Acidithiobacillus ferrooxidans oxidizes sulfide ores and promotes metal solubilization. The efficiency of this process depends on the attachment of cells to surfaces, a process regulated by quorum sensing (QS) cell-to-cell signalling in many Gram-negative bacteria. At. ferrooxidans has a functional QS system and the presence of AHLs enhances its attachment to pyrite. However, direct targets of the QS transcription factor AfeR remain unknown. In this study, a bioinformatic approach was used to infer possible AfeR direct targets based on the particular palindromic features of the AfeR binding site. A set of Hidden Markov Models designed to maintain palindromic regions and vary non-palindromic regions was used to screen for putative binding sites. By annotating the context of each predicted binding site (PBS), we classified them according to their positional coherence relative to other putative genomic structures such as start codons, RNA polymerase promoter elements and intergenic regions. We further used the Multiple EM for Motif Elicitation algorithm (MEME) to further filter out low homology PBSs. In summary, 75 target-genes were identified, 34 of which have a higher confidence level. Among the identified genes, we found afeR itself, zwf, genes encoding glycosyltransferase activities, metallo-beta lactamases, and active transport-related proteins. Glycosyltransferases and Zwf (Glucose 6-phosphate-1-dehydrogenase) might be directly involved in polysaccharide biosynthesis and attachment to minerals by At. ferrooxidans cells during the bioleaching process.The biomining bacterium  Acidithiobacillus ferrooxidans is an acidophilic Gram-negative autotrophic γ-proteobacterium that obtains its energy through the oxidation of ferrous iron and reduced inorganic sulfur compounds. At. ferrooxidans, along with other bacterial, archeal and eukaryotic microorganisms forms structured biofilm communities in metal-rich low pH environments, where chemolithotrophic metabolism is the primary form of energy acquisition [At. ferrooxidans interacts with its energetic substrate is an important step towards rational design strategies to improve bioleaching efficiency [uisition . The biouisition . Severaluisition ,4. Howevficiency .N-Acyl homoserine lactone (AHL) mediated QS (AHL-QS) is the most intensively studied system. It has been characterized in great detail in Gram-negative bacteria. Molecular components of this system include LuxR-family transcriptional regulators, 18–20 nucleotide cis-acting elements called lux-boxes, AHLs and LuxI-family AHL synthases. LuxR-family proteins are AHL-responsive global transcriptional regulators that activate or repress transcription upon interaction with lux-boxes [Bacterial cell-cell communication is based on the regulation of gene expression of interacting bacteria through the exchange of low-weight organic compounds or small peptides that function as signals. Signaling mechanisms have a prominent role in the emergence of cooperative behaviors in bacterial populations and communities . Quorum ux-boxes . QS reguux-boxes  or as a ux-boxes ,11.At. ferrooxidans strain ATCC 23270 possesses a functional AHL-based QS system [luxI/luxR homologs termed afeI/afeR. Here, the QS locus comprises the divergently organized afeI and afeR genes and a third gene (orf3) located in the intergenic region between these two and with the same orientation as afeI. The protein product of orf3 is conserved among some bacterial species but its function is unknown. The synthase AfeI drives the synthesis of at least nine different AHL molecules with medium or large acyl side chains [afeI. This was demonstrated by using a heterologous biosensor construct in E. coli where AfeR promotes AHL-dependent transcription of afeI [afe-box regulatory element located upstream of afeI is an 18–20 bp palindromic sequence with nearly perfect dyad-symmetry. It overlaps the -35 region of a sigma 70 promoter consensus sequence, as it is in many other bacteria. However, the afe-box has the particularity of being the central fraction of a larger 30 bp palindrome with which it shares the same center of symmetry. Moreover, each half of the 30-bp sequence shows palindromy. Hence, two alternative centers of symmetry can be found in the total sequence [S system ,13. Thissequence .At. ferrooxidans cells to solid energetic substrates such as sulfur and pyrite [At. ferrooxidans, we have used a bioinformatic strategy to identify genes whose expression might be regulated by AfeR, as it has been described for other transcriptional regulators including CepR, a LuxR-family protein [afeI operator we constructed Hidden Markov Models (HMMs) to screen for putative binding sites for AfeR in the At. ferrooxidans ATCC 23270 genome. Results revealed that at least 75 genes corresponding to 2.6% of the identified ORFs from At. ferrooxidans could be direct targets for AfeR.Recently, it has been described that AHL molecules improve the attachment of d pyrite . As a st protein ,16. BaseafeI and orf3 genes of the At. ferrooxidans QS locus (afe-box) has an unexpected sequence complexity  were defined as “a fragment of the genome sequence where one or more HMM hit is found in either strand”. Thus, a total of 273 PBSs were identified. As expected, the A and B type PBSs were mainly overlapped. These two kinds of PBS were grouped in the “SP” set. The same kind of overlap occurred with the C and D HMM-models and therefore, these two kinds of PBSs were grouped in the “MP” set. The third set comprises the E model PBSs and thus it was named “LP”. Overlapping between the three kind of PBS sets was infrequent. Only five double sites (1 SP∩MP plus 2 MP∩LP plus 2 LP∩SP) and 2 triple sites (SP∩MP∩LP) were found. These overlapping sites were merged and each merge was considered a single PBS. Finally, the complete operation yielded a total of 265 unique PBSs.i.e. the DNA region between the stop codons of converging genes is not a “sense” IG region). The Type II set consists of the PBSs that are found 200 bp upstream and 50 bp downstream from a predicted start codon and are not part of an IG region. Type III set consists on all PBS that are not part of Type II or Type III sets  was calculated to determine if the binding site density was homogeneously distributed along the whole sequence or if it was biased towards IG regions. The 265 PBSs were further classified in three different sets according to their proximity to a predicted start codon . The TypIII sets . ApproxiIII sets . An arbiafeI upstream palindromic sequence as an input for this search. The results show that the most frequent and highest scoring motif was an 18-mer that corresponds to the MP based models present in each input sequence. The MEME program found the 18 bp motif in most of the MP-containing input sequences. On the other hand, the SP based PBSs were not detected as a MEME common motif. Interestingly, some of the SP containing input sequences possessed the MEME 18 bp motif near or overlapping the corresponding SP sequence. These new motifs were not detected as MP-based PBSs in the previous HMM search. Nevertheless, the MEME tool mainly confirmed the results obtained with the HMMs. Then, the gene functions associated with all these identified PBs were noted . All PBSs belonging to the Type III set were discarded as probable binding sites due to their location. Applying this filter yielded a total of 62 PBSs . Moreovere noted . The ideafeI palindrome. Two out of eight motifs were located in intergenic regions between converging ORFs. Two previously identified motifs were those located upstream the afeR gene and AFE_2100 (annotated as a virulence-associated protein). One new motif was found in the intergenic region upstream the AFE_1504 ORF (annotated as “nifW protein”). Also a new motif was located between the diverging ORFs AFE_2800 and AFE_2799, which are a glyoxalase family protein and an Ada family-DNA-3-methyladenine glycosylase II transcriptional regulator, respectively. The positions of PBSs that where found using the MEME algorithm are summarized in Selected PBSs that also shared the MEME 18 bp common motif, including the new ones located in the SP-containing inputs, were aligned and a consensus sequence was generated using MultAlin . Eight mlux-box consensus-based search, the upstream 250 nucleotides from the four most identical sequences to the product of afeI in the genbank database  were used as input for the MEME discovery tool . In all four sequences a 18 bp motif that corresponded to a palindromic sequence similar to the afe-box was found (Results not shown). The PSSM generated was put into the MAST tool and the intergenic region database from At. ferrooxidans was screened for common motifs. The result from this procedure showed that only the afeI 18-bp motif was found. On the contrary, when the same database was screened using the PSSM generated from the MEME analysis of the MP-based PBSs, the number of hits was broader (see results). This suggests that using lux-box like sequences for the generation of HMMs in the At. ferrooxidans search would have yielded fewer hits, and hence a narrower search.To evaluate the effectiveness of an alternative At. ferrooxidans are unknown. Here, we report results obtained from a bioinformatic approach to identify possible binding sites of the AfeR protein in the At. ferrooxidans ATCC 23270 genome sequence with the aim of defining a putative QS regulon for this bacterium.Functions regulated by QS in afeI gene as a template. Alignments of known lux-box type sequences to generate the HMMs were not performed because of the little overall conservation of these sequences [afe-box because different modes of binding are expected depending on the organization of sequence modules, as it has been experimentally tested for the fur binding site [lux-box is only a fraction of a larger structure suggests that non-AfeR transcriptional regulators might also bind to the afeI operator.HMMs were constructed based on alignments of hypothetical sequences that took the palindromic structure located upstream of the equences . The HMMing site . The faccis-acting regulatory motifs. Also, it is possible that this preference is due to the overlapping of the -35 element of a sigma 70 promoter from afeI with the modeled SP structure, thus implying that at least some of the SP hits may actually be -35 elements. Supporting the hypothesis mentioned above, the Bprom predictor found some Type I SP hits overlapping -35 elements but it also found hits overlapping -10 elements and hits with no overlapping at all. On the other hand, only MP model-based PBSs were found as common motifs when all PBSs were searched with the MEME tool, making MP PBSs more likely to be the correct regulatory motif for AfeR. Thus, as SP and MP based models did not show significant overlapping in the HMM search, the determinants for the AfeR interaction with DNA are likely to be enclosed within the central 18–20 nucleotides of the large 30 bp palindrome and that the SP PBSs may be binding sites for a distinct regulator. It is possible then that SP based PBSs may be part of a distinct regulon that overlaps the AfeR regulon in the transcriptional regulation of afeI. There are examples of multiple regulatory cis-acting elements upstream of various QS-controlled genes, including the regulatory zones of I and R genes [lux-box like elements as is the case of the binding site for the regulator RsaL, upstream of lasI from P. aeruginosa [luxI gene from V. fischeri [The search yielded a total of 265 PBSs. SP model-based PBSs were preferentially located on intergenic regions whereas MP model-based PBSs were not . This ca R genes ,23. Thesruginosa  or the Mlux-box like sequence is found upstream of QS-controlled genes. This is also the case for the lasB, hcn and pqsA genes from P. aeruginosa [Among the ORFs presented in the PBS table , five ofruginosa –27.afeI promoter in the presence of AHL [70 promoters. Some of them overlap -35 elements, others overlap -10 elements and others do not overlap either. Previously described LuxR family activators of transcription like TraR, LasR and LuxR itself generally bind a lux-box like element centered in position -43 (overlapping the -35 element of the σ70 promoter) in the presence of AHL. Some transcriptional regulators of the LuxR family bind their target sequences in the absence of and self-dissociate in the presence of AHL, as is the case of EsaR of Pantoea stewartii or ExpREcc of Erwinia carotovora [lux-box like sequence overlaps the -10 element of the σ70 promoter. The mentioned regulators can also function as activators of transcription in artificial genetic constructs where the position of the cis-acting element is changed to a -35 element overlap (maintaining the dependence on absence of AHL substrate) [lux-box like element position. Moreover, it has been demonstrated that genuine LasR binding sites are located as far as 383 bp from the translation start codon (phzA gene) [The type of regulation exerted over the AfeR targets remains unknown. AfeR can activate transcription from the e of AHL , but therotovora ,29. Bothbstrate) . This shzA gene) , thus maafeI upstream sequence nor perfect dyad symmetry. Dyad symmetry is not a prerequisite for the lux-box like elements to interact with their cognate regulator as demonstrated for various QS-controlled genes in P. aeruginosa [None of the PBSs found had similar palindromic complexity to neither the ruginosa .orf3 gene. The position of the afe-box located upstream of orf3 and afeR overlaps predicted -35 elements in both cases, suggesting an activator role for AfeR at this locus. Other LuxR-like proteins are also subject of self-regulation, as is the case for the positive feedback loop experienced by PhzR from Pseudomonas fluorescens [Pantoea stewartii [The gene functions associated to the 18 bp MEME motif have been related to several biological processes . One of orescens  and the tewartii .There were two other transcriptional regulators forming part of the predicted targets: first, a helix-turn-helix transcriptional regulator that also contains a LexA motif and second, a Sigma 54-dependent transcriptional regulator. This is consistent with the fact that AfeR is a global regulator of transcription that controls the expression of many genes indirectly. The S24/LexA-like domain is related to the bacterial SOS response; specifically it catalyzes its own proteolysis separating the DNA-binding domain and the rest of the protein. (CD number: cd06529) The Sigma 54-dependent transcriptional regulator has also an HTH DNA-binding motif and also an AAA-type ATPase domain (CD number: cd00009).In the same locus as sigma 54-dependent transcriptional regulator, but in the opposite orientation, there is a metallo-β-lactamase protein family gene. Interestingly, there is the second metallo-β-lactamase among the hypothetical regulon. These proteins belong to the same superfamily as the “quorum quenching” lactonase enzymes AiiA and AttM, frequently found in Gram-positive and Gram-negative bacteria, respectively . Quorum Another target related to β-lactam antibiotics is a protein that contains an ErfK/YbiS/YcfS/YnhG domain  which codes for a transpeptidase that functions as an alternative pathway for peptidoglycan cross-linking. This pathway provides resistance to β-lactam antibiotics because it functionally replaces the sensitive penicillin-binding protein (PBP).Pseudomonas aeruginosa. There, TonB mutants show an impairment in AHL production independently of its iron transport activity [We found four transport-related putative targets. The first two are the divergently organized genes encoding a TonB-dependent receptor and an ABC transporter (of the ABC_DR_subfamily_A). The TonB-dependent receptor is involved in the specific transport of substances across the membrane, of which iron-carrying siderophores are the best characterized ones (Conserved domain database (CD) number: cd01347). Nevertheless, a role in QS has been demonstrated for TonB in E. coli). TruB catalyzes the isomerization of specific uridines in tRNA. The third RNA-related gene is the aspartyl-tRNA synthase. Finally, the RibonucleaseT (RNAseT) protein in E. coli RNAseT is involved in tRNA turnover. It has a 3′-5′exonuclease activity, also related with DNA polymerase III. RNAseT is only found in gamma-proteobacteria (CD number: cd06134).Five genes related to RNA are also predicted targets. The first one is a TrmH-family protein, which is known to methylate rRNA. rRNA methylation in bacteria strongly correlates with resistance to ribosome-targeted antibiotics . A seconAgrobacterium tumefaciens [Several proteins with diverse activities where found to be part of this putative regulon. First, a conjugal transfer protein (TrbF), thought to be part of the pilus required for the transfer of DNA. DNA transfer is a major QS-regulated phenotype in efaciens . The reszwf a gene encoding for glucose 6-phosphate-1-dehydrogenase (G6PDH) has been also identified as part of this regulon. G6PDH is part of the pentose phosphate pathway and is directly related to the cellular pool of glucose 6-phosphate that is involved in the biosynthesis of EPS precursors UDP-glucose and UDP galactose [zwf leads to an approximately 90% reduction in alginate, one of the EPS produced by Pseudomonas aeruginosa [At. ferrooxidans is capable of synthesizing the EPS precursors UDP-glucose and UDP-galactose [zwf and afeI are increased in the presence of a tetrazolic AHL-analogue [Finally, two glycosyl-transferase activities were found among the putative regulon. These activities are usually related to polysaccharide biosynthesis. The two genes belong to different groups. The “Group 1” glycosyl-transferase protein transfer activated nucleotide-sugar molecules to several substrates, including protein, lipids and other sugar residues. The “Group 2” glycosyl transferase has the same activity but structurally it belongs to a different family (CD number: cd04186). It is possible that these proteins play a role in the biosynthesis of the exopolymeric substances (EPS) or the lipopolysaccharide. In addition, alactose . On the ruginosa . It is walactose  and EPS alactose ,41. Moreanalogue ,43 suggeafeI gene and the orf3 open reading frame were used to generate HMMs. First, four defined-length palindromes were chosen  .Fractions of the 30 bp palindrome located in the intergenic region between the indrome) . Second,www.softberry.com) Type I and Type II PBS sequences with additional 100 bp flanking each side ware used as input for Bprom. All Type I PBSs were further characterized. On the other hand, Type II PBSs were selected for further characterization only if a Bprom prediction occurred within the Bprom input sequence. Shared motifs were identified using the MEME motif discovery tool [At. ferrooxidans genome. The input for the MAST program was the position specific scoring matrix (PSSM) generated from the common motif sequences derived from MEME analysis. Multiple alignments of selected PBSs and consensus sequence (Available ORF sequences contain limited annotated information. For this reason, they were further annotated and organized in sets using ARTEMIS . The conery tool ,52. The ery tool  was usedsequence  were concis-regulatory elements. Among this set of genes, the afeR response regulator and the divergently organized conserved unknown gene orf3 were identified. For both genes, the cis-regulatory element overlaps the -35 promoter elements suggesting that auto-regulation of AfeR may take place, as it occurs in Vibrio fischeri and several other gram-negative bacteria.Our bioinformatic prediction allowed the identification of several gene functions associated with the presence of upstream putative quorum sensing At. ferrooxidans might be involved in biofilm formation and directly correlates with the increase of adhesion to pyrite and sulphur by At. ferrooxidans obtained by the addition of AHL molecules [At. ferrooxidans. However, it has been previously described that zwf is activated by QS in P. aeruginosa [zwf gene [At. ferrooxidans QS regulon.The identification of genes involved in the biosynthesis of exopolysaccharides strongly indicates that the QS pathway of olecules . Furtherruginosa . Moreovezwf gene , the biozwf gene . This su


Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses. The innate immune response is a complex biological program that is configured to allow host cells to rapidly respond to infection and tissue injury. An essential feature of this response is the sequential activation of large numbers of genes that play roles in amplification of the initial inflammatory response, exert anti-microbial activities, and initiate acquired immunity. Here, we use a combination of genome-wide approaches to characterize the basal and activated states of promoters that drive the expression of genes that are turned on at immediate/early or late times in macrophages following their stimulation with a mimetic of bacterial infection. These studies identify genetically encoded features that establish basal levels of expression and distinct temporal profiles of signal-dependent gene activation required for effective immune responses. The general features of immediate/early and late genes defined by these studies are likely to be instructive for understanding how other high-magnitude, temporally orchestrated programs of gene expression are established. GAS elements) recognized by STAT1 homodimers and genes containing interferon-stimulated response elements (ISREs) recognized by STAT1/STAT2/IRF9 complexes Precise control of gene expression in response to external cues is essential for normal development, homeostasis and immunity. In the case of the innate immune system, which provides initial protection against bacterial and viral pathogens through the utilization of germ line-encoded pattern recognition receptors In addition to sequence-specific transcription factors, several classes of co-activator and co-repressor complexes are involved in the regulation of transcriptional responses. These complexes harbor several enzymatic functions, including nucleosome remodeling and histone modifying activities. Nucleosome remodeling activities play essential roles in controlling the accessibility of DNA regulatory elements to sequence-specific and general transcription factors Histone modifications that include, among others, acetylation, methylation, phosphorylation and ubiquitinylation, have been proposed to represent a code that is interpreted by distinct classes of architectural and regulatory proteins that in turn determine the ability of chromatin to function as a substrate for DNA synthesis, repair and transcription Acetylation of histones H3 and H4 by histone acetyltransferases (HATs) such as CBP, p300 and p/CAF in particular has been linked to transcriptional activation Recent genome-wide analyses In the present studies, we use a combination of genome-wide approaches to test several features of current models and to identify additional determinants of immediate/early and late transcriptional responses to TLR4 ligation. We demonstrate that nearly all immediate/early and late TLR target genes exhibit characteristics of active genes under basal conditions regardless of CpG content and direct detectable levels of expression of mature mRNAs. We provide evidence that basal expression of I/E and late genes is initially established by signal-independent transcription factors, exemplified by PU.1, that we suggest are involved in the initial recruitment of histone-modifying machinery. We also find numerous differences in enriched sequence motifs that direct basal patterns of TBP and Pol II density, induction of H4 acetylation, and enhance transcriptional elongation that we propose collectively contribute to the distinct temporal profiles of immediate/early and late gene activation.To study global relationships between histone modifications and TLR-dependent gene expression, we performed parallel gene expression profiling experiments and ChIP-Sequencing (ChIP-Seq) analysis in elicited mouse peritoneal macrophages (EPM) and bone marrow-derived macrophages (BMDM) treated with the highly specific TLR4 agonist, Kdo2 lipid A (KLA) 6 unique, mappable sequence tags were collected for each antibody and treatment condition. Tags were assigned to a specific promoter if they occurred within 1 kb of the transcriptional start site (TSS) based on the global frequency distribution of these marks . Representative genome browser images for the I/E gene Tnf and the late gene Isg20 are illustrated in ChIP-Seq experiments initially evaluated trimethylation of histone H3 lysine 4 (H3K4me3), acetylation of histone H3 lysines 9 and 14 (H3K9/K14ac), and total RNA polymerase II. A total of 5–8×10In resting macrophages, the H3K4me3 mark was enriched over background on 89% of I/E promoters and 78% of late promoters. This pattern was observed in both elicited peritoneal macrophages and bone marrow derived macrophages , indicatvida infra). Upon KLA stimulation, H3K9/14ac was largely unchanged, or trended towards a decrease (vida infra).Acetylation of H3K9/14 exhibited a very similar distribution on both classes of promoters and was highly correlated with H3K4me3 . This isdecrease  even thoThe gene expression program elicited by TLR4 agonists differs between cell types. For example, murine embryonic fibroblasts (MEFs) are capable of responding to TLR4 signaling Taken together, these data indicate that most I/E and late inflammatory genes in macrophages are characterized by the presence of H3K4me3 and H3K9/K14ac marks under basal conditions, exhibiting histone modifications characteristic of actively transcribed genes. These findings are in line with the observation that the basal levels of mRNA transcripts for the majority of I/E and late genes are well within the range of detection of the microarray platform or RNA sequencing approaches used for analysis , . Even Examination of Pol II enrichment profiles revealed that I/E genes but not late genes exhibit a striking enrichment for promoter-associated Pol II under basal conditions, with a peak located immediately adjacent to the first H3K4me3-positive nucleosome downstream of the transcriptional start site . As expe6 uniquely mapped reads for both untreated and KLA-treated EPMs indicated that while there were a small number of transcripts that exhibited low splicing efficiencies under basal conditions, the great majority of I/E transcripts were effectively spliced under both basal and KLA-stimulated conditions  and an unknown motif were the most highly enriched sequences in the late promoters , suggesting direct roles of PU.1 in establishing H3K4me3 and basal expression. The fraction of PU.1 promoter occupancy observed at new TLR4-responsive promoters was similar to that of the new promoters overall (82/212 new promoters exhibited PU.1 binding within 500 bp of the TSS). While the specific factors that build new TLR4-responsive promoters that do not have promoter bound PU.1 remain to be identified, these promoters are nevertheless established during macrophage differentiation by mechanisms that are independent of TLR4 signaling.The majority of TLR4-responsive genes that were selectively activated in PUER cells exhibited basal H3K4me3 in PU.1-null cells, but were associated with distal PU.1 binding sites (>500 bp from the TSS), consistent with roles of PU.1 in establishing signal-dependent enhancers required for gene activation by TLR4 agonists Slc15a3 , Cxcl2 aromoters , and theThe present studies indicate that nearly all immediate/early and late TLR4-responsive promoters direct expression of measurable levels of mature mRNA transcripts under basal conditions and exhibit basal patterns of histone modifications  that are associated with actively transcribed genes. Previous studies suggested that late genes might not exhibit these features Tnf) and exert anti-microbial activities , as well as the broad range of basal expression of genes that contribute to general cellular functions . Although it is generally considered that genes directing the expression of factors that amplify inflammatory responses must be kept in a tightly repressed state under basal conditions to prevent deleterious chronic inflammation, it is also possible that the low but detectable levels of expression of at least some of these factors serve biological functions, such as maintenance of the immune system in a state of readiness. In addition, the present studies examine populations of cells, which average out the established heterogeneity in gene expression observed at the single cell level These findings suggest that rather than representing off/on transitions, regulation of most TLR4-reponsive genes results from a two-step process in which signal-independent factors establish basal levels of gene expression that can then be amplified by signal-dependent transcription factors, such as NF-κB, AP-1 and SRF. These signal-dependent factors can act at the promoter itself, consistent with the enrichment of their motifs within promoters, as well as at distal enhancers that may be established in a cell-specific manner Using a combination of genome-wide approaches, we confirm and extend the recent observation that I/E genes are enriched for a ‘paused’ form of Pol II immediately downstream of the TSS that rapidly transitions to an elongating form upon TLR4 ligation. Indeed, increased elongation efficiency in response to TLR4 ligation is observed for nearly all I/E genes. As expected, the distinct temporal responses of I/E and late genes are highly correlated with response elements for distinct sets of signal-dependent transcription factors, with I/E genes exhibiting a high degree of enrichment for NF-κB, AP-1 and SRF binding elements, and late genes exhibiting enrichment for ISRE elements. The finding that TATA boxes are preferentially enriched in I/E genes, where they are associated with highest levels of TBP and paused Pol II, provides genetic and experimental evidence for a signal-independent mechanism that enables specific promoters to become poised for rapid activation. While the TATA box can efficiently recruit the machinery for transcriptional initiation, signal-dependent elements appear necessary to recruit factors necessary for productive transcriptional elongation. As 62% of the I/E and 89% of the late promoters analyzed in these studies do not contain obvious consensus TATA motifs, other factors must also play roles in recruitment of TFIID, including signal-independent factors such as ETS proteins and SP1.ChIP-Seq experiments also confirmed the recent observation of TLR4-induced increases in promoter-associated H4K5/8/12ac at I/E genes The observation that the fully activated expression levels of some TLR4-responsive genes do not reach even the basal levels of expression of other TLR4-responsive genes  further http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE23622.All ChIP-Seq, RNA-Seq, GRO-Seq and Microarray data sets have been deposited in the NCBI GEO database  of UC San Diego (Protocol Number: S01015), and every effort was made to minimize suffering.−/− and PUER cells were propagated and the PU.1-ER fusion protein was activated with 100 nM 4-hydroxy-tamoxifen as described All animal work has been conducted according to relevant institutional, national and international guidelines. Peritoneal macrophages were harvested by peritoneal lavage with 10 ml ice-cold PBS 3 days after peritoneal injection of 3 ml thioglycolate. Peritoneal cells were washed once with PBS, and seeded in 10% fetal calf serum (FCS)/DMEM containing 100 U penicillin/streptomycin in tissue culture-treated petri dishes overnight. Non-adherent cells were washed off with room temperature PBS. Fresh media was applied and cells were subjected to KLA treatment or solvent control 24 h later. RAW264.7 cells were cultured in 10% FCS DMEM. Thioglycollate-elicited macrophages were isolated as previously described Mouse hematopoietic progenitor cells were obtained by enrichment of bone marrow cells for lineage-depleted cells using the StemSep murine progenitor enrichment cocktail and StemS device  as previously described RNA was extracted using RNeasy kit  and washed twice with ice-cold PBS. Cells were resuspended in swelling buffer , 1 mM PMSF) for 5 minutes. Cells were spun down and resuspended in 500 µl lysis buffer , 1 mM PMSF)) and chromatin was sheared to an average DNA size of 300–400 bp by administering 6 pulses of 10 seconds duration at 12 W power output with 30 seconds pause on wet ice using a Misonix 3000 sonicator. The lysate was cleared by centrifugation , and 500 µl supernatant was diluted 2.5-fold with 750 µl dilution buffer ). The diluted lysate was pre-cleared by rotating for 2 h at 4°C with 120 µl 50% CL-4B sepharose slurry . The beads were discarded, and 1% of the supernatant were kept as ChIP input. The protein of interest was immunoprecipitated by rotating the supernatant with 2.5 µg antibody overnight at 4°C, then adding 50 µl blocked protein A-sepharose CL-4B  and rotating the sample for an additional 1 ½ to 2 h at 4°C. The beads were pelleted , the supernatant discarded, and the beads were transferred in 400 µl wash buffer I (WB I)  into 0.45 µm filter cartridges , spun dry , washed one more time with WB I , and twice each with WB II , WB III , and TE. Immunoprecipitated chromatin was eluted twice with 100 µl elution buffer each  into fresh tubes for 20 min and 10 min, respectively, eluates were pooled, the Na+ concentration was adjusted to 300 mM with 5 M NaCl and crosslinks were reversed overnight at 65°C in a hybridization oven. The samples were sequentially incubated at 37°C for 2 h each with 0.33 mg/ml RNase A and 0.5 mg/ml proteinase K. The DNA was isolated using the QiaQuick PCR purification kit  according to the manufacturer's instructions. Antibodies against PU.1 (sc-352) and Pol II (sc-899 X) were purchased from Santa Cruz Biotech . Antibodies against H3K4me3 (ab8580) were from Abcam , and antibodies recognizing H4K5/6/12ac  and H3K9/14ac (06-599) were from Millipore . Information on antibody, cell type, treatment and mapped reads is provided in ChIP was performed as described previously 7), serum starved and treated with KLA/DMSO. For RNA-Seq cells were scraped and RNA purified using TRIZOL reagent . Ribosomal RNA was depleted from DNase-treated samples using Ribominus Eukaryote Kit (Invitrogen) followed by ethanol precipitation. cDNA synthesis and library preparation then followed the protocol described in Peritoneal macrophages were plated onto 15 cm plates  was adapter-ligated and PCR amplified according to the manufacturer's protocol (Illumina). ChIP fragments were sequenced for 36 cycles on an Illumina Genome Analyzer according to the manufacturer's instructions. The first 25 bp (32 bp for RNA-Seq/GRO-Seq) for each sequence tag returned by the Illumina Pipeline was aligned to the mm8 assembly (NCBI Build 36) using ELAND allowing up to 2 mismatches. Only tags that mapped uniquely to the genome were considered for further analysis. Analyzed ChIP-Seq experiment that were published previously can be found in the GEO database under accession numbers GSE21512 and GSE19553 or from http://biowhat.ucsd.edu/homer/, described in Heinz et al. Motif discovery was performed using HOMER Click here for additional data file.Figure S2Global and gene-specific profiles of H3K4me3, H3K9/14ac, and Pol II in resting and activated macrophages. A. Global distribution of H3K4me3 at the promoters of the indicated classes of genes aligned at the transcriptional start site under basal conditions. B. Global distribution of H3Kme3 of the indicated classes of genes in resting and KLA-stimulated (1 h) macrophages. C. Distribution of total RNA sequencing reads/kb for all genes, I/E (Early) and late genes based on ChIP-Seq reads from elicited macrophages obtained under basal conditions.(TIF)Click here for additional data file.Figure S3Global distribution of H4K5ac, H4K8ac and H4K12ac at E/I (A) and late (B) gene promoters under control and after 1 h KLA treatment in elicited peritoneal macrophages.(TIF)Click here for additional data file.Figure S4Cxcl2 (A), Il1rn (B) and Clptm1 (C). Tracks from top to bottom are H3K4me3 tags in PU.1 KO cells, H3K4me3 tags in PUER cells cultured for 24h with tamoxifen, H3K4me3 tags in PUER cells cultured for 48 h with tamoxifen, PUER (PU.1 binding activity) tags in PUER cells cultured for 24 h with tamoxifen, C/EBPβ tags in PU.1 KO cells, C/EBPβ tags in PUER cells cultured for 24 h with tamoxifen, mRNA-strand specific GRO-Seq tags in untreated PU.1 KO cells, mRNA-strand specific GRO-Seq tags in PU.1 KO cells treated with KLA for 1 h, mRNA-strand specific GRO-Seq tags in PUER cells cultured in tamoxifen for 24 h, and mRNA-strand specific GRO-Seq tags in PUER cells cultured in tamoxifen for 24 h and treated with KLA for 1 h.PU.1 establishes promoter H3K4me3 and basal expression required for KLA activation of a subset of TLR4-responsive genes. ChIP-Seq and GRO-Seq experiments were performed in PU.1-null hematopoietic progenitor cells (PU.1 KO) and in PUER cells treated with tamoxifen for the indicated times. Genome browser images are shown for (TIF)Click here for additional data file.Table S1Summary table for ChIP-Seq, RNA-Seq, and GRO-Seq experiments. The antibody column indicates the specific target and source of antibody for ChIP-Seq experiments, or designates the experiment as an RNA-Seq or GRO-Seq experiment. The Cell Type/Treatment column indicates the cell type and treatment conditions. The Total Mapped Reads column indicates the total mapped reads used for analysis for each experiment.(DOC)Click here for additional data file.Dataset S1Cumulative ChIP-Seq, RNA-Seq, GRO-Seq and microarray data for the I/E genes displayed in (XLS)Click here for additional data file.Dataset S2Cumulative ChIP-Seq, RNA-Seq, GRO-Seq and microarray data for the Late genes displayed in (XLS)Click here for additional data file.


Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.The asexual cycle of the parasite  Toxoplasma gondii is an obligate intracellular parasite capable of infecting a wide range of species, including all warm-blooded mammals tested. This parasite is transmitted primarily by two stages within its lifecycle. The sexual cycle occurs in felines, the definitive host, and results in the shedding of copious environmentally stable oocysts in the feces. The asexual life cycle occurs within intermediate hosts, where the infectious form of the parasite differentiates to rapidly replicating tachyzoites, capable of disseminating through the body. Signals from the host likely trigger conversion to the slow growing bradyzoite form, which is harbored in brain and muscle tissue within cysts. These largely quiescent cysts can remain for the life of the host, and provide the likely route of transmission by which most humans contract the infection, via consumption of undercooked meat T. gondii. In silico surveys have identified cis-acting elements in the upstream regions of tachyzoite and bradyzoite genes T. gondii will be an important future area of study.Although drugs can combat the replicating tachyzoites, no therapy exists to eradicate established bradyzoites. As such, mechanisms of differentiation between tachyzoites and bradyzoites have been the subject of intense research efforts in the hope of isolating key factors as drug targets. These studies are facilitated by the ability to differentiate tachyzoites to bradyzoites in vitro T. gondii, eukaryotic promoters mechanisms combine with epigenetics to regulate developmental gene expression T. gondii uses modification of histones to regulate gene expression, where acetylation is associated with activated transcription SAG1 promoter to allow transcription, while histone deacetylase corepressor (HDAC) TgHDAC3 inhibits transcription at bradyzoite-induced BAG1 and LDH2 promoters T. gondii, while other HATs and HDACs have been identified within the genome but remain unstudied T. gondiiIn T. gondii, including Snf2-related CBP activator protein TgSRCAP TgRSC8, a locus showing homology to yeast paralogs Swi3p and Rsc8p of SWI/SNF and RSC complexes, respectively. The insertion led to a reduction but not an elimination of TgRSC8 protein. In the insertional mutant, transcription of housekeeping genes was unaffected, however, TgRSC8 reduction decreased the steady state transcript levels of some but not all bradyzoite-induced genes analyzed. Collectively, these data indicate a role for ATP-dependent chromatin remodeling complexes in the regulation of differentiation-associated genes in T. gondii.While one class of chromatin remodelers involves the post-translational modifications of histones, another class involves the remodeling of nucleosomes. Chromatin remodeling complexes of the Swi2/Snf2 group, such as the highly similar multiprotein SWI/SNF and RSC complexes, control gene expression by the repositioning of nucleosomes in a manner fueled by ATP hydrolysis. The yeast SWI/SNF complex is recruited to RNA polymerase (Pol) II promoters, while the RSC complex contains several subunits encoded by essential genes, and is recruited to Pol III and specific classes of Pol II promoters Animals were housed under conventional, specific-pathogen-free conditions and were treated in compliance with guidelines set by the Institutional Animal Care and Use Committee of the University of Wisconsin School of Medicine and Public Health (IACUC), according to IACUC approved protocol. The University of Wisconsin is accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care.T. gondii strains used were derivatives of either Pru, or PruΔHPT (Pru with a deletion in the hypoxanthine-xanthine guanine phosphoribosyltransferase gene (HPT)). Strains were grown as tachyzoites in human foreskin fibroblasts (HFF) maintained in Dulbecco's modified eagle medium containing 4.5 g/L D-glucose, supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO2. Strain C9 is Pru transformed with pT230-Tub5/CAT TgPL2  and VC2 was disrupted in TgME49_115700 4/well in 24 well coverslip plates or 2×106/T-25 to HFF monolayers 3 weeks post-seeding and incubating at 37°C, 5%CO2. After 3.5 h, the medium was replaced with RPMI1640 supplemented with 1% FBS and 42 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 100 units/ml penicillin and 100 µg/ml streptomycin, at pH 8.1, and incubating at 37°C, ambient CO2 for 3 days.T. gondii per sample, using tachyzoites at 50 hours or bradyzoites at 3 days post-infection. Parasites were released by passage through a 27 g needle and collected by centrifugation at 425 x g for 10 min. RNA was isolated using 1 ml Ultraspec  according to manufacturer's directions. RNA was separated on formaldehyde/agarose gels for northern hybridization, used with the FirstChoice RLM-RACE kit  or 10 µg was treated with amplification-grade DNase I and 2 µg was used for cDNA generation by Superscript III First-Strand Synthesis System  using random hexamer priming. Probes for northern hybridization include 590 bp of TgRSC8 amplified from T. gondii genomic DNA (gDNA) using RSC8probeF and RSC8probeR , followed by addition of the PacI C fragment TgRSC8 complementation construct pTPR17 was generated by amplification from genomic DNA (gDNA) of upstream sequences to an EcoNI site within exon 1 using primers P1 with added 5′ EcoNI and SpeI sites and P2  under the control of T. gondii TUB1 promoter, and 5′ and 3′ untranslated regions (UTRs), was introduced to Pru in replacement of the HPT locus by 6-thioxanthine selection cat-TgRSC8-LUC, the cat-TgRSC8 fused region was amplified from C9 using primers P16 and P17. A promoterless LUC with T. gondii TUB1 3′UTR was amplified from pTUB1-LUC-5′3′HPT with P18 and P19. For cat-LUC, the cat gene was amplified from pT230-TUB5/CAT using P16 and P20, and LUC was amplified using P21 and P19. Amplified fragments were assembled by SOE-PCR, and XbaI fragments containing the fusions were ligated into the SpeI site of pBC SK+DHFR3, and linearized by ApaI for electroporation. The TgRSC8 coding region was amplified from pTPR17 with P22 and P23, and cloned into NcoI-NotI sites of pET28a+  to create an E. coli expression vector.Plasmid pTUB1-LUC-5′3′HPT , containing firefly luciferase  and antibody reactions and washes were performed in PBS/0.2% Triton X-100 at room temperature. Primary rabbit antisera were provided by I. Coppens, Johns Hopkins Malaria Research Institute, Baltimore, MD (H2Bv), J. Boothroyd, Stanford University School of Medicine, Palo Alto, CA (SAG1) and L.M. Weiss, Albert Einstein College of Medicine, Bronx, NY (BAG1). Murine monoclonal anti-TgRSC8 antibodies were produced for these studies. The TgRSC8 expression vector was transformed into E. coli Rosetta (DE3) pLysS (Novagen), and protein was purified from IPTG-induced cultures under denaturing conditions using TALON Nickel affinity resin , dialyzed to PBS, and quantified by MicroBCA . Murine monoclonal antibodies 3AD6 and 1DE10 were developed against this antigen, as previously described T. gondii serum derived from mice 22 days after infection with Pru, and rabbit anti-BAG1 antibody. One hundred anti-T. gondii-reactive vacuoles were visually scored using a 40X objective and Zeiss Axiovert 100TV phase contrast microscope with FluoArc lamp and Zeiss filter sets 31, 34, 38 and 50. BAG1 expression was scored as complete if all DAPI-reactive parasites within the vacuole also reacted with anti-BAG1 antiserum.Parasites in HFFs on coverslips were fixed in phosphate-buffered saline (PBS)/3% formaldehyde for 20 minutes, neutralized with PBS/0.1 M glycine for 5 minutes, and blocked overnight in PBS/3%BSA/0.2% Triton X-100 at 4°C. 6/well. D-luciferin was added to 0.25 mg/ml, and luminescence was captured by Xenogen IVIS 200, using a background setting of 6000 photons/sec/cm2/sr, medium binning, F/stop = 1, and 5 minute integration time. Data was analyzed using LivingImage v3.2 . Negative control values obtained from strain Pru for each experiment were subtracted from test samples as background.Luciferase activity was quantified as described T. gondii proteins from 48 hr tachyzoites or 3 day induced bradyzoites were separated on 10% polyacrylamide gels, transferred to Immobilon-P  and blocked in 5% skim milk in PBS/0.1% Tween 20. Antibodies used were murine anti-CST1 , mouse monoclonal antibodies 1DE10 and 3AD6, and rabbit anti-TUB2 to detect β-tubulin as a loading control . HRP-conjugated secondary antibodies were used in conjunction with the ECL Plus western blotting detection system and scanning on a STORM860 imager using blue laser excitation, PMT at 900V and 100 mm scale detection . Densitometric values were acquired using ImageQuant v5.2, using local average background subtraction. Normalized values were calculated as (CST1 or TgRSC8/TUB2)test sample × 100.Infected monolayers were washed twice with Dulbecco's PBS (DPBS) to remove debris and extracellular parasites. Three-day induced bradyzoites were isolated by serial passage through 27 g and 30 g needles then fixed in DPBS with 2% formaldehyde. Samples were permeabilized for 1 hour in DPBS with 5 mM EDTA, 2% BSA and 0.2% saponin, then stained with 3AD6 monoclonal anti-TgRSC8 and rabbit polyclonal anti-BAG1 followed by donkey anti-mouse IgG AlexaFluor 488 and donkey anti-rabbit IgG AlexaFluor 647 secondary antibodies . Samples were briefly fixed in 1% formaldehyde then stored in DPBS with 5 mM EDTA and 0.2% BSA at 4°C until analysis. At least 10,000 events per sample were collected on a BD LSR II flow cytometer . Compensation and analyses were performed using FlowJo .m were amplified. Primer efficiencies (E) were determined from triplicate 10-fold serial dilution tests, plotting threshold cycle (Ct) versus log dilution on an increasing linear scale, where E = 10(1/m), and were applied to expression level calculations using the Pfaffl modification approach TUB1 and GAPDH contain paralogs in the T. gondii genome. Transcript levels of TUB1 paralogs TgME49_116400, TgME49_031770 and TgME49_031400, and GAPDH paralogs TgME49_089690 and TgME49_069190 were analyzed in tachyzoites and bradyzoites by Q-PCR, and all but the undetectable TgME49_031770 were stably expressed between both stages (data not shown). The published TUB1 locus (TgME49_116400) and GAPDH TgME49_089690 were selected for these studies TUB1 or GAPDH levels using the following formula (Ehousekeeping geneCt)/(Etest geneCt) × 100. The relative expression of test genes appeared similar when normalized to either locus. Analyses of variation (ANOVA) were performed on resulting data. Significance was defined as p≤0.05 for mutant versus vector controls, and mutant versus at least two of three complemented strains for all but the enolase locus. Primers for this locus showed reduced efficiency (E = 1.69) in comparison to those for other loci (E = 1.83-2.01), possibly contributing to inter-experimental variability. As each replicate showed at least a two-fold difference between mutant and vector controls, and between mutant and two to three complements, we declare the data to be significant.Amplifications occurred in 25 µl volumes using 1 µl cDNA template, 200 nM of each primer  and showed conservation of the yeast W546 and R564 residues  as a loading control . This bacterial-derived 663 bp chloramphenicol acetyltransferase (cat) gene is transcribed from a constitutive T. gondii TUB1 promoter, and employs the TUB1 5′ UTR, and SAG1 3′ UTR and downstream sequence. When probed with the E. coli cat coding sequences, no transcript is detected in PruΔHPT, however, two transcripts are detected in the C9 transformant . To determine if insertion occurred within the transcribed region of f TgRSC8 , a trans control . Sequencsformant . The catanscript . The catTgRSC8 is a downstream gene on a polycistronic transcript in the C9 mutant, we sought to determine if it was translated in T. gondii. A construct was designed in which the TgRSC8 ORF was replaced by a promoterless firefly luciferase gene (LUC), creating a fusion of cat and LUC, with 580 bp of TgRSC8 5′UTR in between  HPT, C9, and complemented strains by western immunoblot. Not surprisingly, CST1 expression was found to be unaffected in the TgRSC8 mutant (data not shown).As BAG1 expression was affected in the TgRSC8 mutant, we analyzed the expression of another marker of bradyzoite development. Cyst wall glycoprotein CST1 is thought to be in part responsible for reactivity of cysts with S. cerevisiae, Rsc8p localizes to the nucleus, and directly complexes to DNA and histones to control gene expression. Using either monoclonal antibody for detection within T. gondii tachyzoites and in vitro developed bradyzoites, TgRSC8 appeared in the same compartment with nuclear DNA as identified by both DAPI and reactivity with antiserum against the major T. gondii H2B histone, H2Bv . As TgRSC8 is expressed in C9, it is possible that substituted TgRSC8 acts in a dominant negative fashion, leading to inviability of recipient parasites and selection for non-expressing clones.Yeast Swi3p with alanine substitutions of amino acids D374 and N392 was expressed and assembled into SWI/SNF complexes, but it did not bind DNA S. cerevisiae Rsc8p is known to impact transcription of many genes in yeast. As BAG1 protein expression in C9 was reduced in bradyzoites, the steady state levels of several transcripts previously described as induced in bradyzoites, as well as two constitutively transcribed genes, were determined for strain C9, three complemented strains and two vector control strains by Q-PCR in bradyzoites. The genes analyzed included bradyzoite-induced genes encoding BAG1 (TgME49_059020), LDH2 (TgME49_091040), SUSA1 (TgME49_078080), ENO1 (TgME49_068860), SAG2X , BRP1 (TgME49_114250), and BGR1 (TgME49_053330), which represent a diverse group from metabolic enzymes to surface markers . As BAG1 is a heat shock protein, we sought an additional heat shock protein target for study. Analysis indicating T. gondii expresses a bradyzoite-induced HSP70 was performed using methods now known to detect three closely related HSP70s  TUB1 and GAPDH, and the gene encoding dense granule protein 2 , predicted to be expressed equivalently in both stages in wild-type T. gondii, were also assessed .Other bradyzoite-induced genes showed a different profile of expression in the strains tested. The transcript levels of surface marker SAG2X and bradyzoite-specific dense granule protein BGR1 were not affected in the  strains . InteresTgRSC8 is translated in the C9 mutant. Similarly, luciferase activity was detected from a promoterless LUC gene placed downstream of cat when within the same sequence context as TgRSC8 in strain C9. While translation of downstream genes does occur in polycistronic transcripts, it is surprising that the non-native message generated by plasmid insertion would create a dicistronic message that's downstream gene is translated. Many transcripts in T. gondii have lengthy 5′ untranslated regions, suggesting this parasite is capable of initiating translation far from the cap site of the message. Removal of intervening sequences between the LUC and cat genes did not impact LUC expression, indicating the lack of an internal ribosomal entry site, or other specific sequence elements to recruit translational machinery downstream of cat. TgRSC8 translation in the C9 mutant must occur either by the translational machinery bypassing a start site within reasonable context to initiate at a downstream site, or the translational machinery must not disengage at the stop codon of cat to allow translation of the downstream ORF. A consensus translation initiation sequence of gNCAAaATGg was identified in T. gondii, shown with the start codon underlined TgRSC8 message, the cat ORF is translated from the first methionine, and has the more favorable context of ATGCGACAAA. However, the context of the start codon of TgRSC8 in C9 is not as favorable, but is identical to the native locus, and is ATGTGCCGCG. The translation of TgRSC8 in C9 will be a topic of future study.Despite its position as the second gene in a polycistronic mRNA, BAG1. Cis-acting sequences were identified within the BAG1 promoter that were sufficient to induce bradyzoite-specific expression when placed within a constitutive promoter BAG1 promoter determined that acetylation states correlate with stage-specific expression. These data suggest the cooperative function of traditional cis-acting sequence elements with both histone-modification and nucleosome remodeling arms of epigenetic control. The intersection of histone-modification and chromatin-remodeling machinery has been uncovered previously in T. gondii as the histone methylation activities of TgCARM increased in the presence of ATP, suggesting a linkage with ATP-dependent chromatin remodeling BAG1 promoter may require the presence of a trans-activating factor to recruit ATP-dependent remodeling machinery or to stabilize chromatin changes, alleviating nucleosomal repression.We show that a suspected component of the ATP-dependent chromatin remodeling system, TgRSC8, facilities the expression of some bradyzoite-induced genes, including T. gondii genome, only TgRSC8 contains both SWIRM and SANT domains, and no paralogs were found within the genome. T. gondii may not contain multiple conserved remodeling complexes as are found in yeast, or alternatively, TgRSC8 could associate with multiple distinct complexes. Attempts at generation of a TgRSC8 knockout in wild-type T. gondii were unsuccessful, indicating this may be an essential gene (data not shown). The inability to derive D337A and N355A TgRSC8 variants suggests that these residues play an important role in functionality or stability of TgRSC8. Expression of dysfunctional copies of TgRSC8 in tachyzoites may impair the function of existing copies of TgRSC8 in strain C9, as yeast Rsc8p forms dimers The RSC and SWI/SNF yeast chromatin remodeling complexes share conserved components, including Rsc8p and Swi3p. These conserved components were proposed to comprise the structural core of their respective complexes TUB1, GAPDH, and constitutively expressed GRA2 were unaffected by the TgRSC8 reduction found within strain C9. This suggests either a lack of necessity for TgRSC8 function at these loci, or that fewer TgRSC8 molecules are required to mediate their transcription than are required for bradyzoite-induced BAG1, LDH2, ENO1 and SUSA1. Genes encoding a bradyzoite-upregulated HSP70 and BRP1 showed a reduction in bradyzoite transcription in the mutant, yet were not significantly increased by introduction of additional TgRSC8. This and the lack of impact of TgRSC8 reduction on other bradyzoite-induced loci may also be due to the dosage of this molecule found in strain C9. Additionally, the level of TgRSC8 protein was reduced in bradyzoites relative to tachyzoites in wild-type strains. This suggests TgRSC8 may act as a negative regulator of transcription of bradyzoite-induced genes in tachyzoites. In this case, decrease of TgRSC8 during bradyzoite development may trigger transcription of bradyzoite-induced genes. However, complementation of the C9 mutant by addition of TgRSC8 restored expression of BAG1, inconsistent with this hypothesis. The purpose of reduction of TgRSC8 during in vitro bradyzoite development remains unclear, but may be involved in the relative timing of the cascade of gene expression during bradyzoite development. These results provide further evidence that bradyzoite gene expression in T. gondii is a complex process governed by multiple mechanisms.Housekeeping genes Figure S1TgRSC8 primers and probe. The TgRSC8 locus is shown with boxed exons (black), introns (white), and 5′ and 3′ UTRs (gray) to scale. Plasmid insertion in strain C9 occurred in a NotI site within the 5′ UTR of TgRSC8 (lollipop). Positions of primers used for generation of complementation constructs are indicated by small arrows Click here for additional data file.Figure S2HA-tagged TgRSC8 is a nuclear protein in both tachyzoites and bradyzoites. Amino-terminally HA-tagged TgRSC8 was detected in strain compN by anti-HA reactivity (green). DNA is detected by DAPI (red). Tachyzoites are indicated by detection of the surface marker SAG1, while bradyzoites are shown by DbA reactivity (blue). At left are DIC images, including a 5 µm scale indicator (black bar), and merged panels are shown on the right.(EPS)Click here for additional data file.Figure S3Analysis of steady-state transcript levels by Q-PCR.A. Transcripts corresponding to housekeeping genes TUB1 and GAPDH were assessed in both tachyzoites (gray bars) and in vitro-developed bradyzoites (black bars) of wild-type strain PruΔHPT. The threshold cycles for transcript detection are indicated on the y-axis. Shown are the averages and standard deviations of three experiments performed on independent samples. B. Effects of TgRSC8 reduction on Q-PCR threshold cycles representing transcript levels of bradyzoite-induced and constitutive genes. Q-PCR was performed on cDNA derived from in vitro bradyzoites of vector control strains VC1 (dark blue bars) and VC2 (yellow bars), TgRSC8 mutant C9 (red bars), and three complemented strains . Threshold cycle values for transcript detection are on the y-axis. Shown are the averages and standard deviations of three experiments, performed on cDNAs derived from independent RNA samples.(EPS)Click here for additional data file.Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.


Candida albicans, the homologue of the Set3C inhibits the yeast-to-filament transition, but the precise molecular details of this function have remained elusive. Here, we use a combination of ChIP–Seq and RNA–Seq to show that the Set3C acts as a transcriptional co-factor of metabolic and morphogenesis-related genes in C. albicans. Binding of the Set3C correlates with gene expression during fungal morphogenesis; yet, surprisingly, deletion of SET3 leaves the steady-state expression level of most genes unchanged, both during exponential yeast-phase growth and during the yeast-filament transition. Fine temporal resolution of transcription in cells undergoing this transition revealed that the Set3C modulates transient expression changes of key morphogenesis-related genes. These include a transcription factor cluster comprising of NRG1, EFG1, BRG1, and TEC1, which form a regulatory circuit controlling hyphal differentiation. Set3C appears to restrict the factors by modulating their transcription kinetics, and the hyperfilamentous phenotype of SET3-deficient cells can be reverted by mutating the circuit factors. These results indicate that the chromatin status at coding regions represents a dynamic platform influencing transcription kinetics. Moreover, we suggest that transcription at the coding sequence can be transiently decoupled from potentially conflicting promoter information in dynamic environments.Despite their classical role as transcriptional repressors, several histone deacetylases, including the baker's yeast Set3/Hos2 complex (Set3C), facilitate gene expression. In the dimorphic human pathogen  C. albicans. In this work we dissect how such a chromatin-modifying enzyme regulates fungal morphogenesis. We surprisingly found that perturbation of chromatin has little influence on steady-state transcription, but modulates transient gene expression changes in differentiating C. albicans cells. Altered transcription kinetics affects a group of transcription factor genes that determine morphology. We thus identified a chromatin modifier that exerts kinetic control of transcription factor genes to control fungal morphogenesis. The results highlight the importance of chromatin to determine the kinetics of transcription changes rather than the steady-state transcript levels.Many human pathogenic fungi are able to change their morphological properties, including their size and shape, in response to their outside environment. This ability, which is key for infection, is not completely understood on the molecular level. We have previously shown that not just DNA–binding transcription factors, but also chromatin-modifying enzymes that interact with DNA–binding proteins, are important regulators of morphogenesis in the model fungus  Cells with identical genomes can adopt various phenotypes, which is a central feature during differentiation of multicellular organisms. During differentiation, the outside stimuli and the cellular machineries that process them are thought to determine the resulting cell types. Often, both the original and the resulting cell types are stable and display defined morphologies and gene expression programs. Understanding how such transitions between two stable cellular states are controlled and achieved on the molecular level is of central importance for understanding development and the relationship of organisms with their environment.Candida albicans display diverse morphologies ranging from unicellular yeast-like, to multicellular pseudohyphal, and hyphal structures C. albicans, shared by many other pathogenic fungi of even distantly related taxa such as Histoplasma and CryptococcusUnicellular species such as simple fungi can also undergo differentiation. For instance, pleiomorphic fungi, such as the opportunistic human pathogen C. albicans is responsive to environmental and host stimuli and is controlled by several signal transduction cascades and over thirty transcriptional regulators Hyphal differentiation of Saccharomyces cerevisiae, and sequence homology suggests that it is evolutionarily conserved from fungi to mammals S. cerevisiae, the Set3C occupies the coding sequence of highly transcribed genes and is required for full expression of the galactose-inducible gene cluster C. albicans Set3C acts as a repressor of hyphal differentiation and its function requires functional cAMP/PKA signaling The Set3 Complex (Set3C) was first identified as a repressor of sporulation in C. albicans Set3C controls morphogenesis. First, we used a combination of chromatin immunoprecipitation followed by sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) to define the genome-wide regulatory target genes of the Set3C in yeast and hyphal cells. We found that similar to its S. cerevisiae homologue, the CaSet3C exclusively decorates coding regions and is associated with high transcriptional activity. However, during transient hyphal-inducing conditions, the Set3C delays the establishment of the hyphal-specific gene program by modulating the transcript levels of four phase-specific transcription factors , suggesting that the Set3C can act both as a transcriptional activator and repressor. We also demonstrate that these four factors form a core transcriptional circuit underlying morphogenesis, whose output is restricted by the Set3C. The results provide comprehensive insights into the mechanisms whereby the chromatin layer of regulation superimposes on a core transcriptional factor circuit that controls cellular morphogenesis in C. albicans and possibly in other fungal pathogens.In this study, we set out to identify the molecular mechanism through which the S. cerevisiae, the Set3C is composed of seven different subunits, which show sequence conservation in C. albicans and mammals S. cerevisiae, four of the subunits form a core complex  and are required for structural integrity, while three additional subunits are peripheral  C. albicans strains carrying epitope-tagged alleles of the Set3 and Hos2 subunits. C. albicans is diploid, thus the second alleles in these strains were deleted. Since deletion of the Set3C causes hyperfilamentation S. cerevisiae core complex subunits   , indicatalbicans . In addialbicans  and confalbicans .meta-gene analyses) confirmed the strict localization of binding to coding regions  and RNAPIII-transcribed tRNA loci , arguing that both subunits co-localize on the chromosome  of Set3 and Hos2 in exponentially growing yeast-phase cultures. We first identified binding peaks using Model-based Analysis of ChIP-Seq (MACS)  regions , and sug regions . Set3 anromosome .in vivo, we performed ChIP experiments of acetylated histone H4. The ratio of acetylated H4 to total histone H3 was increased in a set3Δ/Δ strain when compared to wild type at the Set3C-bound positions  are both highly occupied and expressed  remained unaffected. In wild type cells, acetylation level of histone H4 at the coding sequences of selected Set3C-targets correlated with RNA expression  of exponentially growing yeast-phase cultures. We found that ll genes , and thaomologue  [17]. Inxpressed . This imic genes . In cont/Δ cells . These dC. albicans, which is repressed by the Set3C r = 0.69, Pearson's correlation) and Hos2   are responsible for Set3C recruitment, and collected the target lists of all transcription factors (TFs) whose genome-wide binding has been analyzed in C. albicans. These candidate TFs have been implicated in several cellular processes, including morphogenesis and biofilm formation set3Δ/Δ cells.Though the Set3C appears to be selectively recruited to a subset of transcribed genes, the differences in steady-state transcription and gene induction patterns in set3Δ/Δ yeast phase cells  are Set3C targets, and three of the factors  showed altered transcription kinetics in set3Δ/Δ cells  also form a regulatory circuit. Remarkably, ChIP experiments revealed that Nrg1 bound its own promoter and the promoters of the other three TFs in yeast-phase cells  and two are enriched in hyphae (TEC1 and BRG1). The four factors form an interwoven network, whereby all four factors regulate themselves, as well as the other three factors. For practical reasons, we consider here the yeast-specific phase of the circuit as the ground state. In yeast cells, Nrg1 represses hyphal-specific genes, and cAMP/PKA signaling-dependent removal of Nrg1 is required for the induction of hyphal genes set3Δ/Δ cells, the circuit remains intact, but shows a “hyper-excited” state shortly after hyphal induction, that is reflected in the transcript levels of all four regulators  Further evidence for the “hyper-excited” state includes the elevated expression of verified Brg1 target transcripts such as UME6 and HGC1 , and three factors are found in the hyphal regulatory circuit . Hence, if the Set3C restricts the excitation state of the hyphal regulatory network, and there exists at least a partial overlap between the two regulatory circuits, the Set3C may also affect the output of the biofilm regulatory circuit and thus modulate biofilm formation. Strikingly, set3Δ/Δ cells produce a strong “rubbery” biofilm with visibly stronger mechanical properties when compared to wild type cells .In cription , but thenduction , and sevnduction , we propS. cerevisiae recently revealed that histone deacetylation at stress-responsive promoters by Rpd3 is required for normal induction and repression kinetics C. albicansWhy would the chromatin at coding regions be used as such a relay platform? A detailed resolution of the transcriptomic response to several stresses in S. cerevisiae, recruitment of the Set3C has been linked to the recognition of H3K4me2 which decorates mainly coding regions C. albicans Set3C does not depend on dimethylation of H3K4  are completely devoid of ChIP-Seq signal . In S. c of H3K4 . Notably of H3K4 . To ruleC. albicans had to adapt to various niches representing a wide spectrum of physical, chemical and immunological parameters. One of its adaptive strategies appears to be controlling phenotypic transitions through small, evolvable transcriptional circuits that are responsive to outside stimuli of broad amplitudes. Our model proposes that the switch between distinct circuit phases proceeds through an intermediate stage characterized by chromatin changes not just at promoters, but also at gene bodies. We postulate that such chromatin-overlayed transcriptional regulatory circuits underlie the morphological diversity of C. albicans, and most likely many other pleiomorphic fungal pathogens.During its coevolution with the human host, S. cerevisiaeWe wish to point out that during the revision of this work, a report was published, demonstrating that the Set3C modulates transcription kinetics in response to carbon source shifts in C. albicans strains were routinely cultured in YPD . For solid media 2% agar was added. Yeast phase cultures were propagated at 30°C. To obtain exponentially growing yeast cells, single colonies were grown in YPD at 30°C overnight, diluted the next day to an optical density at 600 nm (OD600) of 0.1, and incubated on a rotary shaker for exactly five hours after which cultures reached OD600 = 0.8±0.05. For hyphal induction the cultures were split, one aliquot was washed once with distilled water and resuspended in prewarmed (37°C) YPD+20% fetal calf serum (FCS) of an equal volume of the starting culture. Induced cells were shaken at 37°C for 30 minutes, which provides enough time to induce several hypha-specific transcripts and it is before the first nuclear division occurring around 60 minutes after induction (data not shown). Cultures were optionally snap-frozen in liquid N2 after washing steps and stored at −80°C.C. albicans strains, primers and plasmids used in this study are listed in SET3/set3Δ (DHCA401), set3Δ/Δ (DHCA402), HOS2/hos2Δ (DHCA405) and hos2Δ/Δ (DHCA406) strains were previously described SET1, BRG1 and TEC1 alleles were deleted using the fusion PCR strategy with the C.d.ARG4 and NAT1 markers NRG1 and SET3 alleles were deleted using the SAT1 flipping strategy tec1Δ/Δ (CJN2320) strains were previously described NRG1 with GFP was also created by fusion PCR. Transformation was performed via electroporation as described The complete list of IP was performed essentially as described RNA was isolated by the hot phenol method 2. Pellets corresponding to 80 OD600 cells were resuspended in 480 µl ice-cold ChIP lysis buffer . After addition of around 500 µl glass beads , cells were broken at 6 m/s for 60 seconds 8 times on a FastPrep instrument . Samples were cooled on ice for five minutes between cycles. The bottom of the tubes was punctured with a 273/4G needle and lysates were collected by centrifugation at 1500 g for 1 min at 4°C into a fresh tube. The lysates were diluted to 2.4 ml with ChIP lysis buffer. Aliquots of 300 µl were sonicated 15 times with 30s ON/30s OFF cycles at high setting on a Bioruptor (Diagenode). After sonication the aliquots were combined and centrifuged once at 14000 g for 5 min at 4°C. Supernatants were collected and used as input chromatin lysates. Set3-9myc and Hos2-9myc ChIPs were performed using EZ-View anti-myc coupled agarose beads (Sigma). After overnight incubation at 4°C washing and DNA purification was performed exactly as described ChIP was performed essentially as described set3Δ/Δ cells in both yeast and hyphal phases were included. The resulting 51 base reads were mapped onto the Assembly 21 of the C. albicans genome containing only the coding sequences using TopHat with default parameters, and allowing only for uniquely mapping reads Sequencing of fragmented cDNA was carried out on a HiSeq 2000 instrument (Illumina) at GATC Biotech AG . Three biological replicates of wild type and set1Δ/Δ backgrounds in yeast phase cells were sequenced, with two biological replicates of Set3-9myc ChIP and Hos2-9myc ChIP in hyphal phase cells and two biological replicates of Hos2-9myc ChIP in yeast phase cells. ChIP material of six biological replicates of the untagged control strain in the yeast phase was pooled prior to library preparation. One input sample of each genotype in each morphological phase was also sequenced. Samples were multiplexed with custom adapters. Reads (31–32 base) were mapped onto the chromosomal Assembly 21 of the C. albicans genome using Bowtie, allowing only for uniquely mapping reads r>0.9, Pearson's correlation, not shown). We defined the Set3C target set as an ORF having at least 2-fold Set3 or Hos2 enrichment and at least 1.5-fold enrichment of the other  prior to visualization. The complete ChIP-Seq dataset including chromosomal coordinates and RPKM values of all ORFs and tRNA clusters is found in ChIP libraries were sequenced on a GAIIx platform (Illumina). Three biological replicates of the Set3-9myc ChIP in wild type and Data have been deposited at the Gene Expression Omnibus (GEO) under accession number GSE38427.Figure S1C. albicans Set3C is a conserved nuclear complex. (A) Phenotypic analysis of C. albicans strains carrying epitope-tagged SET3 and HOS2 alleles. Colonies were photographed after incubation on YPD medium at 37°C for three days. Scale bar corresponds to 2 mm. (B) Identification of Set3 and Hos2 interaction partners with immunoprecipitation followed by mass spectrometry. The molecular weights of subunits based on homology to S. cerevisiae are shown on the left panel. Set3-3HA and Hos2-3HA were immunoprecipitated from whole cell extracts and the bound complexes were resolved on SDS-PAGE. The bands indicated by an arrow on the Silver stained gel were isolated and identified by mass spectrometry. Aa: aminoacid Sc: S. cerevisiae, Ca: C. albicans, MW: molecular weight (kDa). (C) GFP-tagged Hos2 shows nuclear localization. Nuclei were stained with Hoechst solution. Scale bar corresponds to 10 µm.The (PDF)Click here for additional data file.Figure S2C. albicans genome. Reads were extended with the length of the MACS d parameter (∼150 bp) prior to visualization. The right arm of chromosome 2 is shown. MRS indicates the Major Repeat Sequence, a repetitive element.Snapshot of the Genome Browser. Reads of the indicated ChIP-Seq samples were mapped on the Assembly 21 of the (PDF)Click here for additional data file.Figure S3−5 were included. On the left panel, the total number of bases in the reads that map to the peak regions is shown (first column), as well as the percentage of the bases located within coding regions, promoters (1000 bases upstream of the start codon), tRNA loci or intergenic regions . The composition of the genome as percentage of the four categories is also displayed (third column), as well as the enrichment of bases in each category in the peaks over the genomic composition (fourth column). On the right panel, the percentage of peaks having a summit position located within the four genomic features is shown. The Assembly 21 of the C. albicans genome was used to determine the genome composition. (B) Correlation of MACS fold enrichment values with the enrichment measured by the RPKM method in one representative Set3 and Hos2 ChIP-Seq experiment. Only the peaks that overlap with gene bodies and with P<10−30 were included. “r” denotes a Pearson's correlation coefficient. (C) Genomic segments encoding long 5′ untranslated regions (UTR) are devoid of Set3C ChIP-Seq signal. A snapshot of the Genome Browser is shown. The coding region is displayed in a blue box. RNAPII ChIP-chip track was taken from Set3 and Hos2 primarily localize to coding regions and tRNA loci. (A) MACS analysis of one representative Set3 and Hos2 ChIP-Seq experiment. Peaks were identified based on enrichment over the respective input samples as described at the (PDF)Click here for additional data file.Figure S4r” denotes a Pearson's correlation coefficient. (B) qRT-PCR validation of the downregulation of hexose catabolic (glycolytic) genes in set3Δ/Δ cells. Data are shown as mean+SD of three independent experiments. Statistical significance was determined by two-tailed t-test relative to the control values. *P<0.05, **P<0.01, ***P<0.001. (C) Histone H4 acetalytion is increased at many target loci in set3Δ/Δ cells. ChIP experiments were performed with antibodies against acetylated histone H4 and the C-terminus of histone H3. The qPCR probes were designed to amplify a fragment at the 5′end of the respective genes. The qPCR values at the probe positions were normalized to a fragment of the telomere of Chromosome 7. The ratio of the signal of the acetylated H4 ChIP and H3 ChIP is shown on the y-axis. Data are shown as mean+SD of two independent experiments. Statistical significance was determined by two-tailed t-test relative to the control values. *P<0.05, **P<0.01, ***P<0.001. (D) Acetylation of H4 at the coding region correlates with RNA expression level. Hexose catabolism genes are colored red, TF genes are colored black. “r” denotes a Pearson's correlation coefficient.Set3C is associated with active transcription. (A) Set3C binding correlates with transcriptional activity genome-wide. Average log2 FPKM values of three RNA-Seq experiments are plotted against Hos2 enrichment. Each dot represents the moving average of 100 genes, and sliding step size is one gene, exactly as described at (PDF)Click here for additional data file.Figure S5set3Δ/Δ cells. Transcripts in wild-type and set3Δ/Δ yeast cells induced to differentiate into hyphae 10, 20, 30 and 60 minutes after induction are shown exactly as described on UME6 and HGC1 (in blue box) are major binding targets of the Brg1 transcription factor in hyphae set3Δ/Δ cells suggests Brg1 hyperactivity. Data are shown as mean+SD of four independent experiments. Statistical significance was determined by two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001.qRT-PCR analysis of additional transcripts in differentiating wild-type and (PDF)Click here for additional data file.Figure S6set3Δ/Δ biofilms. Biofilm growth assays were carried out in Spider medium by growing the biofilm directly on the bottom of 6-well polystyrene plates, as follows. Strains were grown overnight in YPD at 30°C, and diluted to an OD600 of 0.5 in 4 ml Spider medium for each well of the 6-well plate. The 6-well plate was then incubated at 37°C for 90 min at 200 rpm agitation for initial adhesion of cells in an ELMI digital thermostatic shaker. The plates were washed with 4 ml PBS, and 4 ml of fresh Spider medium was added. The plate was incubated at 37°C for an additional 48 h at 200 rpm agitation to allow biofilm formation. set3Δ/Δ cells form a strong “rubbery” biofilm that can be completely removed by the tip of a Pasteur pipette.Phenotypic comparison of wild-type and (PDF)Click here for additional data file.Figure S7in vitro. Set3-3HA was immunprecipitated from whole cell extracts and the interaction is probed by Western blot detection of histones H3 and H4. The interaction is lost in set1Δ/Δ cells that lack H3K4 methylation. (D) Set3 occupancy is not lost in set1Δ/Δ cells in vivo. Set3 enrichment values were measured by the RPKM method of three independent ChIP-Seq experiments. Each dot corresponds to one gene. Set3C targets not bound in set1Δ/Δ cells are highlighted blue, de novo Set3C targets in set1Δ/Δ cells are highlighted red. “r” denotes a Pearson's correlation coefficient. (E) Validation of a de novo Set3C binding target orf19.7502 in set1Δ/Δ cells with qPCR. On the top panel a snapshot of the read density profiles from the Genome Browser is shown with the chromosomal coordinates. Data are shown as mean+SD of three independent experiments. Statistical significance was determined by two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001. (F) Deacetylation of histone H4 by Set3C is independent of H3K4 methylation at the PFK1 locus. Top panel: ChIP experiments were performed with antibodies against acetylated histone H4 and the C-terminus of histone H3. The qPCR values at the probe positions were normalized to a fragment of the telomere of Chromosome 7. Bottom panel: Histone H3K4 dimethylation is unaffected in set3Δ/Δ cells. ChIP experiments were performed with antibodies against H3K4me2 and the C-terminus of histone H3. The qPCR values at the probe positions were normalized to a fragment of ADE2. We failed to retrieve usable ChIP DNA from set1Δ/Δ with the H3K4me2 antibody. Data are shown as mean+SD of two independent experiments. Statistical significance was determined by two-tailed t-test relative to the control values. *P<0.05, **P<0.01, ***P<0.001. n.d.: not determined.H3K4 methylation is dispensable for Set3C recruitment. (A) Alignment of the sequence of the CaSet3 PHD domain to other PHD domains. The eight residues forming the Zn-finger are framed in blue boxes. The residues implicated in the binding of methylated H3K4 are highlighted with arrowheads. (B) Comparative analysis of the predicted structure of the CaSet3 PHD domain. The left side shows the crystal structure of the PHD domain of HsBPTF adapted from (PDF)Click here for additional data file.Table S1C. albicans strains used in the study.List of (DOC)Click here for additional data file.Table S2List of primers used in the study.(DOC)Click here for additional data file.Table S3List of plasmids used in the study.(DOC)Click here for additional data file.Table S4ChIP-Seq RPKM values in all samples for all Assembly 21 ORFs and tRNA clusters.(XLS)Click here for additional data file.Table S5Set3C target gene set in yeast and hyphal phases.(XLS)Click here for additional data file.Table S6FPKM and coverage values for all RNA-Seq samples.(XLS)Click here for additional data file.


Human T-lymphotropic virus type 1 (HTLV-1) encodes the viral protein Tax, which is believed to act as a viral transactivator through its interactions with a variety of transcription factors, including CREB and NF- B. As is the case for all retroviruses, the provirus is inserted into the host DNA, where nucleosomes are deposited to ensure efficient packaging. Previous studies using immunoprecipitation from Tax-expressing cells, 2-dimensional gel electrophoresis, and mass spectrometry analysis have identified the ATPase subunit BRG1 as a Tax-interacting protein. BRG1, is part of at least 8 complexes which include BAF and PBAF, as well as WINAC, NCoR, mSin3A/HDAC, and NUMAC. Nucleosomes act as roadblocks in transcription, making it difficult for RNA polymerase II (Pol II) to proceed toward the 3' end of the genome. Our results using HTLV-1 integrated chromatin indicate that the Tax-activated promoter utilized PBAF complex which appears to be responsible for nucleosome remodeling. Also, the promoter was assembled into an ordered array of translationally positioned nucleosomes. In contrast, the inactive promoter was relatively inaccessible to nuclease and was not assembled into a translationally positioned nucleosomal arrays.


Transcription of numerous mammalian genes is highly pulsatile, with bursts of expression occurring with variable duration and frequency. The presence of this stochastic or ‘noisy’ expression pattern has been relatively unexplored in tissue systems. The prolactin gene provides a model of tissue-specific gene regulation resulting in pulsatile transcription dynamics in both cell lines and endocrine tissues. In most cell culture models, prolactin transcription appears to be highly variable between cells, with differences in transcription pulse duration and frequency. This apparently stochastic transcription is constrained by a transcriptional refractory period, which may be related to cycles of chromatin remodelling. We propose that prolactin transcription dynamics result from the summation of oscillatory cellular inputs and by regulation through chromatin remodelling cycles. Observations of transcription dynamics in cells within pituitary tissue show reduced transcriptional heterogeneity and can be grouped into a small number of distinct patterns. Thus, it appears that the tissue environment is able to reduce transcriptional noise to enable coordinated tissue responses to environmental change. We review the current knowledge on the complex tissue-specific regulation of the prolactin gene in pituitary and extra-pituitary sites, highlighting differences between humans and rodent experimental animal models. Within this context, we describe the transcription dynamics of prolactin gene expression and how this may relate to specific processes occurring within the cell. The temporal dynamics of biological systems is a key component of tissue or organism function. In endocrine tissues, frequency encoding of information via pulsatile hormone secretion has long been appreciated to be important in the regulation of target systems . More reProlactin is an important endocrine hormone, which is secreted primarily from lactotroph cells of the pituitary gland in a circadian manner, with increases in secretion occurring during the pro-oestrous phase of the rat 4-day oestrous cycle, as well as during pregnancy and stress. Prolactin in humans can also be generated locally in numerous tissues and cells, including the endometrium, brain, breast, skin, lymphocytes and adipocytes. Unsurprisingly, therefore, prolactin has been shown to function in numerous processes, including reproduction, metabolism, immunology and behaviour, as well as pathologically in cancer. The diverse expression of the human prolactin gene requires complex regulation, with the use of two independent promoters, which show differential responses to regulatory mediators and cell-type specific activity. This organisation of the human prolactin locus is not conserved across mammalian species and is markedly different from the rat and mouse prolactin loci, which have been used as experimental models of prolactin regulation for decades as a result of sequence conservation of important regulatory elements located proximally to the gene a,b.cis and in trans, indicate that long distance regulation is important and influences the global organisation of DNA within the nucleus  reporter genes. Early research used a construct containing 5 kbp of the human pituitary promoter sequence followed by the luciferase gene, which was transfected into the GH3 somatolactotroph cell line ,77 or adDetailed analysis and mathematical modelling of pulsatile prolactin transcription dynamics have recently been reported a 87). I. I87). Iin vivo in organ systems is highlighted by the different mobility of transcriptional components in cell lines and mammalian tissues . Future developments may aim to increase the sensitivity of these systems and to make them implantable into animal tissues to enable long-term imaging studies.Currently, it is not possible to measure the dynamics of cellular processes, such as transcription, in single cells, ex vivo using a transgenic rat model was developed using the human prolactin locus as a model of complex gene regulation. Reporter genes (luciferase and destabilised GFP) were knocked-in to the first coding exon of the human prolactin locus encoded in a BAC, which enables appropriate regulation of the transgene (Indirect measurement of transcription activity in tissue ransgene 3b. The pransgene 3b (102,1ransgene , indicatransgene  and funcransgene .In adult pituitary tissue, adjacent cells show heterogeneous transcription patterns but the summation of these patterns enables coordinated behaviour across the tissue . It is uIn pituitary development, the transcription patterns of the prolactin gene are altered between nascent lactotroph cells in the embryonic pituitary and more mature cells in neonatal pituitaries 3b. This The major influence of chromatin on nuclear processes, such as transcription and replication is well established and, as mentioned above, chromatin has a large affect on the transcription dynamics of mammalian genes, including the prolactin gene. Chromatin is the complex of DNA and all associated protein and RNA and is mainly comprised of histone proteins. H2A, H2B, H3 and H4 form the nucleosome around which DNA is wrapped, and H1, the linker histone, binds in between nucleosomes aiding folding and chromatin stabilisation. Modification of chromatin structure occurs through the post-translational modification of histones, the substitution of canonical histones with histone variants and by remodelling of nucleosome position by ATP-dependent chromatin remodelling proteins . ChromatIn general, the kinetics of chromatin remodelling and correlation with transcriptional activity are relatively unknown. Inducible transcription reporter systems have been used to assess changes to chromatin with the onset of transcription . Howeverin vivo, independently from its function in transcriptional activation (Nucleosome positioning may affect transcription dynamics through competition with transcription factors for DNA binding sites. The positioning of nucleosomes has a large impact on transcription activity, with remodelling being required to expose transcription factor and RNA polymerase-binding sites in the promoter and also to allow RNA polymerase to transit through the gene. Nucleosome positioning is defined through sequence preferences and competition with other DNA binding proteins; thus, nucleosome positioning is plastic, reflecting equilibrium with DNA binding protein concentrations. This is demonstrated in genome-wide mapping studies of nucleosome position, which report that approximately 80% of the genome shows no preferential positioning of nucleosomes . Stochastivation . These dtivation .Nucleosome positioning may also be affected by Z-DNA. Z-DNA formation is favoured by alternating purine–pyrimidine sequence repeats, generating a nonclassical DNA structure with a left-handed helix. The biological role of Z-DNA has not been fully elucidated, although it may have roles in transcription initiation and chromatin remodelling . There iThe structure and function of chromatin is also modulated by the post-translational modification of histone proteins by phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, ADP ribosylation, deimination and proline isomerisation. Modification of histones affects the interaction of the DNA with the nucleosome and also generates binding motifs enabling the recruitment of regulatory proteins. The modification of histones, along with protein constituents, is grouped to define a small number of chromatin domains that can be linked to gene activity . The modAt the prolactin locus, Pit-1 and ER may be able to alter chromatin structure by histone modification via the recruitment of co-repressor and co-activator complexes and remodelling enzymes –125. ChrDespite the wealth of data on the regulation of the prolactin gene, it is still unclear how cell-specific activity is conferred on the prolactin gene in both pituitary and extra-pituitary sites and a full understanding of how regulation is conferred upon the gene by numerous proteins along with the spatial organisation of the locus within the nucleus is incomplete. Emerging technologies will hopefully provide the resources needed to shed light on these unresolved issues. In particular, the increasing tractability of BAC transgenesis  will ena