluorescence with a method as described (TIF)Click here for additional data file.Figure S4Western analysis of Brg1 in YPD medium with or without rapamycin at 25°C or 37°C. (A) Wild-type cells carrying Brg1-Myc (HLY4059) were diluted into the indicated media and conditions, and cells were collected at 0 min, 1 h, 3 h, and 5 h for Western analysis. (B) Brg1 protein stability is not regulated by rapamycin. Western of wild-type cells carrying Myc-Brg1 under the MAL2 promoter inoculated from overnight culture into fresh YPD medium at 25°C or 37°C in the presence or absence 10 nM rapamycin.(TIF)Click here for additional data file.Figure S5Constitutively expressed NRG1 blocks BRG1 activation. Cells of wild type or the nrg1 mutant carrying MAL2p-Nrg1-Myc, from overnight cultures in YEP Maltose at 30°C, were inoculated at 1∶20 dilution into YEP Maltose at 37°C in the presence of 10 nM rapamycin and grown for 1 h. BRG1 mRNA levels were determined by RT-PCR as (TIF)Click here for additional data file.Figure S6ChIP of Nrg1 and Brg1 at different regions of the BRG1 promoter. Wild-type cells carrying Nrg1-Myc or Brg1-Myc were grown in YPD medium at 25°C or YPD+10 nM rapamycin at 37°C, respectively, for 6 h. SC5314 was used as a no tag control. The enrichment over that of untagged controls is shown. Locations of specific sequence elements are marked Click here for additional data file.Figure S7Ectopically expressed Brg1 could not bind to hyphal promoters in yeast cells. ChIP of Myc-Brg1 under the MAL2 promoter in wild-type cells grown in YEPMaltose medium at 25°C or 37°C for 6 h.(TIF)Click here for additional data file.Figure S8ChIP of Brg1 at different regions of hyphal promoters. Wild-type cells carrying Brg1-Myc were grown in YPD+10 nM rapamycin at 37°C for 6 h. SC5314 was used as a no tag control. The enrichment over that of untagged controls is shown. Locations of specific sequence elements are marked  (TIF)Click here for additional data file.Figure S9ChIP of Brg1 at different regions of UME6 and HGC1 promoters. Wild-type cells carrying Brg1-Myc were grown in YPD+10 nM rapamycin at 37°C for 6 h. SC5314 was used as a no tag control. Locations of specific sequence elements are marked  UME6 (A) and HGC1 (B) over that of untagged control is shown.(TIF)Click here for additional data file.Table S1C. albicans strains used in this study.(DOC)Click here for additional data file.Table S2Primers used in this study.(DOC)Click here for additional data file.