Development of prefrontal and other higher-order association cortices is associated with widespread changes in the cortical transcriptome, particularly during the transitions from prenatal to postnatal development, and from early infancy to later stages of childhood and early adulthood. However, the timing and longitudinal trajectories of neuronal gene expression programs during these periods remain unclear in part because of confounding effects of concomitantly occurring shifts in neuron-to-glia ratios. Here, we used cell type–specific chromatin sorting techniques for genome-wide profiling of a histone mark associated with transcriptional regulation—H3 with trimethylated lysine 4 (H3K4me3)—in neuronal chromatin from 31 subjects from the late gestational period to 80 years of age. H3K4me3 landscapes of prefrontal neurons were developmentally regulated at 1,157 loci, including 768 loci that were proximal to transcription start sites. Multiple algorithms consistently revealed that the overwhelming majority and perhaps all of developmentally regulated H3K4me3 peaks were on a unidirectional trajectory defined by either rapid gain or loss of histone methylation during the late prenatal period and the first year after birth, followed by similar changes but with progressively slower kinetics during early and later childhood and only minimal changes later in life. Developmentally downregulated H3K4me3 peaks in prefrontal neurons were enriched for Paired box (Pax) and multiple Signal Transducer and Activator of Transcription (STAT) motifs, which are known to promote glial differentiation. In contrast, H3K4me3 peaks subject to a progressive increase in maturing prefrontal neurons were enriched for activating protein-1 (AP-1) recognition elements that are commonly associated with activity-dependent regulation of neuronal gene expression. We uncovered a developmental program governing the remodeling of neuronal histone methylation landscapes in the prefrontal cortex from the late prenatal period to early adolescence, which is linked to cis-regulatory sequences around transcription start sites. Prolonged maturation of the human cerebral cortex, which extends into the third decade of life, is critical for proper development of executive functions such as higher-order problem-solving and complex cognition. Little is known about changes of post-mitotic neurons during this prolonged maturation period, including changes in epigenetic regulation, and more broadly, in genome organization and function. Such knowledge is critical for a deeper understanding of human development, cognitive abilities, and psychiatric diseases. Here, we identify 1,157 genomic loci in neuronal cells from the prefrontal cortex that show developmental changes in a chromatin mark, histone H3 trimethylated at lysine 4 (H3K4me3), which has been associated with regulation of gene expression. Interestingly, the overwhelming majority of these developmentally regulated H3K4me3 peaks were defined by rapid gain or loss of histone methylation during the late prenatal period and the first year after birth, followed by slower changes during early and later childhood and minimal changes thereafter. The genomic sequences showing these dynamic changes in H3K4me3 were enriched with distinct transcription factor motifs. Our findings suggest that there is highly regulated, pre-programmed remodeling of neuronal histone methylation landscapes in the human brain that begins before birth and continues into adolescence. Prolonged maturation of the human cerebral cortex, which extends into the third decade of life, is critical for proper development of executive functions, such as higher order problem solving and complex cognition Human postmortem brain studies have revealed robust transcriptional changes during the transition from the prenatal period to childhood and into adulthood, with broad implications for inhibitory and excitatory neurotransmission, myelination, metabolism, and various other cellular functions Quantifying developmentally regulated changes in neuronal transcriptomes and epigenomes, however, is confounded by the rapidly occurring and dramatic shifts in cell composition during maturation of the human cerebral cortex. For example, there is a post-natal increase of several fold in the number of astrocytes generated by cell division from local precursors In an earlier study, we presented high-resolution maps of H3K4me3 in neuronal and non-neuronal nuclei collected from the PFC of 11 individuals ranging from 0.5 to 69 years We analyzed 36 datasets of H3K4me3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) of sorted nuclei from the human prefrontal cortex. Thirty-one of these datasets were for neuronal NeuN+ nuclei and as control, the other five datasets were for non-neuronal (NeuN–) nuclei. The neuronal (NeuN+) samples covered an age range from 34 gestational weeks (gw) to 81 years (yr), including three prenatal samples (C1–C3) and three infant samples . The NeuN– samples spanned an age range of 40 gw to 69 yr, including one prenatal sample and one infant sample. Among these datasets, 16 neuronal samples  and two NeuN– samples were newly generated and sequenced for this study, and the remaining 18 datasets were taken from our previous publications http://zlab.umassmed.edu/zlab/publications/ShulhaPLOSGen2013.html. On average, 50% uniquely mapped reads in ChIP-seq samples were in promoters (within 2 kb of the TSS) of RefSeq genes , and 4% reads of the input sample were in promoters, consistent with the prior knowledge that H3K4me3 is strongly enriched around TSS Samples were sequenced using Illumina GAII and a total of 390 million (M) 36-bpreads were obtained, where 318 M reads mapped to unique locations in the human genome . The datTo investigate promoter H3K4me3 occupancy profiles in neurons, we constructed a vector for each neuronal sample with 21,084 elements , each element being the normalized number of reads in a RefSeq promoter. Then we computed the Pearson correlation coefficient for all pairs of samples and presented the results as a heatmap . All 31 To determine how the overall population of H3K4me3 peaks (regardless of their association with any annotated TSS) differ across the neuronal samples, we performed principal components analysis on all H3K4me3 peaks as previously described To identify H3K4me3 peaks subject to age-dependent regulation, we systematically compared the three prenatal samples with the 25 samples older than 1 year of age and found 742 (415) peaks with at least two-fold higher or lower levels in the prenatal samples , encoding an actin-associated protein enriched in dendritic spines and postsynaptic densities These up and down peaks were within 2 kb of the TSSs of 247 and 508 RefSeq genes respectively, and using the DAVID tool, we asked whether these two groups of genes were enriched in any gene ontology (GO) categories SHANK2, encoding a synaptic scaffolding protein that when mutated is responsible for mono- or oligogenic causes of autism and other neurodevelopmental disease LFC/ARHGEF2, encoding a Rho-specific guanine nucleotide exchange factor important for neurogenesis and dendrite spine morphology PLEKHG5, a Pleckstrin homology domain-containing gene that is responsible for an autosomal recessive form of motor neuron disease CABP1,which regulates voltage-gated Ca2+ channels and synaptic short term plasticity SEPT9, a member of the cytoskeleton-related septin family, which is responsible for hereditary neuropathic syndromes such as amyotrophic neuralgia NOTCH3, which is essential for preventing premature death of young differentiating neurons WNT7B, which shows distinct regional expression patterns during human brain development, including progenitor cells and sub-layers of the developing cortical plate DPYSL3, which encodes for a dihydropyrimidinase-like protein important for neuronal differentiation and regulated by NMDA glutamate receptors Some of these genes contain multiple promoters that overlapped with multiple age-dependent H3K4me3 peaks. The first group contained eleven genes whose alternative promoters overlapped H3K4me3 peaks with opposing developmental changes (one up peak and one down peak). Some of these genes are essential for normal brain development, including nervous system development, neurogenesis, neuron differentiation, axonogenesis, neuron projection, synapse, and postsynaptic density among others . To test whether our dataset harbors genomic loci with a different, or non-monotonic age profile, we performed k-means clustering, an unsupervised learning technique to separate all H3K4me3 peaks into five clusters (k = 5) so that peaks within each cluster have similar age profiles. In We also performed GO enrichment analysis on the genes near Cluster 1 and 2 peaks, and the results are shown in et al. performed microarray experiments to assay the genome-wide transcription levels in the prefrontal cortex of 269 subjects spanning the majority of the human lifespan, including 38 prenatal samples (14–20 gws) and 18 infants (<1 yr) H3K4me3 level is a good indicator of gene expression, and we wanted to further test whether the genes near the age-dependent H3K4me3 peaks show similar age-dependent expression patterns. Colantuoni Similarly, we plotted the average expression patterns for the genes near each of the H3K4me3 ChIP-seq peaks in the five clusters determined by k-means clustering . Indeed,et al.Note that the prenatal gene expression data in Colantuoni et al. were for the 14–20 gw period increase in H3K4me3 during development showed a significant decrease in methyl-CpG levels (FDR<0.05), while only 6.2% genes associated with declining H3K4me3 levels also showed a significant decrease in methyl-CpG levels  are enriched in the up or down peaks using the Clover algorithm 10(FDR). Both gene sets were highly enriched in five GO categories related to development . These results indicate that similar functional pathways are pruned epigenetically between neuronal and non-neuronal cells even though the genes that belong to these pathways differ between the two cell types.To further explore which, if any, of the above mentioned developmental H3K4me3 changes are specific to PFC neurons, we explored H3K4me3 landscapes in non-neuronal (NeuN–) nuclei obtained from one prenatal, one infant and three adult specimens . SimilarSimilar analysis for H3K4me3-up genes revealed a different picture. The genes whose TSSs are proximal to the NeuN– H3K4me3-up peaks overlapped partially with the genes that are proximal to the NeuN+ H3K4me3-up peaks . However, these two sets of genes were enriched in mostly non-overlapping GO categories compare  and S11.The present study provides detailed analyses into the developmental regulation of a transcriptional mark, H3K4me3, in neuronal and non-neuronal chromatin during the extended course of PFC maturation. There were 1157 loci, including 768 TSS-proximal and many other regulatory sequences that showed evidence for the developmental remodeling of H3K4me3. Strikingly, these peaks showed similar kinetics as defined by an unidirectional course with the largest decline or increase occurring within the first 1–2 years of postnatal life, followed by a gradual slowing of age-related changes that apparently continue at least until early adolescence or even beyond. We show that these developmentally regulated H3K4me3 peaks at transcription start sites are associated with age-related changes in the expression of the corresponding RNA. We further showed that a subset of regulatory motifs, including Pax and AP-1 transcription factor recognition sites, are overrepresented among the developmental regulated peaks showing a decrease , or increase (AP-1) respectively, during the course of PFC maturation. Finally, the developmental remodeling of H3K4me3 landscapes in PFC is cell type specific. Collectively, our results draw a connection between cis-regulators of chromatin structure and function and the molecular mechanisms governing maturation of the human prefrontal cortex.The findings presented here, when taken in conjunction with studies exploring developmental changes in DNA methylation th week of pregnancy) with those of infant and older brains Our study faces several important limitations. Given that no technique with single cell resolution exists to map histone methylation levels at specific loci, our assays by design only inform about H3K4me3 profiles averaged across millions of cell type-specific nuclei that are required for the ChIP-seq assays. Thus, the cell population-based developmental kinetics of H3K4me3 with the simple and unidirectional exponential curves, as presented here, leave open whether individual PFC neurons would show a more dynamic interplay between H3K4 methylation and demethylation. Furthermore, our prenatal specimens were limited to the mid- and late stages of the third trimester, and it remains possible that brains of an earlier prenatal age could show a more complex or multi-layered regulation of the H3K4me3 marks, compared to what is reported here. For example, there are four groups of genes reported by Colantuoni et al., who compared RNA expression at earlier stages of gestation  tissues from the rostral prefrontal cortex of subjects ranging in age from the 34in banks . Tissue 6 sorted nuclei were prepared for subsequent chromatin immunoprecipitation with anti-H3K4me3 antibody (Upstate/Millipore), and ChIP-seq libraries prepared from the immunoprecipitated DNA by blunt-ending, A-tailing and ligation to adaptors  and PCR amplification and sequencing on an Illumina Genome Analyzer II platform, as described Mononucleosomal preparations from at least 1×10All of our sequencing libraries contained single-end 36-bp reads and we mapped them to the human genome with Bowtie (version 0.11.3) To construct the heatmap in The MACS software (version 1.3.5) Principal Component Analysis was performed on a matrix that contains peaks unique to each sample. Each sample was compared against every other sample using the MACS software with parameters mentioned above. The H3K4me3 peaks thus obtained were further filtered using the following criteria: (1) MACS p-value must be less than 1e-20, (2) read density ratio between the two samples must be greater than 4, and (3) normalized read density must be greater than 0.005.To search for age-dependent H3K4me3 peaks, all peaks from NeuN+ samples were combined and overlapping peaks merged, resulting in 47,281 peaks. The 742 down peaks (≥1 k bp) were defined as: (1) the average read density in prenatal samples must be greater than or equal to 0.01, (2) the ratio of average read density of prenatal samples and the 25 samples older than 1 year must be greater than or equal to 2, and (3) the t-test p-value for comparing the three prenatal samples with the 25 samples older than 1 year must be less than or equal to 0.05. The reciprocal criteria were used for defining the 415 up peaks.We performed k-means clustering on the age profiles of all H3K4me3 peaks. We limited our calculations to regions that were ≥1 k bp and had an average H3K4me3 read density ≥0.01 in prenatal samples or in the 25 samples older than 1 year. The small number of peaks in chrY was excluded from this analysis. This resulted in 14,708 regions that were further normalized by the strongest signal  for each region. We then used the “k-means” procedure from the R software with 5 clusters. We averaged the H3K4me3 levels across the regions in each cluster and plotted the resulting average H3K4me3 profile for each cluster in We used the DAVID web-server for detecting enriched Gene Ontology categories. For each set of H3K4me3 peaks, genes whose TSSs were within 2 k bp of an H3K4me3 peak are included in the analysis. We used false discovery rate (FDR) to quantify statistical significance, because it accounts for multiple testing correction.http://braincloud.jhmi.edu/downloads.htm).The dataset contains methylation level for CGs in a set of gene promoters. CGs in every gene that matched our proximal H3K4me3 peaks were analyzed for age-dependent changes Click here for additional data file.Figure S2th and 75th percentiles; and the whiskers indicate the 10th and 90th percentiles.Box plots showing the distributions of H3K4me3 levels of up genes (A) and down genes (B) in each sample, in correspondence with (PDF)Click here for additional data file.Figure S3Average age profiles for H3K4me3 level for five clusters obtained by k-means clustering. (A) Cluster 1; (B) Cluster 2; (C) Cluster 3; (D) Cluster 4; (E) Cluster 5.(PDF)Click here for additional data file.Figure S4Average age profiles for expression level for five clusters obtained by k-means clustering for H3K4me3. (A) Cluster 1; (B) Cluster 2; (C) Cluster 3; (D) Cluster 4; (E) Cluster 5.(PDF)Click here for additional data file.Figure S5Average age profiles for H3K4me3 level for four groups of genes defined by (PDF)Click here for additional data file.Table S1The up (A) and down (B) H3K4me3 peaks. Numbers of TSS-proximal peaks and genes are different as several peaks can be located near the same gene and vice versa.(XLSX)Click here for additional data file.Table S2GO categories enriched in the genes whose TSS are within 2 kb of age-differential H3K4me3 peaks, with one sheet for up gene sets and another sheet for down gene sets. GO categories that are enriched in both down and up gene sets are colored in blue.(XLSX)Click here for additional data file.Table S3Genes with H3K4me3 regulation at alternative promoters (spaced up to 100 kb apart).(XLS)Click here for additional data file.Table S4Number and proportion of H4K4me3 peaks extending over >500 bp, >1 kb, >2 kb, >3 kb, >4 kb, and more than 5 kb.(XLS)Click here for additional data file.Table S5Gene ontology enrichments for H3K4me3 peaks >4 kb in nucleosomal preparations of NeuN+ PFC nuclei.(XLS)Click here for additional data file.Table S6GO categories enriched in the genes whose TSS are within 2 kb of age-differential H3K4me3 peaks for Cluster 1 and Cluster 2 derived from k-means clustering. GO categories that are enriched in both clusters are colored in blue.(XLSX)Click here for additional data file.Table S7CpG methylation levels at promoters of genes subject to developmental up-regulation of H3K4me3 levels in PFC neurons.(XLS)Click here for additional data file.Table S8CpG methylation levels at promoters of genes subject to developmental down-regulation of H3K4me3 levels in PFC neurons.(XLS)Click here for additional data file.Table S9Statistics on the CpGs that are in promoters of the genes near developmentally up- and down-regulated H3K4me3 peaks in PFC neurons that also show a statistically significant change in DNA CpG methylation between prenatal samples and samples older than 1 year. Two tables are provided for two cutoffs that define statistical change of methylation levels: top for FDR<0.05 and bottom for FDR<1e-10.(XLSX)Click here for additional data file.Table S10The transcription factor motifs enriched in the up or down H3K4me3 peaks.(XLSX)Click here for additional data file.Table S11GO categories enriched in the NeuN– genes whose TSS are within 2 kb of age-differential H3K4me3 peaks for Clusters corresponding to up and down groups derived from k-means clustering.(XLSX)Click here for additional data file.


Arabidopsis thaliana, the components negatively regulating this pathway are less well understood. Here, we demonstrate that Oryza sativa LEAF and TILLER ANGLE INCREASED CONTROLLER (LIC) acts as an antagonistic transcription factor of BRASSINAZOLE-RESISTANT 1 (BZR1) to attenuate the BR signaling pathway. The gain-of-function mutant lic-1 and LIC–overexpressing lines showed erect leaves, similar to BZR1–depleted lines, which indicates the opposite roles of LIC and BZR1 in regulating leaf bending. Quantitative PCR revealed LIC transcription rapidly induced by BR treatment. Image analysis and immunoblotting showed that upon BR treatment LIC proteins translocate from the cytoplasm to the nucleus in a phosphorylation-dependent fashion. Phosphorylation assay in vitro revealed LIC phosphorylated by GSK3–like kinases. For negative feedback, LIC bound to the core element CTCGC in the BZR1 promoter on gel-shift and chromatin immunoprecipitation assay and repressed its transcription on transient transformation assay. LIC directly regulated target genes such as INCREASED LEAF INCLINATION 1 (ILI1) to oppose the action of BZR1. Repression of LIC in ILI1 transcription in protoplasts was partially rescued by BZR1. Phenotypic analysis of the crossed lines depleted in both LIC and BZR1 suggested that BZR1 functionally depends on LIC. Molecular and physiology assays revealed that LIC plays a dominant role at high BR levels, whereas BZR1 is dominant at low levels. Thus, LIC regulates rice leaf bending as an antagonistic transcription factor of BZR1. The phenotypes of lic-1 and LIC–overexpressing lines in erect leaves contribute to ideal plant architecture. Improving this phenotype may be a potential approach to molecular breeding for high yield in rice.Brassinosteroids (BRs) regulate rice plant architecture, including leaf bending, which affects grain yield. Although BR signaling has been investigated in  lic-1 with the phenotype of the ideal rice plant architecture. The C3H-type transcription factor LIC antagonizes BZR1 to repress BR signaling in rice. We used BR to induce the negative regulator LIC and found that it functioned at high BR level, which may restrain plant development. LIC was phosphorylated by GSK3–like kinases. Phosphorylated LIC mainly localized in cytoplasm, whereas dephosphorylated LIC was in nucleus, which was regulated by BR treatment. LIC regulated transcription patterns of the downstream genes in an opposite direction to BZR1. BZR1 activated BR signaling, but the brake module of LIC repressed BR cascade amplification. LIC and BZR1 may balance BR signaling to control growth and development in rice.Brassinosteroids (BRs) are phytohormones mediating multiple biological processes, such as development and stress response. They have been used in crops to produce high yield. In rice, the ideal plant architecture for high yield includes effective tillers, as well as height and leaf angle, which is modulated by BRs. Activation of BRI1–mediated BR signaling is well understood, but much less is known about its inactivating mechanism. Here, we found a gain-of-function mutant  Oryza sativa), leaf-angle response to BRs is a specific physiological process. For example, the erect leaves of BR-deficient rice allow for greater growth density and higher grain yield Brassinosteroids (BRs) are plant steroid hormones that have been used to increase the yield of crops Arabidopsis. Most of the signaling components of this pathway, from the BR receptor BRI1 and co-receptor BAK1 to nuclear transcription factors BZR1 and BES1/BZR2, have been identified CPD by directly repressing transcription CGTGT/CG) and mainly represses gene expression. BES1 binds to E-box by interacting with BIM1 or MYB30 to promote target gene expression The BR signaling pathway has been well studied in Arabidopsis share primary BR biosynthesis and signal transduction pathways. A series of Arabidopsis orthologs of biosynthetic genes identified in rice include D2, D11, BRD1, BRD2, and CPDArabidopsis, only a few targets have been identified in rice Arabidopsis orthologs INCREASED LEAF INCLINATION (ILI1) and ILI1 BINDING bHLH (IBH1), function downstream of OsBZR1 to regulate cell elongation and lamina joint bending Arabidopsis. BZR1 is a key component of the transcription pathway that activates BR signaling in both species. However, how to halt BR signaling at the node of transcription factors including BZR1 remains unclear.Rice, as a model monocot plant and one of the major crops, has been used to study the BR action mechanism. Both rice and LEAF AND TILLER ANGLE INCREASED CONTROLLER (LIC) is involved in sterol homeostasis in rice LIC gain-of-function mutant lic-1 and LIC-overexpressing rice lines to explore a novel mechanism of BR signaling. Both groups showed erect leaves and reduced BR sensitivity as compared with antisense lines. LIC was further characterized as an antagonistic transcription factor of BZR1 in regulating rice architecture. Furthermore, LIC is phosphorylated by GSK1/BIN2 (GSK3-like kinases), which affect translocation from the nucleus to cytoplasm. LIC may mediate a novel mechanism that represses the BR signaling pathway.A CCCH-type zinc finger protein, lic-1 was obtained from the Rice Mutant Database (http://rmd.ncpgr.cn) LIC and was predicted to cause the deletion of 110 amino acids  ratio of lic-1 mutants to the wild-type, which indicates that lic-1 is a dominant mutant. The crossed progenies of lic-1 and the LIC antisense lines showed increased leaf angles that were similar to those of the antisense lines  were similar to leaf angle patterns  LIC  was added to the system, the shifted band was suppressed. In contrast, neither mutated P1  nor P2 (TGCGTG) shifted under the same conditions (LIC promoter containing the P1 binding element significantly enriched as compared with the reference gene promoter (UBQ5) and control fragments  LIC . EMSA wanditions . We perfnditions . Real-tincreased . Thus, BBZR1 RNAi lines with erect leaves to the LIC antisense lines with increased leaf bending to explore the genetic relationship of the lines. By molecular identification  for both lic-1 and the WT. At the total protein level, the signal intensity of LIC in the WT and lic-1 was not significantly different after treatment  and promotes its cytoplasmic retention in Yeast two-hybrid assay revealed that LIC but not forms LICm and LICp interact with GSK1 and SKETHA, as well as AtBIN2. The mutated form did not interact with them . WesternWhen plants were treated with 24-eBL (1 µM), the phosphorylated form of LIC was suppressed , whereasGFP-tagged LIC showed the N/C ratio of LIC with digital fluorescence signals was 3.0  and activation at high  and decreased with high levels   . In the se lines . Thus, LLIC antisense lines in microarray data were re-sorted. In previous microarray analysis LIC antisense lines. We extracted 1 kb of upstream sequences of the genes with altered expression patterns as the predicted promoters and then used MEME (http://meme.sdsc.edu/meme/cgi-bin/meme.cgi) to locate the recurrent motifs. We extracted 14 motifs representing the potential regulatory cis-elements from the altered genes  was consistently found with high values (CTCGC (ATCGCG) and M2 (TCTCGC) led to decreased intensity of the shifted band. In contrast, mutated M3 (AAAAGC) caused the band to disappear.We used MEME to locate the recurrent motifs among the multiple region sequences identified on ChIP. The motif h values . EMSA des , ILI1 expression was higher than in the WT, whereas IBH1 transcription was not significantly altered  in binding in diverse regions such as a, d, e, f and n, but not in regions such as b, c, h, i, j and l . We found that LIC protein recognizes the BZR1 gene through the core element (CTCGC) to repress its transcription. The expression of both genes may be induced by BR treatment at various concentrations. BR treatment at low concentrations (10−9 M) induced the expression of BZR1 and promoted the dephosphorylation of BZR1 protein as an activation mechanism. However, BR treatment at high concentrations (up to 10−7 M) induced LIC expression. The repression of BZR1 transcriptional expression by LIC is enhanced by high BR levels. Therefore, LIC and BZR1 antagonize each other in controlling BR-mediated leaf bending.BZR1 is a key component positively regulating BR signaling, whereas LIC plays a negative role in the signaling pathway. With BR treatment, LIC and BZR1 accumulate in the nucleus and regulate downstream genes. BZR1 represses BR-downregulated genes through the downregulation of BIN2 phosphorylation and decreases cytoplasmic retention mediated by 14-3-3 proteins during BR-mediated induction CCCH domain, binds DNA or RNA in vitro  plants were grown in the field or in the greenhouse at 30°C/25°C (day/night) cycles. For the analysis of BR induction in leaf bending and root growth, rice seeds were sterilized with 1% NaClO and grown in half-strength Murashige and Skoog (MS) medium with the indicated concentrations of 24-eBL  at 30°C under continuous light. Seedlings were examined 7 days after germination. For every transgenic rice plant, 3 lines were used.Rice . The first-strand cDNAs were synthesized by use of M_MLV reverse transcriptase (Promega) and used as RT-PCR templates. Quantitative real-time PCR analysis involved an Mx3000P (Stratagene) with a SYBR green detection protocol. RT-PCR was repeated at least 3 times for each harvested samples with gene-specific primers and gene see . The datLIC, GSK1, SKETHA, and AtBIN2 without the stop codon were amplified by PCR from rice or Arabidopsis and cloned into pGADT7 or pGBDT7 vectors. All binary vector constructs were transformed into Agrobacterium tumefaciens strain GV3101 or EHA105, then transformed into rice calli by A. tumefaciens-mediated transfection The cDNA of LIC from a rice cDNA library was amplified by PCR and ligated into pUN1301 binary vectors for overexpression. Full-length cDNAs of LIC and GSK1 were ligated into pBI121 and pRT105-3×flag vectors A. tumefaciens strain GV3101 and then transformed into tobacco by A. tumefaciens-mediated transfection.For tobacco transformation, full-length cDNAs of LIC sequence was inserted into the pBI221 vector to generate pBI221-LIC. To generate the BZR1pro:LUC reporter gene, the BZR1 promoter was amplified with the rice genomic DNA used as a template and then inserted into the pGEM-T Easy vector to produce pGEM-BZR1p. The BZR1 promoter was released from pGEM-BZR1p by digestion with HindIII and BamHI and inserted into the corresponding sites of the YY96 vector BZR1pro:LUC. The ILI1pro:LUC and IBH1pro:LUC reporter genes were constructed as for BZR1pro:LUC.Full-length Arabidopsis protoplasts and PEG-mediated transfection were as described BZR1pro:LUC, ILI1pro:LUC and IBH1pro:LUC; effector plasmid; and 35S:GUS construct  were co-transformed into protoplasts. After transformation, the protoplasts were incubated at 23°C for 12–15 h, then pelleted and resuspended in 100 µL of 1× CCLR buffer (Promega). For the ß-glucuronidase enzymatic assay, 5 µL extract was incubated with 50 µL 4-methylumbelliferyl ß-d-glucuronide assay buffer  at 37°C for 15 min, and the reaction was stopped by adding 945 µL of 0.2 M Na2CO3. For luciferase activity assay, 5 µL extract was mixed with 50 µL luciferase assay substrate (Promega), and activity was detected with use of a Modulus Luminometer/Fluometer with a luminescence kit (Promega). The reporter gene expression was expressed as relative ratio of LUC to ß-glucuronidase.Isolation of LIC was cloned into the pGADT7 vector. The cDNAs of GSK1, SKETHA, and AtBIN2 were cloned into pGBDT7 (Stratagene) and then transformed into yeast strain AH109. Transformants were screened for growth on medium lacking Leu, Trp, and His. Recovered clones were then assayed for LacZ activity by a filter lift assay. For the transactivation activity assay, LIC, AtBIN2, GSK1, and SKETHA were cloned into the pGBDT7 vector and co-transformed with pGADT7 into yeast cells. Yeast that could grow on SD/-Leu/-Trp/-His medium with ß-galactosidase activity exhibited transactivation activity.The cDNA of Western blot analysis involved extracts prepared from yeast cells as described 5′-TATCCAACATGAATGCCACA-3′ and 5′-CAGCACGAGATGAGTAAAACAA-3′. Sequences used in bioinformatics analysis are in Chromatin immunprecitipation (ChIP) was performed as described 5′-CTCGGTACCGGAGCTGGTGGGTATGACGTC-3′, and OsBES1CHind3, 5′-CGCAAGCTTTCATTTCGCGCCGACGCCGAGC-3′. The recombinant MBP–OsBZR1 was purified from Escherichia coli with amylose resin  according to the manufacturer's instructions LIC was cloned into the expression vector pGEX-4T-1 E. coli BL21 (DE3). Cells were grown at 30°C and induced by the addition of isopropyl β-d-thiogalactopyranoside at a final concentration of 1 mM when the optical density (OD)600 of the cultured cells was 0.5–0.9. The fusion protein was purified with Glutathione Sepharose 4B . The nucleotide sequences of the double-stranded oligonucleotides for EMSA were for LIC P1 (5′-CGA CGT CGT GCG GCC GCG-3′ and 5′-CGC GGC CGC ACG ACG TCG-3′) and LIC P2 (5′-CGG GCG CGT GTG TGG CGG-3′ and 5′-CCG CCA CAC ACG CGC CCG-3′). The oligonucleotides were annealed and then labeled with the Biotin 3′ End DNA Labeling Kit (Pierce). Standard reaction mixtures (20 µL) for EMSA contained 2 µg purified proteins, 2 µL biotin-labeled annealed oligonucleotides, 2 µL 10×binding buffer , 1 µL 50% glycerol, 1 µL 1% NP-40, 1 µL 1 M KCl, 1 µL 100 mM MgCl2, 1 µL 200 mM EDTA, 1 µL 1 µg/µL poly (dI-dC) and 8 µL ultrapure water. The reactions were incubated at room temperature (25°C) for 20 min and loaded onto a 10% native polyacrylamide gel containing 45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.3. The gel was sandwiched and transferred to an N+ nylon membrane (Millipore) in 0.5× TBE buffer at 380 mA in a 4°C refrigerator for 60 min. The detection of biotin-labeled DNA by chemiluminescence followed the manual of the LightShift Chemiluminescent EMSA Kit (PIERCE).EMSA was performed essentially as described http://rsb.info.nih.gov/ij). To measure the ratio of nuclear to cytoplasmic signals (N/C ratio) for LIC-GFP for each cell, small areas were drawn, and measurements of integrated densities were taken from representative areas within the nucleus, cytoplasm, and background  of each cell. Each sample of at least 20 cells was measured 3 times; the average N/C ratio were then calculated GFP fluorescence was visualized under a confocal microscope  equipped with an argon laser (488 nm). GFP was excited by an Argon laser at 488 nm, and images were acquired using a 512b Roper Cascade EMCCD camera and MetaMorph software . Images of LIC-, LICm-, and LICp-GFP were obtained with identical image acquisition settings. A series of images at different points along the z-axis were collected from the top to the bottom. Projection of the z-series of images results in a 3D view of the cell. To quantify the effect of 24-eBL on OsLIC-GFP localization in the time-course experiment, images were obtained with a 500-ms exposure time. Quantification of the fluorescent protein signal involved use of ImageJ  and fractionated by centrifugation at 3000×g. The supernatant was taken as the cytosolic fraction. The pellet was further washed with a resuspension buffer  and reconstituted as the nuclear fraction. All proteins were separated on SDS-PAGE gels, transferred to a nitrocellulose membrane, and probed with anti-LIC antibody. For the in vitro kinase assay, purified LIC-GST protein was incubated with BIN2/GSK1 protein or λ-phosphorylase 1 at 30°C for 30 min and loaded onto SDS-PAGE gels. SDS-PAGE gels of 8% or 15% were used to analyze the total LIC level or its phosphorylation, respectively. The proteins were transferred to a nitrocellulose membrane until the 35-kDa protein marker ran out of the gel during electrophoresis.Total protein samples were extracted from 2-week-old rice seedlings with 2× SDS loading buffer; cytoplasmic and nuclear fractions were extracted as described LIC with the primers 5′-GGATCCGCAAGTACGGAGCGCAGTG-3′ and 5′-AAGCTTTTCG CAGGACCAGGAGCA-3′, subcloned into the pGEM-T-easy vector (Promega), and used as a template for RNA probe synthesis. A fragment of BZR1 was amplified with the primers 5′-ATCAGGAAGCCGGACTGGG-3′ and 5′-GGTTGACGAGGTTGTAGGTGGG-3′. Hybridization in situ with digoxigenin-labeled sense or antisense RNA of LIC and BZR1 was conducted as described Tissues were fixed in 4% (w/v) paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer; samples were vacuum-infiltrated for 30 min and then stored overnight at 4°C. The dehydrated samples after a graded ethanol series were embedded in Paraplast Plus . A fragment of 232 bp was amplified from the second exon of http://meme.sdsc.edu/meme/cgi-bin/meme.cgi) was used to find recurrent motifs among multiple sequences in Affymetrix microarray data for the LIC antisense lines that were up- or downregulated by at least 2-fold MEME software  A diagram of the T-DNA insertion site in the lic-1 mutant and the primers used in the identification of the mutant. LB represents the left border primer in T-DNA, LP and RP represent the left and right primers for LIC respectively. P1+P2 represent primers used to amplify the N-terminal fragment of LIC and P1+P3 represent primers used to amplify full-length LIC. (B) PCR of genomic DNA to amplify T-DNA with primers LB+RP and LIC with primers LP+RP. Italicized numbers 9, 17 and 22 indicate homozygous mutants. (C) PCR of cDNA to amplify full-length LIC and the N-terminal fragment of LIC in the lic-1 mutant. (D) Quantitative RT-PCR analysis of LIC RNA levels in antisense lines and overexpressing lines. Data are mean ± SD (n = 3).Identification of (TIF)Click here for additional data file.Figure S2lic-1 mutant and the transgenic lines. (A) Gross morphologic features of LIC overexpressors and the lic-1 mutant (40 days old). LIC-overexpressing lines (OX1) and the lic-1 mutant showed dwarfism and erect leaves. The antisense line 2 (AS2) and BR-deficient mutant d2 are controls. Bar = 20 cm. (B) Quantification of leaf angles in the wild type, lic-1 mutant and OX1; AS2 and d2 are controls. Data are mean±SE of 50 measured plants.Comparative morphology of the (TIF)Click here for additional data file.Figure S3lic-1 mutant and LIC transgenic lines. lic-1 mutant and LIC-overexpressing line 2 (OX2) show erect leaves and antisense line 3 (AS3) an increased leaf angle.Phenotypes of (TIF)Click here for additional data file.Figure S4D2, D11, BRD1 and the receptor gene BRI1 in the wild type, LIC antisense line 2 (AS2) and lic-1 mutant. Data are mean ± SD (n = 3). *P<0.05 and **P<0.01 compared with the wild type as determined by Student's t test.BR marker genes expression in transgenic lines. Quantitative RT-PCR analysis of the mRNA level of BR synthetic genes (TIF)Click here for additional data file.Figure S5lic-1 mutant and the LIC-overexpressing lines in root growth. The upper panel represents treatment without BR, and the bottom panel represents 1 µM BR treatment; OX1, LIC-overexpressing line 1; AS2, LIC antisense line 2. Bar = 1 cm. (B) Quantification of primary root length under different concentrations of BR. Data are mean ±SD of root length in 30 plants. (C) BR promoted root growth at low levels (<1 nM) and restrained root elongation at high levels (>100 nM). Bar = 2 cm.Rice root growth at different concentrations of BR. (A) BR sensitivity of the (TIF)Click here for additional data file.Figure S6LIC antisense line and a BZR1 RNAi line. (A) Identification of the BZR1 RNAi vector and the LIC antisense vector in hybrid generations. H1, H2 and H3 represent hybrid generations and CK indicates the BZR1 RNAi line or the LIC antisense line as a positive control. (B) Quantitative RT-PCR analysis of LIC and BZR1 RNA levels in parent lines and hybrid generations. Data are mean ± SD (n = 3).Identification of hybrid generations of a (TIF)Click here for additional data file.Figure S7Western blot analysis of protein expression in the yeast cells. (A) LIC interacted with AtBIN2 and rice orthologs in yeast cells. Left panel, LIC interacted with AtBIN2, OsGSK1 and OsSKETHA in a yeast two-hybrid assay; pGADT7-DWF1– and pGBDT7-GSR1–co-transformed yeast served as a positive control (TIF)Click here for additional data file.Figure S8AAAATGG), MS2  and MS3 (AAAATCT) respectively, eliminated the shifted bands. (C) Putative DNA motifs to which LIC binds as predicted from ChIP sequences. Letter probability of every site is shown on the right. (D) LIC bound to the sequence CTCGC marked as S. M1 (ATCGCG), M2 (TCTCGC) and M3 (AAAAGC) were the mutated probes. Co represented the competitive unlabeled S sequence.EMSA to test LIC binding to the predicted motifs. (A) Putative DNA motifs to which LIC binds (denoted as S1–14) as predicted by use of microarray chip gene promoters and MEME software see . (B) EMS(TIF)Click here for additional data file.Figure S9Specificity of the anti-LIC antibody and the anti-BZR1 antibody used in the ChIP assay. Left, western blot analysis with the LIC antibody displayed one specific band for the total protein fraction; LIC protein was decreased in antisense lines and increased in overexpressing lines. Right, western blot with the BZR1 antibody displayed one specific band for wild-type proteins.(TIF)Click here for additional data file.Figure S10LIC and BZR1. (A) Expression patterns of LIC and BZR1 in various organs in rice . Data are mean ± SD (n = 3). (B) LIC and BZR1 expression patterns during seed development and in leaves . The color scale illustrates the microarray signal level. YL, young leaf; ML, mature leaf.Expression patterns of (TIF)Click here for additional data file.Figure S11LIC was used in BLAST searches of NCBI databases . Midpoint-rooted neighbor-joining trees were constructed with full-length protein sequences by use of MEGA 3.1 (http://www.megasoftware.net/index.html) Phylogenic tree of rice LIC (Os06g49080) and related proteins in other model species. The sequence of (TIF)Click here for additional data file.Table S1lic-1 hybrid lines.Number of seeds per panicle and leaf angle for the progenies of antisense line 2 and (DOC)Click here for additional data file.Table S2LIC transgenic rice lines and gain-of-function mutants.Phenotypes of (DOC)Click here for additional data file.Table S3Sequences from the ChIP assay for motif searches.(DOC)Click here for additional data file.Table S4Primers used in this study.(DOC)Click here for additional data file.


A fundamental question in biology is how an organism's genome controls cellular differentiation and identity to produce numerous distinct cell types. At the epigenome level, this is partially influenced by cell type specific regulatory regions marked by accessible chromatin. Recent genome-wide maps of chromatin modifications and DNA associated proteins have identified over a million candidate regulatory elements in the human genome. These DNA sequence elements are proposed to drive the activation or repression of nearby genes, however new tools are needed to do a thorough and systematic functional analysis of these elements in an endogenous context.To directly test distal elements in a loss of function manner, we have fused the chromatin modifying enzyme lysine specific demethylase 1  to transcription activator-like (TAL) effector DNA binding proteins. The TAL:LSD1 fusions are designed to specifically bind target DNA sequences and alter the nearby chromatin state, specifically demethylation of histone H3 lysine 4 di and mono-methylation (H3K4me2/1), a mark of distal enhancers. We have designed TALs to target over 50 putative distal enhancers. These constructs were individually transfected into the erythroleukemic cell line K562, after which we examined changes in H3K4me2 and H3K27acetylation by ChIP, or examined gene expression changes by RNA-seq. Additionally we designed TALs fused to a 3X FLAG epitope to do ChIP-seq analysis and determine the specificity of TAL targeting.We show using TAL:3XFLAG ChIP-seq that binding of TALs is virtually exclusive to their target sequence. We show the TAL:LSD1 fusions have the ability to specifically decrease H3K4me2, as well as H3K27acetyl, another mark of distal enhancers, at their intended target sites. We find roughly 50% of TALs significantly reduce the chromatin modifications at the target enhancer, but not at non-target control enhancers. For a subset of these TAL:LSD1 fusions, we used RNA-seq to identify a nearby gene that is downregulated upon loss of the chromatin state at the enhancer. These nearby target genes show a range of decreased expression, allowing us to relatively quantitate the contribution on transcriptional activation of the target enhancer.By utilizing TAL DNA binding proteins fused to chromatin modifying enzymes, we can precisely alter the endogenous chromatin state of a locus. We use this to direct enzymes that remove active chromatin marks. This is in effect, creating a method to do “epigenetic knockdowns” analogous to the commonly used method of genetic knockdowns. The removal of active chromatin marks disrupts the function of distal enhancers, thereby allowing us to identify the promoters they regulate, and quantify the enhancer’s contribution to its target gene’s expression.


Rhizobium leguminosarum bv. trifolii is required for the establishment of an effective symbiosis with the host plant Trifolium spp. EPS biosynthesis in rhizobia is a very complex process regulated at both transcriptional and post-transcriptional levels and influenced by various nutritional and environmental conditions. The R. leguminosarum bv. trifolii rosR gene encodes a transcriptional regulator with a C2H2 type zinc-finger motif involved in positive regulation of EPS synthesis. In silico sequence analysis of the 450-bp long rosR upstream region revealed the presence of several inverted repeats (IR1 to IR6) and motifs with significant identity to consensus sequences recognized by PhoB and LysR-type proteins associated with phosphate- and flavonoid-dependent gene regulation in R. leguminosarum. Using a set of sequentially truncated rosR-lacZ transcriptional fusions, the role of the individual motifs and the effect of phosphate and clover root exudates on rosR expression were established. In addition, the significance of IR4 inverted repeats in the repression, and P2–10 hexamer in the activation of rosR transcription, respectively, was found. The expression of rosR increased in the presence of phosphate (0.1–20 mM) and clover root exudates (10 μM). PHO boxes and the LysR motif located upstream of the rosR translation start site were engaged in the regulation of rosR transcription. The synthesis of EPS and biofilm formation decreased at high phosphate concentrations, but increased in the presence of clover root exudates, indicating a complex regulation of these processes.The acidic exopolysaccharide (EPS) secreted in large amounts by the symbiotic nitrogen-fixing bacterium  Trifolium, Pisum, Vicia and Medicago spp., which form indeterminate-type nodules [Rhizobium leguminosarum and Sinorhizobium (Ensifer) meliloti are impaired in the invasion of nodule cells and nitrogen fixation [R. leguminosarum EPS is a multi-step process requiring the activity of several enzymes, most of them encoded by pss genes located in the large chromosomal EPS cluster I [R. leguminosarum plays a significant role in biofilm formation, being the major component of the matrix [prsD, plyB, and plyBplyA mutants, forming significantly longer molecules of this polymer than the wild type, are impaired in biofilm formation [S. meliloti, galactoglucan (EPS II)  plays a crucial role in biofilm formation [Exopolysaccharide (EPS) production is a wide spread feature among bacteria, and several functions are ascribed to this polymer, including protection against environmental stress and host defense responses, nutrient gathering, attachment to surfaces, and biofilm formation ,2. In ni nodules –5. EPS-dfixation –9. Acidifixation ,5,10,11.luster I ,13. Simie matrix ,15. Alsoormation . Similarormation .S. meliloti, which produces two types of EPSs, several regulators of succinoglycan (EPS I) and galactoglucan (EPS II) synthesis have been identified on the chromosome  and megaplasmid pSymB  [exoR, exoS, exsB, and exoX genes negatively influence EPS I synthesis, and expR and mucR negatively regulate EPS II synthesis [S. meliloti as a positive regulator of EPS I and a negative regulator of EPS II synthesis [S. meliloti [exp genes involved in EPS II synthesis by the action of the phosphate-dependent WggR and PhoB regulatory proteins [S. meliloti and several other species of proteobacteria [The regulation of EPS biosynthesis in rhizobia is controlled at both transcriptional and post-transcriptional levels and influenced by various nutrient sources and stress conditions. In nd exoX) ,5. The mynthesis –20. ExpGynthesis . MucR plynthesis . The bioynthesis –25. On tynthesis ,25,26, iproteins ,27. PhoBproteins . The PHObacteria . Regulatbacteria .S. meliloti. SyrM increases the expression of some exo genes involved in the synthesis of this polymer via positive stimulation of syrA expression [Under nitrogen limitation, two proteins, SyrM and NtrC, act as positive regulators of EPS I production in pression ,31.S. meliloti, the regulatory mechanisms and external factors influencing EPS biosynthesis in R. leguminosarum have not been extensively studied. Two genes, psiA and psr, identified on the symbiotic plasmid pSym of R. leguminosarum bv. phaseoli, are involved in the regulation of EPS synthesis [psiA copies inhibit EPS synthesis, but this effect is overcome in the presence of additional psr copies. EPS production is also negatively regulated by exoR, which shows extensive similarity to S. meliloti exoR [R. leguminosarum bv. trifolii, a negative regulatory effect of pssB, encoding inositol monophosphate phosphatase, on EPS synthesis has been described [In contrast to ynthesis ,33. Multoti exoR . In R. lescribed .rosR encoding a positive transcriptional regulator of EPS synthesis has been identified in R. leguminosarum bv. trifolii [mucR of S. meliloti [rosR of Rhizobium etli [rosAR of Agrobacterium radiobacter [ros of Agrobacterium tumefaciens [2His2 type zinc-finger motif and are required for positive or negative regulation of EPS biosynthesis. Mutation in R. leguminosarum bv. trifolii rosR results in substantially diminished amounts of EPS and ineffective symbiosis with clover, whereas multiple copies of this gene cause a nearly two-fold increase of EPS production [rosR upstream region decreases rosR transcription, demonstrating autoregulation of its own gene [E. coli consensus sequence binding the cAMP-CRP complex have been found in the promoter region [rosR transcription initiation from the P1 promoter was confirmed [rosR transcription has been significantly decreased showing the possibility of regulation of rosR expression by catabolic repression. It has been established that mutation in rosR resulted in several pleiotropic phenotypes, such as quantitative alterations in the polysaccharide constituent of lipopolysaccharide, changes in membrane and secreted protein profiles, and higher sensitivity to surface-active detergents and some osmolytes [rosR mutant has been shown to exhibit dramatically decreased attachment to and colonization of clover root hairs.Recently, trifolii . It sharmeliloti , rosR ofium etli , rosAR oiobacter , and rosefaciens . All theoduction ,41. RosRown gene . Besidesr region . Also, tonfirmed . In the smolytes . In addiR. leguminosarum bv. trifolii rosR upstream region in the expression of this gene via transcriptional fusions of the rosR upstream region with lacZ. The level of rosR expression and its correlation with EPS production and biofilm formation are assessed in the presence of phosphate and clover root exudates.Here, we examine the significance of several sequence motifs identified in the rosR expression was driven from two separate promoters: the distal strong P1 promoter and the proximal, weaker, P2, and two rosR transcripts of different lengths were identified [In silico sequence analysis of the rosR upstream region revealed the presence of many inverted repeats of different lengths (named IR1 to IR6), and among these IR5 was the longest, with 12-bp inverted repeats . This transcriptional terminator was a 39-base long sequence containing two 12-nucleotide inverted repeats separated by 5 nt, which formed a very stable stem structure with an energy of −20.3 kcal/mol , a 770-nt long transcript was generated. RNA synthesis from the TS2 (position −240 bp) resulted in a 737-nt long transcript. Downstream of the TGA stop codon of kcal/mol .rosR RNA transcripts initiated at TS1 and TS2 revealed the presence of several additional sequences, which stabilized their structures, especially in the upper part of both transcripts. Three stem structures were generated from the first 120 nucleotides of transcript 1 with a total energy of −41.4 kcal/mol, which were substantially stronger than the very stable structure of the transcriptional terminator , located just downstream of the P1 promoter, resulted in a very high increase of rosR transcription [in silico analyses.Previously, we established that deletion of a short DNA fragment in the cription . In thisrosR transcription, we used the previously described pEP13 and pEP14 rosR-lacZ fusions [rosR upstream region which was 36-bp shorter at the 3′ end than the insert of pEP13. β-galactosidase activity in pEP14 was ca. three-fold higher than in pEP13 when studied in Rt24.2. In contrast, rosR transcription for both fusions was at the same level in E. coli DH5α, suggesting that some element(s) specific for the rhizobial background could be engaged in the repression of rosR expression  resulted in a 4-fold decrease of rosR-lacZ expression in relation to pEP13 . It is plausible that the first part of the IR4 motif functions as a target site for some unidentified repressor protein, and its role in the rosR RNA secondary structure stabilization is rather minor. The second part of IR4 that matches the P2–10 hexamer, primarily functions as a binding site of RNA polymerase. Taken together, the results described suggest that the mRNA transcript of this region forming an IR4 loop structure could modulate the level of rosR expression.Three stem structures generated from the first 120 nucleotides of the wild type transcript 1 had a total energy of −41.4 kcal/mol, whereas the mutation of the first part of IR4 (corresponding to the pEP21 and pEP22 fusions) resulted in a moderate decrease of RNA secondary structure stability . The mutation of the second part of IR4 slightly affected the S. meliloti mutants defective in phosphate transport did not fix nitrogen [S. meliloti [R. leguminosarum has not been investigated. Computational analysis revealed the presence of phoB  in R. leguminosarum bv. viciae 3841 genome [R. leguminosarum bv. trifolii WSM1325 PhoB, 93% identity to S. meliloti PhoB and 54% identity to E. coli PhoB [Phosphate is one of the most important nutrients for bacteria and is often limited in the environment . S. melinitrogen , suggestnitrogen ,28,44,46meliloti ,44, the 1 genome , encodinoli PhoB , indicatin silico played a role in the regulation of rosR expression, rosR-lacZ transcriptional fusions were investigated in vivo. R. leguminosarum bv. trifolii 24.2 wild type (Rt24.2) harboring the pEP1 carrying the longest promoter region (403-bp) or its derivatives containing the promoter region truncated at the 3′- or 5′-end were cultured in the presence of phosphate, and β-galactosidase activities were measured  was observed in the presence of 20 mM K2HPO4 (2HPO4 (data not shown).In general, M K2HPO4 . TranscrrosR transcription, strain Rt24.2 bearing fusions with different deletions was cultured in the individual concentrations of phosphate, and β-galactosidase activities were determined , the transcription in pEP9 was lower in relation to pEP1 in the wild type strain , but this difference disappeared in the phoB mutant. For pEP14 and pEP15, similar profiles of rosR expression were found in both genetic backgrounds. Moreover, a significant decrease of rosR-lacZ expression was observed for pEP14 in comparison to pEP1. The pEP15 fusion, lacking all three PHO box-like sequences, showed a very low rosR expression independent of phosphate in the wild type and the phoB mutant 3, possibly interacting with proteins of the LysR family, was identified in close vicinity of the RosR-box and two PHO boxes in the rosR upstream region . In the case of pEP9, lacking the LysR motif, rosR expression was similar in the wild type and the nodD mutant, and almost negligently responsive to exudates. These data suggest that the LysR motif present in the rosR upstream region might be functional, recognized by NodD and may influence rosR transcription.For ANU843(pEP1) growing in the presence of 10 μM exudates, a moderate increase of β-galactosidase activity (1.37-fold) was found. In the ANU851(pEP1) S. meliloti, SyrM, also belonging to LysR-type transcription factors, is involved in the activation of the EPS I synthesis genes [R. leguminosarum genome.The LysR motif was described as a binding site for several transcription factors containing a helix-turn-helix domain, among them NodD . In S. mis genes ,31, but R. leguminosarum was positively controlled by RosR binding to the RosR-box in the promoter region of rosR and pssA encoding the first IP-glycosyl transferase [rosR upstream region on a low copy plasmid pMP220, Rt24.2 carrying the pEP9-pEP15 fusions, and Rt24.2(pBR1) harboring additional copies of rosR on the pBBR1MCS-2 plasmid  (rosR transcription (pEP1 fusion) was stimulated by up to 20 mM phosphate. Introduction of multiple copies of the rosR upstream region containing the RosR-box, being the target site for RosR binding, caused a significant reduction of the amount of EPS (pEP1 fusion). Because in the case of pEP14, which almost totally lacked transcriptional responsiveness to phosphate, the strain carrying this fusion still produced more EPS under phosphate limitation than phosphate-rich condition, these results suggest that other chromosomal genes, besides rosR, involved in EPS production might also be regulated by phosphate. This demonstrates complexity of phosphate-dependent regulation of EPS synthesis in R. leguminosarum. The EPS synthesis in this bacterial species seems to be much more sensitive to phosphate concentration than EPS I production in S. meliloti, being efficiently synthesized under very high concentration of phosphate (above 150 mM). The expression of S. meliloti exoYFQ operon engaged in EPS I synthesis was affected by phosphate concentration and was PhoB-dependent, although PHO box-like sequence identified in its promoter had a relatively low sequence identity with the PHO box consensus [i starvation [Among tested growth conditions, the most effective production of this polysaccharide was observed in M1 with 0.1 mM phosphate, and the wild type strain produced 1.94-fold more EPS under phosphate limitation (0.1 mM) than in the presence of high phosphate concentration 20 mM). The stimulation of EPS synthesis in the phosphate starvation was even more visible for Rt24.2 (pBR1) bearing additional ld type) . EPS proonsensus . In low-onsensus , and bot mM. The rosR transcription and EPS production  and ANU843 (1.28-fold) wild type strains cultured in M1 supplemented with 10 μM exudates, and full congruence was observed between oduction . This inrosR mutant, Rt24.2 harboring pEP1 with additional copies of the rosR upstream region, and Rt24.2 carrying pBR1 with multiple rosR copies  formed about 70% of control biofilm, and the Rt2472 nditions . PhospharosR were higher than those produced by the control strain, especially under low-phosphate. This was in accordance with the earlier observed increase of EPS production under these conditions, confirming the positive role of EPS in biofilm formation .Recent studies of rhizobial species showed that they form biofilms either on abiotic surfaces and on host plant roots –16,53. Fot hairs ,16, confed by us , which dt 7-fold  and 20-fS. meliloti, a key role of EPS II in mature biofilm formation for the correct interaction with the host plant has been shown [, common nodD1ABC genes, whose products synthesize core Nod factor, were required for the establishment of a mature biofilm [S. meliloti [In en shown . Also, c biofilm . Moreovemeliloti .R. leguminosarum bv. trifolii. Similarly, phosphate limitation only slightly enhanced biofilm formation by the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system [In this work, we found that phosphate starvation and the presence of clover root exudates slightly increased biofilm development by y system .rosR mutant was significantly decreased [pssA mutant, which did not produce EPS, induced a 3-fold lower number of nodules than the wild type, and those nodules were hardly occupied by bacteria. This resulted in a significant decrease in the wet shoot mass (60%) of clover plants in comparison to plants inoculated with the wild type strain, confirming the significant role of EPS in host plant infection and effective symbiosis. On the other hand, we observed that multiple copies of rosR and pssA genes significantly enhanced EPS production, competitiveness and clover nodulation in R. leguminosarum bv. trifolii [Previously, we established that the symbiotic efficiency of the ecreased . This mutrifolii .rosR copies and Rt24.2 harboring the pBBR1MCS-2 vector  carrying additional 2 vector . Nitroge2 vector . This ne2 vector .To assess the effect of exudates on symbiosis of Rt24.2 and its derivatives, bacteria were treated for 3 h with flavonoids before plant inoculation . This prThe bacterial strains, plasmids, and oligonucleotide primers used in this study are listed in R. leguminosarum bv. trifolii strains were grown in 79CA medium with 1% glycerol as a carbon source [−1 vitamin stock solution [E. coli strains were grown in Luria-Bertani (LB) medium at 37 °C [rosR expression, EPS production, and biofilm formation, the strains were cultured in M1 medium containing 20 mM morpholinopropane sulfonate (MOPS) supplemented with appropriate concentrations of K2HPO4. To establish the influence of clover root exudates on the expression of rosR-lacZ fusions, EPS and biofilm formation, the strains were grown in 5 mL M1 medium supplemented with vitamins and 10 μM clover root exudates. When required, antibiotics for E. coli and R. leguminosarum were used at the following final concentrations: kanamycin, 40 μg mL−1; ampicillin, 100 μg mL−1; tetracycline, 10 μg mL−1; and nalidixic acid, 40 μg mL−1.n source , in trypn source  containisolution  at 28 °Cat 37 °C . To studR. leguminosarum bv. trifolii as templates and Pfu DNA polymerase  or Taq DNA polymerase  were used. The primers used in this study are listed in R. leguminosarum bv. trifolii rosR were performed with the “Malign” [mfold version 3.2 [Standard techniques were used for plasmid isolation, restriction enzyme digestion, agarose gel electrophoresis, cloning, and transformation . For PCR“Malign”  and “Fuz“Malign”  programssion 3.2  using throsR promoter fragments, the broad-host-range plasmid pMP220 carrying a promoterless lacZ gene was used. A set of promoter deletions was generated on the basis of the 1.2-kb insert of pB31 bearing the entire rosR promoter. For construction of the pEP21 fusion, two primer pairs, pROS2/pREW2 and pRBAM1/pREW3, were used  via biparental conjugation.To construct plasmid fusions containing specific ere used , giving nd pEP23 . The finrosR-lacZ fusions were grown for 24 h in 79CA or M1 medium supplemented with tetracycline. E. coli VH1000 wild type and phoB mutant strains bearing pEP fusions were cultured for 24 h in M1 medium supplemented with tetracycline and different concentrations of phosphate. The assay for β-galactosidase activity was carried out as described previously [Rt24.2 derivatives containing eviously .R. leguminosarum bv. trifolii strains were grown in M1 medium supplemented with 1% glycerol and Dilworth’s vitamins for 3 days at 28 °C in a rotary shaker. To study the effect of the different factors on the level of EPS produced by R. leguminosarum bv. trifolii, the strains were cultured in M1 medium supplemented with 10 μM root exudates, 1% glycerol as a carbon source, and appropriate concentrations of K2HPO4. EPS was precipitated from culture supernatants with 4 volumes of cold 96% ethanol, and then collected by centrifugation, lyophilized, resolved in water and analyzed for carbohydrates according to Loewus [For EPS isolation, 5-mL cultures of o Loewus . Total sR. leguminosarum bv. trifolii strains were grown in M1 medium supplemented with Dilworth’s vitamins and different concentrations of the examined compounds at 28 °C for 48 h. The cultures were diluted to an OD600 of 0.3, introduced in 100 μL aliquots into microplate wells, and incubated at 28 °C for 48 h without shaking. Then, bacterial growth was quantified by measuring the OD600. The liquid was removed, and each well was washed three times with 150 μL of 0.85% NaCl, stained for 15 min with 150 μL of 0.1% crystal violet, and rinsed three times with 150 μL of water. After drying, biofilm formation was quantified by the addition of 150 μL of 95% ethanol and measurement of OD560 using a Benchmark Plus™ microplate reader .The biofilm formation assay was done in microtiter-plates as described by Rinaudi and Gonzalez . Briefly2, 3 min, 70% ethanol, 3 min) were shaken in 1 L sterile water (proportion: 1 mL of water per 2 seeds) in darkness for 4 days at 28 °C [Clover seeds after surface sterilization  seeds were surface sterilized, germinated, and grown on Fåhraeus medium slants [600 of 0.2 (200 μL per plant) and grown under natural light supplemented with artificial light (14 h-day at 24 °C and 10 h-night at 18 °C) in a greenhouse. After 4 weeks, the number of nodules formed on clover roots was counted, and wet mass of clover shoots was estimated. For nodule occupancy, 4-week-old nodules were surface-sterilized, and each nodule was crushed in 100 μL of 79CA medium containing 0.3 M glucose; the obtained suspensions were plated in dilutions on plates containing 79CA agar.Red clover  found in this region. The transcripts initiated from the distal P1 and proximal P2 promoters could form very stable stem-loop secondary structures, which could be involved in post-transcriptional regulation, affecting the stability of rosR transcripts. mRNA degradation is an important mechanism for controlling gene expression in bacteria and the lifetimes of the individual transcripts can differ significantly depending on various environmental conditions [rosR transcripts initiated at TS1 and TS2 contain a long 5′-untranslated region (5′-UTR)  that might be potentially engaged in the regulation of transcription and translation initiation in response to specific proteins or signals [In this work, we found that the expression of nditions ,69. The  signals ,71.rosR expression driven from the P1 promoter (pEP14 in relation to pEP13). Site-directed mutagenesis of the first part of the IR4 motif confirmed that this sequence negatively regulates rosR transcription in Rt24.2, and the second part of the IR4 overlapping the P2–10 sequence plays a significant role in the activation of this process, primarily by binding of RNA polymerase. Because introduced mutations did not significantly affect the stability of RNA secondary structures, we concluded that the IR4 motif could be a potential target for some as yet unidentified rhizobial trans-acting factor, which negatively regulates rosR expression.In this study, we found that a deletion of a 36-bp fragment located downstream of TS1 and containing 6-nt inverted repeats IR4 caused a three-fold increase of rosR expression and its link with EPS production and biofilm formation. Our data indicate that EPS synthesis and biofilm formation in R. leguminosarum bv. trifolii was stimulated in the presence of clover root exudates and under phosphate limitation. Root exudates positively affected EPS production, which was in concordance with a moderate increase of rosR transcription in the presence of flavonoids and the LysR motif in pEP1. In the case of phosphate, a negative effect of this compound on EPS production was observed. The EPS synthesis in R. leguminosarum seems to be much more sensitive to phosphate concentration than EPS I production in S. meliloti, being efficiently synthesized under very high concentration of phosphate (above 150 mM).Here, we studied the influence of phosphate and root exudates on rosR transcription by binding to specific DNA consensus sites and, consequently, modulate EPS production in the presence of suitable external nutritional signals. These results are in agreement with published data demonstrating that the activity of most bacterial promoters depends on multiple nutritional signals and is controlled by two or more transcriptional factors with each factor relaying one environmental signal [Taken together, the presented data indicate that at least four proteins  influence l signal .


Electron microscopy of single gene molecules and mathematical modeling shows that a promoter stochastically transitions between transcriptionally favorable and unfavorable nucleosome configurations, providing a mechanism for transcriptional bursting. PHO5 gene molecules from yeast, we show that the “activated” promoter assumes alternative nucleosome configurations at steady state, including the maximally repressive, fully nucleosomal, and the maximally non-repressive, nucleosome-free, configuration. We demonstrate that the observed probabilities of promoter nucleosome configurations are obtained from a simple, intrinsically stochastic process of nucleosome assembly, disassembly, and position-specific sliding; and we show that gene expression and promoter nucleosome configuration can be mechanistically coupled, relating promoter nucleosome dynamics and gene expression fluctuations. Together, our findings suggest a structural basis for transcriptional bursting, and offer new insights into the mechanism of transcriptional regulation and the kinetics of promoter nucleosome transitions.The number of mRNA and protein molecules expressed from a single gene molecule fluctuates over time. These fluctuations have been attributed, in part, to the random transitioning of promoters between transcriptionally active and inactive states, causing transcription to occur in bursts. However, the molecular basis of transcriptional bursting remains poorly understood. By electron microscopy of single  PHO5 gene molecules from yeast cells and analyzing their structure by electron microscopy, we provide evidence that the “nucleosomes” completely unwrap and then re-wrap in an intrinsically stochastic manner. Only nucleosomes that wrap the regulatory sequences of the gene (promoter) were observed to unspool; no such unspooling was found across the body of the gene. Random unwrapping and re-wrapping generates an ensemble of alternative promoter nucleosome configurations, some conducive to transcription, others not. Mounting evidence suggests that transcription occurs in bursts, where transcripts are released in close succession, interrupted by intervals of transcriptional inactivity; this may lead to significant stochastic fluctuations in gene expression. Although the mechanism of this behavior is not understood, our findings now provide a structural basis for it, suggesting that spooling and unspooling of promoter DNA from the nucleosomes determines the fundamental frequency of transcriptional bursting.In eukaryotes, such as plants, fungi, and animals, the DNA is wrapped around basic protein cores called nucleosomes at more or less regular intervals. This wrapping discourages transcription, the first step in gene expression. By isolating  Gene expression may be viewed as a sequence of molecular transitions; two noise components are then distinguishable: “intrinsic noise,” which derives from the random choice between alternative transitions and the statistical distribution of dwell times between transitions, and “extrinsic noise,” which arises from fluctuations in the cellular concentrations of the biochemical factors that promote the transitions The number of gene product molecules fluctuates over time and between cells Stochastic models have been proposed to account for the intrinsic noise of gene expression. An essential component of such models is the assumption that genes randomly transition between states that are either transcriptionally active (“ON state”), or inactive (“OFF state”) in vivo studies support the notion that nucleosomes are general repressors of transcription PHO5 promoter of yeast has served as a classical paradigm for studies of the relationship between promoter chromatin structure and transcription PHO5 promoter pointed at the possibility of alternative nucleosome configurations for the induced PHO5 promoter, however, without directly demonstrating their existence The wrapping of DNA into nucleosomes limits access of activators and general transcription factors to promoter DNA and impedes the transcribing RNA polymerase PHO5 gene molecules by electron microscopy (EM). Our data demonstrate the existence of alternative promoter nucleosome configurations at steady state PHO5 expression, including the maximally repressive, fully nucleosomal, and maximally non-repressive, nucleosome-free configuration. We show that the observed configurational probability distribution of PHO5 promoter nucleosomes is obtained by a simple stochastic process of nucleosome assembly, disassembly, and position-specific sliding. Our analysis thus provides a molecular basis for transcriptional bursting; and we confirm that PHO5 expression indeed bears the signature of such bursting, with bursting frequency, rather than burst size, as the parameter that responds to transcriptional activators. We demonstrate the possibility of an integrated model of promoter chromatin dynamics and gene expression that quantitatively accounts for measurements of gene expression noise and EM data. The model allows us to predict the life times of microscopically observable promoter nucleosome configurations under repressing and activating conditions, and identifies specific promoter nucleosome transitions as essential for activated transcription.Here, we report the analysis of the promoter nucleosome configuration of single PHO5 gene molecules, we isolated chromatin rings encompassing the promoter nucleosome positions N-3 to N-1 and the open reading frame of the yeast PHO5 gene (in vivo by induction of site-specific recombination between recombination sequences (RS) flanking the PHO5 locus  light, resulting in inter-strand crosslinking of the DNA double helix in nucleosomal linkers, but not core particle DNA PHO5 rings isolated from repressed cells, separated by only short segments of crosslinked double stranded DNA . The steady state probabilities p0,…, p7 of nucleosome configurations 0,…, 7 of such a process obey the following matrix equation pho80Δ cells (see above). The task at hand is to find p0,…, p7), or a vector very similar to it.How can the observed configurational probability distributions of the promoter nucleosomes be explained? In the following we shall demonstrate that the observed probability distributions can be explained as the result of an intrinsically stochastic process, i.e., a process where the future configuration and the configuration's life-time can be predicted only probabilistically. Specifically, we assume that the probability of finding the promoter in state Transitions between nucleosome configurations may be due to assembly, disassembly, or sliding of a nucleosome. Accordingly, we may distinguish three different kinds of transitions. We call the stochastic process “simple” if transitions of the same kind have the same kinetic parameter value, The assumption of a simple process reduces the task of finding the desired matrix ng point .PHO5 induction This topology, with analysis , accountanalysis . The diser than  , the obser than  , with  dtion N-2 , suggestPHO5 tata box mutant (PHO4 or PHO2 (PHO5.) All observed distributions were well explained by the unidirectional sliding topologies of pho4[85–99] mutant, where topology F enjoyed greater statistical support   . The kincription . . The average number of Pho5 molecules, rom FISH , an estiLike our model of promoter nucleosome dynamics, the stochastic model of gene expression  is basedFor given Arbitrary values for of  and  . Howeverarameter . The 0.6PHO5 expression would exclusively be controlled by modulation of the kinetic parameters for nucleosome disassembly and sliding; and steady state PHO5 induction  Assumption of a random process: At steady state, the promoter stochastically transitions between alternative nucleosome configurations; allowed transitions include nucleosome assembly and disassembly transitions between configurations that differ by one nucleosome, and sliding transitions that move nucleosomes out of, but not into, the central N-2 position of the promoter. (AS) Assumption of a simple process: Nucleosome transition probabilities per time and molecule depend only on the kind of transition—assembly, disassembly and sliding—rather than nucleosome position or configuration. (AC) Conduciveness hypothesis: Removal of nucleosome N-2 is necessary for transcription. This specifies the nucleosome configurations that are conducive to transcription. (AD) Assumption of nucleosomal dominance: Nucleosomes exclude transcription factors from promoter DNA AF) Regulatory assumption: The transcriptional activator of PHO5, Pho4, stimulates promoter nucleosome disassembly and sliding and, following nucleosome removal, assembly of a scaffold complex of general transcription factors that supports transcription The chromatin structure of eukaryotic promoters is subject to poorly understood “remodeling” upon transcriptional activation. To clarify the relationship between the structural dynamics of promoter chromatin and gene expression, we here proposed a stochastic model for the of yeast . This moAR, AC, and AD imply that transcription occurs in the form of stochastic “bursts” (AF), rather than steric exclusion (AD), means that nucleosomes are an integral part of the regulatory mechanism for transcription, and not passive repressors whose inhibitory effect is overcome by mass action. However, activated transcription is not explained by accelerated nucleosome removal alone (AF).Together, “bursts” ; the funThis model has withstood our critical testing, while plausible alternatives could be refuted. Importantly, our findings provide direct structural support for the hypothesis of transcriptional bursting, a concept that has proved useful to account for and mechanistically interpret measurements of gene expression noise AR required the establishment of methods that allowed us to investigate the nucleosome configuration of single PHO5 gene molecules. Previous analyses of in vivo chromatin remodeling relied on endonucleolytic cleavage and averaging over large numbers of gene molecules; both averaging and DNA cleavage erased the information necessary to provide a test of our hypothesis: the probability distribution of promoter nucleosome configurations.A critical test of the random process assumption AR and AS, and hence two degrees of freedom only, predicted each of the experimentally observed probability distributions with surprising accuracy when based on either the topology of Remarkably, a simple stochastic process in accordance with assumptions AR. The topology of It may be argued that the observed configurational probability distributions of promoter nucleosomes are the product of a deterministic process driven by an extrinsic oscillation, such as the cell cycle, with randomly distributed phase difference between cells, rather than an intrinsically random process, as claimed by Some data support the assumption that Pho4 binding stimulates disassembly of only the most proximal nucleosome AF implies that loss of promoter nucleosomes is a cause of activated transcription, rather than its consequence. Nucleosome disassembly can be strongly dependent on Swi2, the catalytic subunit of the ATP-dependent remodeling enzyme SWI/SNF AF, therefore, requires the distinction between cause and effect of nucleosome loss. The two-state promoter model . Neither mutation of the TATA box, nor absence of the Pho2 transcription factor caused an increase in promoter nucleosome occupancy , was imposed by our finding, from noise analysis (PHO4 pho80Δ cells), providing a lower bound for the total probability of conducive nucleosome configurations. Our EM data indicated that the only set of configurations consistent with this lower bound, and united by a common structural feature, was the set of configurations with a nucleosome-free position N-2, and not N-1, contrary to previous conjectures The central role for transcription of the nucleosome in position N-2  by summing over all m and n and applying the steady state assumption, j. For the two-state model . Thus, that parameter hypothesis R given the stochastic process model (ii) and topology T.Values of the kinetic parameter for nucleosome disassembly, support, T against topology H by data R, isT and H given R.The statistical All calculations were performed using Mathematica 8 (Wolfram).Figure S1Psoralen crosslinking of naked and nucleosomal PHO5 DNA. (A) Plasmid pM70.1, which contains the PHO5 gene ring construct, was cut with restriction enzymes BamHI and NcoI. The 2.25 kb fragment containing the PHO5 promoter and ORF was purified and used for psoralen crosslinking trials. The orientation of the molecules is unknown due to the absence of LexA adaptor protein and therefore lack of forked 3′-end. (B–F) Examples of single PHO5 molecules following one to five rounds  of psoralen crosslinking. Note the progressive loss of single stranded DNA bubbles as the number of treatments with psoralen increases. Scale bars are 100 nm. (G) Plasmid pM70.1, which contains the PHO5 gene ring construct, was cut with restriction enzymes BamHI, NcoI, and DraI. These cuts gave two fragments, a 0.65 kb promoter fragment and a 1.6 kb ORF fragment. The orientation of the molecules is unknown (see main text). (H) Naked PHO5 promoter and ORF fragments were treated with psoralen (×5) and visualized. An example of a full field electron micrograph is shown on the left  and two examples each of crosslinked promoter fragment and ORF fragment are shown on the right . Black arrowheads point to single stranded DNA bubbles where there was insufficient crosslinking. (I) Linearized, nucleosome-free PHO5 promoter and ORF fragments were included in each PHO5 ring preparation as a control for crosslinking efficiency. Shown here is an example of a preparation of linearized, repressed PHO5 gene rings with added control DNA. Samples were crosslinked after the addition of the control DNA with psoralen (×7) and prepared as described. An example of a full field electron micrograph is shown on the left  and two examples of crosslinked promoter fragments and ORF fragments are shown on the right . Black arrowheads point to single stranded DNA bubbles that failed to crosslink. White arrowheads point to the forked 3′-ends of naked ORF molecules that were bound by free LexA adaptor proteins in the gene ring preparation. Linearized gene ring molecules are distinguishable from naked PHO5 promoter and ORF DNA by measuring their relative contour lengths. (J) Single stranded DNA bubble sizes for repressed (light gray) and activated (dark gray) TATA-less PHO5 gene rings. Bubbles are from the entire PHO5 molecule including the promoter and ORF regions. The average bubble size for repressed and activated rings was 211 bp and 203 bp, respectively. (K) Single stranded promoter bubble sizes for repressed (light gray) and activated (dark gray) TATA-less PHO5 gene rings. The average promoter nucleosome bubble size for repressed and activated rings was 153 bp and 115 bp, respectively.(TIF)Click here for additional data file.Figure S2Endonuclease accessibility and nucleosome occupancies inferred by EM. Accessibilities of the N-1 and N-2 nucleosome positions were previously measured by restriction enzyme digestion on nuclei preparations in repressed, mutant (pho4:Δ85-99), and activated PHO5 strains (white dots) pho4:Δ85-99, gray bars), and activated (dark gray bars) PHO5 gene rings. No restriction sites exist in the N-3 position that were suitable for accessibility assays.(TIF)Click here for additional data file.Figure S3Excised TATA WT PHO5 rings are fully inducible in phosphate-free media. Cultures of yM2.1 [pSH17] , and yM8.14 [pSH17] , were grown in synthetic complete (SC) media made with raffinose as the carbon source and lacking leucine. Phosphatase activity was assayed as previously described PHO5 is constitutively active in yM8.14, due to the pho80Δ mutation.) The cultures were split in half. To one half galactose was added  to a final concentration of 2% to induce the R-recombinase and excision of PHO5 gene rings. Following 1.5 h of incubation in the presence of galactose, cultures were again assayed for phosphatase activity. Cells were then transferred to phosphate-free SC media containing glucose, rather than galactose, and cultured for another 8 h, during which samples were taken in regular intervals for phosphatase assays. Cells divided approximately once (doubling number PHO5 ring excision efficiency of ∼75% (data not shown) and full PHO5 induction on the excised rings (the expected ratio for full induction is (TIF)Click here for additional data file.Figure S4Error estimation for time scale of promoter nucleosome transitions. The kinetic parameters for the degradation of protein, ζ, and mRNA, δ, determined by measurement of the average cell cycle time, and mRNA half life, respectively, provide the time scale for promoter nucleosome dynamics. For given values of ζ and δ, the kinetic parameter value for nucleosome assembly, 2 for protein noise in the PHO4 wild type, −1) was recalculated to again fit the measured value of data see . Thus, t(TIF)Click here for additional data file.Table S1Nucleosome configuration probabilities and nucleosome loss values. Nucleosome configurations were analyzed in six strains. The number of PHO5 molecules analyzed for each strain is reported in the “Rings” column. The probability of finding PHO5 gene rings with promoter nucleosome configurations 0 through 7 are shown, as are the observed molecule counts . These probabilities, which were determined by counting of nucleosome-size bubbles (PHO5 induction relative to full occupancy (Pro. Loss). “R-value analysis” was also used to calculate nucleosome loss on the promoter and ORF of activated PHO5 gene rings relative to repressed gene rings. The R-value is defined as the ratio of single stranded DNA and the contour length of the DNA molecule. To quantify the apparent nucleosome loss due to PHO5 activation , we determined the average extent of single stranded DNA per molecule in base pairs, inferred from knowledge of the total length of the PHO5 gene ring, promoter, and open reading frame of 2,246, 610, and 1,636 base pairs, respectively, and the EM contour length of the molecule. The R-Value Loss was then calculated by taking the (average) difference in single stranded DNA between molecules isolated from pho4Δ pho80Δ cells (in base pairs) and dividing it by the average bubble size of the repressed promoter (153 bp). The difference of 1.89 nucleosomes between repressed and activated gene rings thus determined closely matched the average linking difference between activated and repressed TATA-less PHO5 rings of +1.85 in vivo, in accord with earlier observations for the SV40 chromosome and synthetic chromatin rings pho4:Δ85-99 cells determined by EM was closely similar to the previously determined linking number difference of +0.92 between rings  bubbles , were us(PDF)Click here for additional data file.Table S2Strain list.(PDF)Click here for additional data file.Table S3Maximum likelihood parameter values. Relative parameter values for nucleosome disassembly, cyt” refers to cytoplasmic Pho4, i.e., PHO4 PHO80 wild type grown in high phosphate. TATA and tata refer to the wild type and mutant PHO5 TATA box, respectively. Parameter values for the integrated model of −1 (transition from active to conducive states), −1 (nucleosome assembly), −1 (PHO4 pho80Δ PHO5TATA); and −1 (pho4Δ pho80Δ PHO5TATA). The values of other parameters were as indicated for −1, −1, −1, −1. These parameter values were determined, as described in the main text, from RNA-FISH, northern blot analysis, and measurements of protein molecule number and noise. With the nucleosome disassembly and sliding parameters from EM, and pho4[85-99] mutant (PDF)Click here for additional data file.Text S1Definiteness of parameter values for integrated model.(DOCX)Click here for additional data file.Text S2Kinetic parameter values.(DOCX)Click here for additional data file.