DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein–DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps, or chemical protein–DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical limitations. Here we describe an optimized DamID method coupled with next-generation sequencing (DamID-seq) in mouse cells and demonstrate the identification of the binding sites of two TFs, POU5F1  and SOX2, in as few as 1000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. POU5F1 DamID-seq in the gastrulating mouse embryo at 7.5 d post coitum (dpc) successfully identified multiple POU5F1 binding sites proximal to genes involved in embryo development, neural tube formation, and mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigation of TF–DNA interactions and GRNs in specific cell types of limited availability in mammals, including in vivo samples.The identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability.  DNA adenine methyltransferase identification (DamID) is a distinct method to investigate protein–DNA interactions, based on the exogenous expression of a protein of interest (POI) tethered to the DNA adenine methyltransferase (Dam) from Escherichia coli (meTC marks on DNA adjacent to the POI binding sites. Subsequent genomic DNA (gDNA) extraction, digestion with the GAmeTC-specific restriction enzyme DpnI, adapter ligation, PCR amplification and microarray (DamID-chip), or next-generation sequencing (DamID-seq) allow identification of the POI binding events. Unlike ChIP-seq, this technique does not require formaldehyde fixation or immunoprecipitation steps that could lead to data biases . We have overcome the aforementioned difficulties by applying translation reinitiation–mediated DamID, recently reported in Drosophila (Genome-wide transcription factor (TF) occupancy is commonly assessed by chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq), but the need for both ChIP-grade antibodies and high numbers of cells limits its use . Althoughia coli . Dam spea biases  or loss  and TFs . However and TFs  since Da and TFs . The det and TFs . Since t and TFs , only a osophila , to a moDrosophila Kc cells  were expressed via plasmid transfection under the ecdysone-inducible (Ec) promoter . The leaKc cells . HoweverS system . TranslaS system , providiS system .Pou5f1 knockout ESC line .To optimize translation reinitiation–mediated DamID in mammalian systems, we initially focused on the binding of the master regulator of pluripotency, POU5F1, in mouse embryonic stem cells (ESCs). Preceding DamID experiments, the functionality of the Dam-POU5F1 fusion protein was confirmed by maintaining an undifferentiated state in an inducible Gt(ROSA)26Sor locus (Gt(ROSA)26Sor promoter with high (∼100%) efficiency via simple plasmid transfection and drug selection.We then generated an ESC line containing a PhiC31 integrase–mediated cassette exchange platform within the or locus A, allowidam-only/dam-Pou5f1 coding sequences downstream from the stop codon of three different ORFs: blasticidin- , neomycin- , and hygromycin-  resistance cassettes . Then we measured the methylation level at the Pou5f1 locus in each cell line by quantitative PCR (qPCR)–based DamID (qDamID) .To identify the optimal length of the first ORF for translation reinitiation–mediated DamID, we placed the Bsd cassette was used, the Dam-only methylation level at many of the tested DNA loci was >70%, indicating that Dam expression was too high, although POU5F1 binding was observed at the previously described target site O4 . The use of Neo cassette successfully decreased the methylation levels, generating a POU5F1 binding signature similar to ChIP-seq data . The longer ORF Hyg cassette made the methylation signals even lower and the overall signals indistinguishable between Dam-only and Dam-POU5F1–expressing cell lines. Thus, we opted to use the Neo cassette as the first ORF, and these cell lines were referred to as Rosa26-Neo-Dam and Rosa26-Neo-Dam-Pou5f1 ESC lines in the subsequent experiments.When the Rosa26-Neo-Dam and Rosa26-Neo-Dam-Pou5f1 ESC lines differentiated similarly well into epiblast-like cells (EpiLCs)  and into fibroblast-like cells in vitro. Since the protein level of Dam-only/POI via translation reinitiation is extremely low, it is unlikely to affect the functionality of the cells. Neo-Dam and Neo-Dam-Pou5f1 mRNA levels remained constant after differentiation as we used the ubiquitously active endogenous Gt(ROSA)26Sor promoter . In contrast, when we placed the Ec promoter-driven Dam-only/Pou5f1 cassettes in the Gt(ROSA)26Sor locus, the transgenes were silenced following differentiation into fibroblast-like cells . Although the Rosa26-Neo-Dam and Rosa26-Neo-Dam-Pou5f1 lines constitutively express Dam-only/Dam-POU5F1, we observed loss and gain of the POU5F1 binding signatures using a 72-h EpiLC differentiation protocol , in agreement with ChIP-seq data. In summary, these results indicated that the translation reinitiation–based DamID system could detect distinct POI binding signal in different cell types, as long as cells divide and dilute the methylation signal generated in previous differentiation stages.One of the big advantages of generating Dam-only/POI–expressing ESC lines is that they can be differentiated into any cell type. The Supplemental Table S1), yet with little modifications of the original DamID-chip protocol  before adapter-mediated PCR amplification. The DpnII digestion step was described in the original protocol to avoid amplification of large fragments that do not contain Dam/Dam-POI–bound sites but are flanked by the DpnI-digested GAmeTC sites. Although this step has been excluded in recent protocols . POU5F1 DamID has been recently used to validate a DamID-seq protocol in a different study .A few studies have described the use of DamID-seq for the analysis of TF binding in mammalian cells . All these steps could be performed in a single tube, and the amplified DNA was then purified using SPRI magnetic beads. The purified Dam-only/POI target DNA was subjected to library preparation for Illumina sequencing with Tn5 transposition . About 40% of the POU5F1 DamID-seq peaks overlapped with 35% of the POU5F1 ChIP-seq peaks . However, both peak size and location of the motif within each peak were distinct between DamID-seq and ChIP-seq . The mode of DamID-seq peak size was 1143 bp , and the distribution of POU5F1 motif was widely spread across ±2 kb from the peak center . The mode of ChIP-seq peak size was 334 bp with the POU5F1 motif in the center of the peak . When we randomly selected the same numbers of genomic regions while maintaining the same size distribution as DamID-seq or ChIP-seq peaks, we found that ∼53% and ∼15% of these random regions contained the POU5F1 motif, respectively . Hence, we concluded that the higher presence of the POU5F1 motif in DamID-seq peaks was due to their bigger size compared with ChIP-seq peaks and that both technologies can enrich for genomic regions containing the POU5F1 motif.We initially performed POU5F1 DamID-seq using 10107 ESCs C. We rea107 ESCs  and usedeq peaks E. This oeq peaks . Motif eeq peaks F. OveralInterestingly, DamID-specific peaks were highly enriched with SOX2 and KLF5 motifs, instead of the POU5F1-SOX2-TCF-NANOG binding motif enriched in the overlapping and ChIP-seq–specific peaks F. POU5F1Pou5f1 expression , similar to the overlapping peaks, but they showed higher levels of H3K4me3 and, to a lesser extent, H3K27me3 and H3K4me1 A. Converpression , while >pression E. The fepression  and, morpression . Notablypression F. Since Rosa26-Neo-Dam and Rosa26-Neo-Dam-Sox2 ESC lines. For the comparison with the 106 NSC DamID-seq data, we used the union of two published NSC SOX2 ChIP-seq data . Similarly, the SOX2 DamID-seq–specific peaks were also devoid of signals from DNase-seq and from H3K4me3 and H3K27me3 ChIP-seq, and the SOX2 ChIP-seq–specific peaks had stronger DNase-seq and H3K4me3 ChIP-seq signals  can identify POU5F1 and SOX2 targets in ESCs and NSCs, respectively, of which 40%–44% were also identified by ChIP-seq. Considering the fundamental differences between the two techniques, as well as the scarcity of strategies to validate ChIP-seq data, this reproducible overlap rate in two different TFs and cell types is encouraging. Importantly, the overlap between ESC POU5F1 DamID-seq and NSC SOX2 DamID-seq peaks was as small as that between ESC POU5F1 ChIP-seq and NSC SOX2 ChIP-seq peaks , indicating that the technique-dependent nonspecific peaks in DamID-seq are as few as those in ChIP-seq. A number of nonoverlapping peaks with distinct features exist in both techniques, and their functional importance needs to be carefully studied in future. Nevertheless, the overlapping peaks have stronger signals in both DamID-seq and ChIP-seq . Hence, a possible strategy to reduce the technique specific peaks and enrich peaks that can be identified by both DamID-seq and ChIP-seq would be increasing the threshold of the peak calling.In summary, we have shown that TF DamID-seq with about 10-fold fewer cells . Consistently, DamID-seq peaks from 104 and 103 are enriched in the overlapping peaks between 106 DamID-seq and ChIP-seq peaks . Thus, even when using lower cell numbers, DamID-seq specifically identifies the most robust binding sites, likely representing functionally relevant TF-genome engagement events. Overall, these data indicate the wide applicability of DamID-seq across different cell types and TFs, even when the starting cell number is limited. To our knowledge, these data represent the lowest number of cells used for the identification of TF binding sites in mammalian cells.Since the DamID protocol does not include any precipitation procedure, which causes a loss of DNA, we reasoned that it could be applied to lower numbers of cells. Accordingly, we performed POU5F1 DamID-seq with 105f1 ESCs A. While   embryos containing approximately 15,000 cells , where e5f1 ESCs A.Nanog-GFP reporter–positive ESC-derived cells in the posterior region of the embryos. The differentiation into multiple cell types within each embryo could further reduce the consistency of POU5F1 binding sites in the bulk samples. Accordingly, the in vivo DamID-seq data showed a higher variability between replicates compared with the POU5F1 ESC and SOX2 NSC data . Despite this variability, we identified 2768 peaks, which consistently had higher reads counts in six Dam-POU5F1–expressing embryos compared to six Dam-expressing embryos (FDR < 0.1), as seen for the 7356 peak regions identified in 1000 ESCs POU5F1 DamID-seq using four replicates each for Dam and Dam-POU5F1 .Each embryo showed a different level of ESC contribution as observed by the amount of We performed further analyses with the 343 high-confidence peaks with a high signal and lower variability among those 2768 peaks B,C and fSupplemental Table S2). It is noteworthy that the expression levels of the peak-associated genes, Mycn, Hand2, Prickle2, Sox4, Prrx1, and Nr2f2, changed approximately twofold 24 h after deletion of Pou5f1 at ∼7.5 dpc using the Cre-loxP system 26Sor promoter with translation reinitiation. This approach has enabled us to detect TF targets in as few as 1000 cells and in developing mouse embryos at the gastrulation stage. The applicability of TF DamID-seq to much smaller cell numbers opens opportunities for new investigations in various biological contexts, even though the spatial resolution of DamID-seq is lower than ChIP-seq as the signal relies on the frequency of the GATC sequences (∼260 bp on average in the mouse genome). To date, ChIP-seq has been almost the only strategy used to uncover TF targets in a genome-wide manner in mammalian cells. Although several technical advances have been made . Therefore, it is unlikely that the competition between lowly expressed exogenous Dam-POI and highly expressed endogenous POI compromises the identification of POI binding sites. In fact, DamID has been proven to be a powerful tool in Drosophila to uncover the binding sites of approximately 100 different chromatin proteins 26Sor promoter is ubiquitously active.DamID has predominantly been used to investigate chromatin proteins . For clo. Another important development of TF DamID-seq would be its application in human cells, in which so far DamID has been used mainly for closed chromatin binding proteins by exploiting the leakiness of Ec promoter to express Dam/Dam-POI following viral delivery of the transgenes. In order to achieve a more controlled expression of Dam/Dam-POI and successful TF DamID-seq, human ES/iPS cell lines with the translation reinitiation system in a safe ubiquitously expressed locus (such as the AAVS1) could be established. Obtaining a large numbers of desired cell types in human ES/iPS cell differentiation is often difficult; thus, DamID-seq could have a large advantage over ChIP-seq in many experimental settings.Despite the constitutive expression of Dam/Dam-POU5F1, we could successfully detect gain and loss of TF binding signatures during cell differentiation since the methylation signal was most likely diluted through cell division. When investigating TF binding dynamics in post-mitotic cells or slowly dividing cells, a limitation of our DamID-seq system is the lack of inducibility of Dam-only/POI protein. A tightly regulated inducible tissue-specific Dam-only/POI protein expression system in adult mouse would further increase the applicability of DamID-seq in vivo . AnotherDrosophila melanogaster  recombination. The dam* and dam*-Pou5f1 (dam* = D181A mutant) sequences were generated via site-directed mutagenesis and transferred into a PB-CAG-pA-DEST  vector to generate CAG-Dam*-IRES-Puromycin-pA and CAG-Dam*-Pou5f1-IRES-Puromycin-pA. All the plasmids are available upon request.The coding sequence of Dam protein was taken from the pIND-V5-EcoDam . The assembly . Two sto6 to 2 × 106) using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer's instructions. Extracted gDNA was treated overnight with and without DpnII enzyme, diluted to a final concentration of 2 ng/µL, of which 5 µL was used for each qPCR using the LightCycler480 instrument (Roche). The qPCR signal was converted to an absolute numerical value based on a standard curve, and the average of three triplicates from digested (+DpnII) and undigested (−DpnII) gDNA was used to calculate the percentage (%) of methylation at GATCX site in Dam-only or Dam-POU5F1 expressing cells:Supplemental Table S3.gDNA was extracted from Dam-only/Dam-POU5F1 protein-expressing cells (about 1 × 103), we performed the experiments in four replicates. For the embryo data, we performed the experiments in six embryos each for Dam-only and Dam-POU5F1.All the DamID-seq experiments were performed in a minimum of three replicates for both Dam and Dam-POU5F1/SOX2. When using smaller number of cells . When number of cells was lower than 10,000, cells were sorted directly into the genomic lysis buffer. The gDNA was extracted according to the manufacturer's instructions and eluted with 10 µL of the elution buffer. The gDNA was transferred into a 0.2-mL PCR tube containing 5 µL of DpnI mix  and digested for 3 h at 37°C before heat inactivation of the enzyme for 20 min at 80°C. Double-strand DamID adapters  were pre2O and quantified by Qubit 2.0 (Invitrogen). The sequencing libraries presented in this study have been prepared using the Nextera DNA sample preparation kit (Illumina), according to the manufacturer's instructions. Briefly, after Qubit quantification, 50 ng of the PCR amplified DNA was used in a tagmentation reaction (5 min) in which the Tn5 enzyme fragments the DNA and simultaneously inserts the preloaded Illumina adapters. After a DNA purification step using the DNA clean and concentrator kit (Zymo Research), the tagmented DNA was amplified by PCR for five cycles using the program displayed in Samples were purified using SPRI magnetic beads, resuspended in 30 µL HPrimers used in the PCR amplification are barcoded, so a different pair of primers is used for each sample. After the PCR, DNA is purified using SPRI magnetic beads (0.8× DNA volume) and eluted in 30 µL of water. The purified DNA was used for Qubit 2.0 quantification and Tapestation analysis before pooling of the libraries and submission to the sequencing facility. The concentration of the library pool was usually ∼25–50 nM; 50-bp single-end sequencing was performed at Edinburgh Genomics on the Illumina HiSeq 2500 (chemistry v. 4) system or at BGI on the Illumina HiSeq 4000 system. A list of all the reagents and volumes required for each step of the newly optimized DamID-seq protocol (from gDNA extraction to next-generation library preparation) is summarized in Supplemental Figure S5 were downloaded from GEO with the GEO accession number GSE64008.POU5F1 DamID-seq data in ESCs used for the comparison in http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and these charts were aggregated and visualized using MultiQC (version 0.9)  (ion 0.9) . The “peion 0.9) .The trimmed reads were aligned to the UCSC mm10 assembly of the mouse genome  using BWTo identify binding sites from DamID-seq data, we measure how frequently each GATC fragment  is bound by the Dam-only and Dam-POI proteins. Fragments that contain genuine binding sites will be bound by the Dam-POI protein more often than the Dam-only protein. Binding frequency is measured by sequencing fragments that have been methylated by the Dam protein, and differential binding is assessed by testing for significantly different read counts for each fragment between the Dam-only and Dam-POI samples.Count matrices were created by counting the number of reads mapped fully within fragments, using the featureCounts command from Subread (version 1.5.0) . Low abuTo address the high variability between DamID-seq replicates (particularly from low cell numbers), we applied smooth quantile normalization . Unlike Differentially bound fragments were identified by testing for differential abundance between the Dam-only and Dam-POI samples. The normalized count matrices were given as input to the limma-trend function from the limma package (3.30.13). The trend and robust arguments were set to TRUE, fitting an intensity dependent trend to prior variances and applying the robust empirical Bayes procedure . DiffereRead coverage across the genome was calculated with the genomecov command from BEDTools (version 2.26.0) . Coveraghttp://jura.wi.mit.edu/bioc/tools/venn.php). Box-plots and scatter plots were generated using the package ggplot2 in the R statistical environment (version 3.3.2). The deepTools suite  . A propols suite  was usedhttp://www.ncbi.nlm.nih.gov/geo/) under accession number GSE98092.All DamID-seq data from this study have been submitted to the Gene Expression Omnibus (GEO;


Caenorhabditis elegans, focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in C. elegans, and should inform our understanding of repressed chromatin in other animals.Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in  EUKARYOTIC DNA is organized and compacted in the nucleus through its association with histones and nonhistone proteins, forming a complex called chromatin. The N-terminal tails of all histones, as well as the C-terminal tails of histones H2A and H2B, are subject to post-translational modifications that selectively impact many aspects of nuclear function. The most common histone modifications include methylation, acetylation, ubiquitination, sumoylation, and phosphorylation.Two broad classes of chromatin, euchromatin and heterochromatin, can be distinguished based on protein composition, characteristic post-translational modifications on histones, and transcriptional activity. Euchromatin is either potentially or actively transcribed, and is enriched for RNA polymerase, histone tail acetylation, and trimethylation on histone H3 lysines 4 and 36 (H3K4me3 and H3K36me3). In contrast, heterochromatin tends to be transcriptionally repressed and is associated with histone methylations such as H3K9me3 or the Polycomb-deposited modification H3K27me3. The proteins that recognize these modified histones and the association of heterochromatin with the nuclear envelope help hold chromatin in a compact conformation, and promote the spread of the repressed chromatin state . DespiteAlthough there are many types of repressed chromatin, reflecting a range of modifications and ligands, heterochromatin was traditionally split into two classes that largely reflect the properties of the underlying DNA. On one hand, constitutive heterochromatin covered regions of the genome that are repeat rich and gene poor, and that are kept in a silent state throughout cell division and cell differentiation by H3K9me2/3 and its ligand, Heterochromatin Protein 1 (HP1) .Caenorhabditis elegans have shown that much of the H3K9me3-marked chromatin coincides with H3K27me3  analyses in Su(var)3-9, Enhancer of zeste, and Trithorax, the first HMTs known to carry this domain . HMTs typically contain a conserved catalytic domain called SET, which stems from s domain . The C. ive HMTs . A loss-hylation  modify lC. elegans has 67 proteins containing such domains, which are predicted to be readers of histone modifications , plant homeodomain (PHD)PHD, WD40 repeat (∼40 amino acid terminating in Trp-Asp) 14-3-3, and BRCT (BRCA1 C Terminus) domains (ications .C. elegans is separated into five autosomes and an X chromosome (C. elegans Sequencing Consortium 1998). Hermaphrodites are diploid for all six chromosomes, whereas males have five pairs of autosomes and only a single X chromosome. The regulation and characteristics of the autosomal chromosomes differ from the X chromosome, in part due to dosage compensation in the soma, which represses gene expression approximately twofold on the two hermaphrodite X chromosomes to match expression on the single male X. Here, we do not cover dosage compensation, but refer the reader to reviews of this subject (The 100-Mbp genome of C. elegans chromosomes are holocentric (C. elegans Sequencing Consortium 1998). These differences are reflected in the distributions of chromatin modifications. For example, average levels of modifications associated with gene activity are higher in central regions -methylation (6mA) has been reported on DNA in C. elegans , as well as minisatellite repeats (10–200 bp) that are distributed rather than clustered in large head-to-tail arrays , that forms a stable meshwork underlying the nucleoplasmic side of the nuclear envelope together with a number of well-characterized lamin-associated proteins (LEM-2 (MAN1) and EMR-1 (Emerin), the SUN domain proteins UNC-84 and SUN-1, the accessory protein BAF, and four KASH domain proteins that form a bridge to the cytoskeleton . This functional redundancy reflects the inherent complexity found in chromatin regulatory mechanisms, and argues for redundancy in repression mechanisms. Although synMuv genes were identified based on their roles in the repression of vulval development, most are widely expressed, and the single mutants often show pleiotropic developmental defects and genetic interactions in nonvulval processes. Common phenotypes among them are impaired fertility, altered regulation of repetitive transgenes, L1 arrest at high temperature, and ectopic expression of germ line genes in somatic tissues  targets in young adults . Moreover, the nuclear MET-2 ChIP pattern in adults is very similar to that of its product H3K9me2  neurons. Loss of the H3K9 methyl-transferase MET-2, HPL-2, LIN-61, or CEC-3 leads to the ectopic expression of unc-4 in all VC neurons, while loss of the JMJD-2 histone demethylase reduces this unscheduled expression  is the founding member. These have been shown to be able to demethylate mono- and dimethylated lysine residues, but they are unable to act on trimethylated lysine. The second class contains the Jumonji C (JmjC) domain, which can act on all three methylation states.Histone methyl marks are removed by demethylases that generally fall into two structural classes . One claC. elegans contains 13 JmjC domain-containing proteins (JMJD-2/JMJD2a and JMJD-1.2/CeKDM7a) are involved in H3K9 demethylation , and an accumulation of RAD-51 foci in the germ line, indicative of DNA breaks  and MEC (Mi-2 and MEP-1) complexes , and it bound methylated H3K23 in a peptide pull-down assay  derepressed genes and repetitive elements of all classes and families, including satellite repeats, simple repeats, and RNA and DNA transposons .The observed derepression of repetitive elements is temperature-dependent, with many more repeat families being transcribed at 25° than at 20° . For simple repeats, transcriptional start sites are not yet mapped, but it is possible that cryptic transcription factor binding sites become exposed by the loss of repressive heterochromatin. Importantly, the loss of individual HMTs, i.e., single met-2 and set-25 mutants, shows that different classes of repeats become expressed in the two mutants, consistent with their differential effects on H3K9me marks .The detection of reproducible but low levels of repeat element transcription in met-2set-25 double mutants 3-9 3-9 . ImportaLIN-61 mutants are sensitive to ionizing radiation and LIN-61 is needed for double-strand break repair by homologous recombination, but not by nonhomologous end-joining or single-strand annealing -type HMT, that also bears PHD and post-SET domains, and methylates H3K36me in a transcription-independent manner. . Mass spectrometry of histones isolated from L1 larvae shows that the loss of MET-1 eliminates 95% of the H3K36me3, while the full complement of me1/me2 is retained. RNAi depletion of MES-4 in met-1 mutants reduces H3K36me1/me2 and the residual H3K36me3 in an additive manner .In addition to components of PRC2, the mes-4 mutants revealed an intimate relationship between H3K27 and H3K36 methylation. Genome-wide profiling in embryos and L3 larvae showed that H3K36me3 and H3K27me3 occupy mutually exclusive domains on autosomes, consistent with the finding that methylation of H3K36 inhibits EZH2 activity  embryos causes prolonged sensitivity to ectopic expression of developmental regulators that can cause cell fate transformations  that directs transcriptional silencing, whereby small RNAs provide sequence specificity that directs heterochromatin assembly at target loci and downregulation of RNA polymerase activity (NRDE-3 in the soma or HRDE-1 in the germ line) recruit NRDE-1, NRDE-2, and NRDE-4 to target loci, leading to an accumulation of H3K9me3 in a MET-2- and SET-25-dependent manner, and the stalling of RNA polymerase II  pathway, a small RNA pathway active in the germ line, which is important for the repression of transposable elements as well as endogenous genes . piRNAs ytoplasm . These derm line .nrde-2 mutants, whereas a bias for DNA transposons was observed for prg-1 and heterochromatin mutants. Moreover, redundancy appears to underlie at least part of this apparent separation of function, because nrde-2 genetically interacts with let-418, lin-13, and hpl-2. Additionally, nrde-2; let-418 double mutants derepress many more repetitive elements than either single mutant (Interestingly, loss of the Nrde pathway leads to the derepression of a different set of repetitive elements than loss of the piRNA pathway or of the above subset of heterochromatin factors (e mutant . ConsistC. elegans is no exception, as indicated by the diverse physiological defects of mutants lacking the enzymes that deposit heterochromatic marks or the proteins that recognize them. Repressive chromatin is essential for nuclear organization, genome domain structure, repression of repetitive elements, genome stability, and regulation of protein-coding genes. Cross talk and redundancy between the proteins and pathways involved has been observed, but these interactions are poorly understood. Furthermore, the specificity of most HMTs and histone mark readers remains to be determined, as does the nature and importance of the interactions observed between heterochromatin and small RNA interference mechanisms. Above all, the role of heterochromatin in controlling or shaping developmental programs is still an open question, and the genetics and rapid development of C. elegans are particularly useful for its investigation. As demonstrated in the past, C. elegans is an excellent organism for gene expression studies, given its genetic flexibility, rapid developmental time, and well-defined cell differentiation pathways.Heterochromatin fulfills many important functions in the genome of complex organisms. In this respect,


Saccharomyces cerevisiae, core promoters of class II genes contain a TATA element, either a TATA box (TATA[A/T]A[A/T][A/G]) or TATA-like element (1 or 2 bp mismatched version of the TATA box). The TATA element directs the assembly of the preinitiation complex (PIC) to ensure accurate transcriptional initiation. It has been proposed the PIC is assembled by two distinct pathways in which TBP is delivered by TFIID or SAGA, leading to the widely accepted model that these complexes mediate transcription mainly from TATA-like element- or TATA box-containing promoters, respectively. Although both complexes are involved in transcription of nearly all class II genes, it remains unclear how efficiently SAGA mediates transcription from TATA-like element-containing promoters independently of TFIID. We found that transcription from the TATA box-containing AGP1 promoter was greatly stimulated in a Spt3p-dependent manner after inactivation of Taf1p/TFIID. Thus, this promoter provides a novel experimental system in which to evaluate SAGA-mediated transcription from TATA-like element(s). We quantitatively measured transcription from various TATA-like elements in the Taf1p-dependent CYC1 promoter and Taf1p-independent AGP1 promoter. The results revealed that SAGA could mediate transcription from at least some TATA-like elements independently of Taf1p/TFIID, and that Taf1p-dependence or -independence is highly robust with respect to variation of the TATA sequence. Furthermore, chimeric promoter mapping revealed that Taf1p-dependence or independence was conferred by the upstream activating sequence (UAS), whereas Spt3p-dependent transcriptional stimulation after inactivation of Taf1p/TFIID was specific to the AGP1 promoter and dependent on core promoter regions other than the TATA box. These results suggest that TFIID and/or SAGA are regulated in two steps: the UAS first specifies TFIID or SAGA as the predominant factor on a given promoter, and then the core promoter structure guides the pertinent factor to conduct transcription in an appropriate manner.In  In eukaryotes, general transcription factors (GTFs), Mediator, and RNA polymerase II (pol II) assemble on the core promoter to form a preinitiation complex (PIC) that directs accurate transcriptional initiation –5. In thin vivo and in vitro, regardless of whether they contain the TATA box or not   or AGP1 [-135 – -1 bp] (TK12517-TK8268/BY4741) were first amplified by PCR using the primer pair/template (genomic DNA or plasmid) as described above in parenthesis, and then fused with TK8267-TK8268 to generate a 0.84 kb fragment (AGP1 promoter was replaced with a different one (TATATAAA) derived from the CYC1 promoter. Subsequently, the other two sub-fragments containing VTC1 [-320 – -11 bp] + LEU2 [VTC1 [+1 – +220 bp] (TK9030-TK9171/BY4741) were amplified by PCR. Finally, these two sub-fragments and the aforementioned 0.84 kb fragment were fused with TK7872-TK9171 to generate a 3.6 kb fragment that was used for transformation of YTK2741 and YTK3778, yielding YTK17952 and YTK17974, respectively , or VTC1 [+1 – +220 bp] (TK9030-TK9171/BY4741) were first amplified by PCR, and then fused with TK7872-TK9171 to generate a 3.6 kb fragment that was used for transformation of YTK2741 and YTK3778, yielding YTK17590 and YTK17603, respectively , LEU2 , or thes screen ) were fiVTC1 [-400 – -11 bp] + LEU2 marker [CYC1 promoter [-400 – -124 bp] (TK8260-TK10257/YTK16396), or the CYC1 promoter [-123 – -1 bp] + VTC1 [+1 – +320 bp] (TK12922-TK7875/YTK16396), were first amplified by PCR, and then fused with TK8260-TK7875 to generate a 3.4 kb fragment that was used for transformation of YTK2741 and YTK3778, respectively  to generate a 3.3 kb fragment that was used for transformation of YTK2741 and YTK3778, respectively. Similarly, YTK17750/17766/17769 and YTK17786/17802/17804 were generated from YTK2741 and YTK3778, respectively, using pM8011 (AGP1UAS+CYC1core [TAGCGCAA])/ pM8019 (CYC1UAS+AGP1core [TATATAAA])/ pM8020 (CYC1UAS+ AGP1core [TAGCGCAA]) as a template instead of pM8010.To create YTK17748 and YTK17784, the fragments containing ] + LEU2  + AGP1UATAF1) and Y04228 (Δspt3) were crossed and dissected to generate YTK11871 (TAF1 spt3). Similarly, YTK11708 (taf1-N568Δ) and Y04228 (Δspt3) were crossed and dissected to generate YTK11873 (taf1-N568Δ Δspt3). YTK13039 was generated from YTK11871 by replacing HIS3-marked pM4770/TAF1 with URA3-marked pYN1/TAF1. To create YTK18955 and YTK18964, the fragments containing VTC1 [-170 – -11 bp] + LEU2 [AGP1 promoter [-780 – +1 bp] + VTC1 [+1 – +150 bp] were amplified by PCR (TK2496-TK10408/YTK17952) to generate a 3.3 kb fragment that was used for transformation of YTK11411 and YTK13039, respectively. YTK18974 and YTK18986 were generated from YTK18955 by replacing pYN1/TAF1 with pM4770/TAF1 and pM4773/taf1-N568Δ, respectively. Similarly, YTK18998 and YTK19010 were generated from YTK18964 by replacing pYN1/TAF1 with pM4770/TAF1 and pM4773/taf1-N568Δ, respectively.The strains used in 08 taf1-N8Δ and Y0AGP1UAS+CYC1core [TATATAAA], the two sub-fragments containing VTC1 [-320 – -11 bp] + LEU2 [AGP1UAS [-780 – -301 bp] (TK13578-TK13612/YTK17590), or CYC1core [-230 – -1 bp] + VTC1 [+1 – +814 bp] (TK13619-TK13579/YTK16396), and a linearized vector (TK12873-TK13577/pBluescript II KS+) were first amplified by PCR, and then ligated together using the In-Fusion HD cloning kit (TaKaRa). pM8011 containing AGP1UAS+CYC1core [TAGCGCAA] was created in the same way, except that YTK16398 was used as a template instead of YTK16396.pYN1, pM4770, and pM4773 were described previously , 57. To ] + LEU2  + AGP1UACYC1UAS+AGP1core [TATATAAA], the two sub-fragments containing VTC1 [-320 – -11 bp] + LEU2 [CYC1UAS [-400 – -231 bp] (TK13578-TK13618/YTK16396), or AGP1core [-300 – -1 bp] + VTC1 [+1 – +814 bp] (TK13614-TK13579/YTK17952), and a linearized vector (TK12873-TK13577/pBluescript II KS+) were first amplified by PCR, and then ligated together using the In-Fusion HD cloning kit. pM8020 containing CYC1UAS+AGP1core [TAGCGCAA] was created in the same way, except that YTK17954 was used as a template instead of YTK17952. These plasmids  were used as PCR templates for the construction of eight yeast strains  as described above.To create pM8019 containing ] + LEU2  + CYC1UANorthern blot analysis was performed as described previously . SpecifiVTC1, CYC1, AGP1, RPS5, and SCR1, DNA fragments were amplified by PCR from yeast genomic DNA, purified, and 32P-labeled by random priming with the Klenow fragment (TOYOBO). The PCR primers used were as follows: VTC1, TK9030-TK9013; CYC1, TK9727-TK9745; AGP1, TK8195-TK8196; RPS5, TK493-TK494; and SCR1, TK9507-TK10081.For detection of CYC1 promoter to measure the activities of SAGA-dependent transcription from various CE sequences isolated by a random screen, in which this promoter was used as a backbone , 62AGP1 cription . The resme cells . We alsor strain . Furtherch other . It shouYC1 mRNA . AlthougCYC1 promoter more strongly prefers active CE sequences belonging to class II or class V  and TATAα [TATATAAA], were shown to be functionally different , GA [II-#1A], or CA [VI-#1CA] for the CYC1 promoter; and NN = TG [I-#4], GG [II-#1G], or CG [VI-#1CG] for the AGP1 promoter. Thus, the most 3’-terminal nucleotide (A or G) was replaced by the original one (NN: AA in CYC1 and AG in AGP1)  or impaired (taf1) . The TATin AGP1) . Note thN: AG I-#, AA [I-#, AC II-#C, AT [IICYC1 promoter was Taf1p-dependent, while that from those in the AGP1 promoter was Taf1p-independent. Namely, no TATATANN sequences, as far as we examined here, could alter the Taf1p-dependence or -independence of transcription from these two promoters. Therefore, we conclude that Spt3p/SAGA can mediate transcription from the AGP1 promoter carrying CE sequences belonging not only to class V (AGP1 promoter the TATATACGWVI-#1CG sequence (lanes 24 and 32) exhibited weak activity that was nonetheless significantly stronger than that of the negative control (TAGCGCAAW) (lanes 19 and 27). TATATAC-containing sequences were rarely isolated in the original screen [CYC1 promoter the TATATACAWVI-#1CA sequence (lanes 8 and 16) did not exhibit stronger activity than the negative control (lanes 3 and 11). These observations suggest that TATATAC might be a Spt3p/SAGA-specific CE motif.The results, categorized by class  , showed  class V  but also class V  independl screen , as infeCYC1 or AGP1 promoters  or impaired (taf1) . However, it was unclear whether the CYC1UAS+AGP1core promoter was expressed in a Taf1p-dependent manner (lanes 5–6), similar to the CYC1 promoter (lanes 13–14), because the activity of the CYC1UAS+AGP1core promoter was weaker than that of the CYC1 promoter under these experimental conditions.Previous work showed that the Taf1p-dependence of transcription could be conferred by the core promoter , 66, theromoters . Hence, ectively , 69. Trad (taf1) . The resCYC1UAS was stimulated by raffinose, presumably due to release from glucose repression (compare lanes 1–4 and 9–12 in CYC1UAS+AGP1core promoter was measured again in cells in which Taf1p/TFIID function was intact (WT) or impaired (taf1) . These observations suggest that activation of either AGP1core or CYC1core by CYC1UAS is Taf1p-dependent, but the AGP1core requires more integral Taf1p function than the CYC1core. More importantly, the results obtained after optimizing the experimental conditions  clearly showed that the CYC1UAS+AGP1core promoter could be expressed in a Taf1p-dependent manner  . UnexpecAGP1 promoter was stimulated to a greater extent in the taf1 strain than in the wild type after the temperature shift  in a Taf1p-independent manner, unless the TATA sequence was mutated [CYC1 promoter belongs to a SAGA-dependent and TFIID-independent class [taf1 strain) [CYC1 promoter is TFIID-dominated and not SAGA-dominated [CYC1 promoter was Taf1p-dependent at the VTC1 reporter locus, but not at the endogenous CYC1 locus [CYC1 mRNA derived from the CYC1 locus obtained at that time, cited as “data not shown” in the original reference [SCR1 RNA due to insufficient washing of the Northern blot prior to reprobing. After repeating the same experiment several times, we are now convinced that the CYC1 promoter is Taf1p-dependent even at the endogenous CYC1 locus  . On the ominated . In a moC1 locus . During eference , were coC1 locus , whereasand SAGA , 71, 72. plasmid  but not  plasmid , suggest plasmid , 75, depVTC1 reporter system using the AGP1 promoter to test for Spt3p/SAGA-dependent transcription from various types of CE sequences without the involvement of Taf1p/TFIID. In this system, following the temperature shift to inactivate the mutated version of Taf1p/TFIID, transcription transiently decreased and then recovered to a much greater extent in the taf1 strain than in the WT in a Spt3p/SAGA-dependent manner  but also to class II , class V , or class VI  (the region of each sequence matching the consensus in each class is underlined) were transcriptionally active in the AGP1 promoter even after inactivation of Taf1p/TFIID. These observations provide strong evidence that Spt3p/SAGA mediates transcription from the TATA-like element independently of Taf1p/TFIID.In this study, we set up a t manner , S3 Fig.zed mRNA , 72, 76 CYC1 and AGP1 promoters would provide useful model systems for this purpose because TFIID and SAGA function very differently on these two promoters, even if they both originally contained TATA boxes  and RPS5UAS-RPS5core (Taf1p-dependent) promoters [RPS5UAS-ADH1core (Taf1p-independent) and RPS5UAS-RPS5core (Taf1p-dependent) promoters [2xGAL4 sites-GAL1core (Taf1p-independent), RPS5UAS-GAL1core (Taf1p-independent), 2xGAL4 sites-RPS5core (Taf1p-dependent), and RPS5UAS-RPS5core (Taf1p-dependent) also supported the latter conclusion that the core promoter is responsible for Taf1p-dependence [Our mapping experiments using chimeric promoters revealed that the Taf1p-dependence and -independence of transcription from the promoter , S6 Fig.romoters . By contromoters , 67. Compendence . Alternapendence , 63.RPS5UAS was observed only for the TATA-less RPS5core [ADH1core [GAL1core [CYC1core [RPG transcription usually recovers within 1 hour after the temperature shift   between romoters . In addiromoters , 84. Conromoters , 21, it AGP1UAS activated CYC1core and AGP1core in a Taf1p/TFIID-independent manner . Therefore, we propose that the functions of TFIID and SAGA are regulated in two steps: first, the UAS specifies TFIID or SAGA as the predominant factor on a given promoter, and then the core promoter structure guides this factor to conduct transcription in an appropriate manner. Future studies should seek to reveal the detailed mechanisms by which TFIID and SAGA mediate transcription from TATA box-containing and TATA-less promoters, as well as the physiological significance of the promoter-specific functions of these two related transcription complexes.In summary, we showed here that t manner , S6 Fig t manner . Similart manner , S6 Fig,S1 FigA. Northern blot analyses to examine expression of CYC1, AGP1, or control genes (RPS5 and SCR1) in four strains: TAF1 SPT3 , taf1-N568Δ SPT3 , TAF1 Δspt3 , or taf1-N568Δ Δspt3 . The analyses were conducted as described in B. Raw data shown in A were quantified and presented graphically as described in (EPS)Click here for additional data file.S2 FigA. Model of the roles of TFIID and SAGA on the CYC1 promoter. Transcription from the CYC1 promoter was significantly weakened in the taf1 strain after the temperature shift (II), whereas it was also weakened in the Δspt3 strain, but in a temperature shift-independent manner (III)  . B. Model of the roles of TFIID and SAGA on the AGP1 promoter. Transcription from the AGP1 promoter was stimulated after the temperature shift in the taf1 strain (II) but not in the Δspt3 strain (III), whereas it was almost abolished in the taf1 Δspt3 strain (IV) (AGP1 transcription in the WT strain (I). Importantly, it is possible to determine whether Spt3p/SAGA mediates transcription from various CE sequences by assaying their transcriptional activities under condition II. C. Two possible models of the function of TFIID and SAGA. SAGA may function upstream of TFIID (left panel) or in parallel to TFIID (right panel). Parallel-type promoters (right panel) were more suitable for our purpose than sequential-type promoters (left panel) because SAGA is more directly involved in transcription on the former than on the latter.er (III) . Notablyain (IV) , indicatain (IV) . These o(EPS)Click here for additional data file.S3 FigA. Northern blot analyses to examine the expression of the reporter (VTC1) or control genes  in four strains: TAF1 SPT3 , taf1-N568Δ SPT3 , TAF1 Δspt3 , or taf1-N568Δ Δspt3 . Analyses were conducted as described in B. Raw data shown in A were quantified and presented graphically as described in (EPS)Click here for additional data file.S4 FigA. Four CE sequences (marked with asterisks at the left) belonging to class II  belonging to class V  isolated in a previous screen [TTAAAWV-#1, GTATTAAAWV-#2, TTTTTAAAWV-#3, TACTTAAAWV-#4, and CATTTAAAWV-#5, respectively. The portion of each sequence matching the consensus TTAAAW is underlined in the preceding sentence or indicated by a red rectangle in the figure. This figure is adapted from s screen ) were teTATATCA #, GCTATAATATAATA # isolateds screen  were tesTATATCA #, GCTATAATATAAAA #, and TAT(EPS)Click here for additional data file.S5 FigA. Nucleotide sequence of the CYC1 promoter. The positions of the two TATA boxes and two TATA-like elements are underlined: #1 (TATAβ), #2 (TATAα), #3, and #4. Each TATA box or TATA-like element was replaced with a specific sequence, denoted as “m”, to disrupt its transcriptional activity [β located at site #2 (marked with a thick underline). In this study, TATAβ was replaced with various CE sequences belonging to class I, II, V, or VI. B. Nucleotide sequence of the AGP1 promoter. The position of the TATA box in the AGP1 promoter is thickly underlined. This element was modified as described in A. Note that it was also replaced with TATAβ (TATATAAA) of the CYC1 promoter and used as a positive control in all experiments, except in A.activity , 65. The(EPS)Click here for additional data file.S6 FigA. Northern blot analyses to examine the expression of the reporter (VTC1) or control genes (RPS5 and SCR1) in the WT strain carrying the reporter driven by the CYC1 , AGP1 , CYC1UAS+AGP1core , or AGP1UAS+CYC1core  promoters, as indicated at the top. All four promoters contain TATATAAA (odd numbered lanes) or TAGCGCAA (even-numbered lanes) sequences at the TATA site, as indicated below the index for “UAS” and “core”. Total RNA (20 μg) was isolated from these eight strains  grown to a logarithmic phase at 25°C (lanes 1–8) or 30°C (lanes 9–16) in rich media containing 2% glucose (lanes 1–8) or 4% raffinose (lanes 9–16), blotted onto the membrane, and hybridized with the gene-specific probes indicated at the right. B. Raw data shown in A were quantified and presented graphically. Values for each transcript  were normalized against the level of SCR1 (pol III transcript) and presented relative to the value for the TATA box of the CYC1 promoter (lane 1). C. Northern blot analyses to examine the expression of the reporter (VTC1) or control genes (RPS5 and SCR1) in WT or taf1 strains carrying a reporter driven by the chimeric CYC1UAS+AGP1core promoter in which the TATA box was substituted with TATATAAA  or TAGCGCAA  as indicated. Total RNA (20 μg) was isolated from these four strains  grown to a logarithmic phase at 25°C in rich media containing 2% glucose  or 4% raffinose  (indicated as “25” above the blot), or further incubated at 37°C for 2 hours in each type of media , blotted onto a membrane, and hybridized with the gene-specific probes indicated at the right. Autoradiogram hybridized with a VTC1 probe and exposed for a longer time is also presented with an asterisk at the left. D. Raw data shown in C were quantified and presented graphically. Values for each transcript  derived from WT (black bars) or taf1 (gray bars) strains were normalized against the level of SCR1 (pol III transcript) and are presented relative to the value for the TATA box in the WT strain cultured in glucose-containing media at 25°C .(EPS)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Brd4 deletion prevents the enrichment of Mediator and RNA polymerase II transcription machinery, but not that of LDTFs, MLL3/MLL4-mediated H3K4me1, and CBP/p300-mediated H3K27ac, on enhancers. Consequently, Brd4 deletion prevents enhancer RNA production, cell identity gene induction and cell differentiation. Interestingly, Brd4 is dispensable for maintaining cell identity genes in differentiated cells. These findings identify Brd4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation.The epigenomic reader Brd4 is an important drug target for cancers. However, its role in cell differentiation and animal development remains largely unclear. Using two conditional knockout mouse strains and derived cells, we demonstrate that Brd4 controls cell identity gene induction and is essential for adipogenesis and myogenesis. Brd4 co-localizes with lineage-determining transcription factors (LDTFs) on active enhancers during differentiation. LDTFs coordinate with H3K4 mono-methyltransferases MLL3/MLL4 (KMT2C/KMT2D) and H3K27 acetyltransferases CBP/p300 to recruit Brd4 to enhancers activated during differentiation.  Despite being an important cancer drug target, the role of epigenetic reader Brd4 in cell differentiation and development remains unclear. Here, the authors provide evidence that Brd4 plays an important role in adipogenesis and myogenesis by binding to active enhancers to regulate gene expression. Enhancers possess recognition motifs for sequence-specific lineage-determining transcription factors (LDTFs), which bind to and activate enhancers2. LDTFs recruit epigenomic regulators that remodel the chromatin landscape by adding epigenetic modifications  to the histone tails of the associated nucleosomes, after which RNA polymerase II (Pol II) is recruited and transcription of enhancer RNA (eRNA) and nearby genes occurs2. Enhancers are enriched with histone H3K4 mono-methylation (H3K4me1), which is mainly deposited by H3K4 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D)3. H3K4me1 precedes the addition of the active enhancer mark H3K27ac by histone acetyltransferases CBP/p3005. CBP/p300 binding identifies active enhancers that control cell-type-specific gene expression6. The acetyl marks on histones act as docking sites for epigenomic readers that have conserved bromodomains7.Differentiation is controlled by cell-type-specific gene expression, which is under the control of transcriptional enhancers9. Brd4 is enriched on active enhancers and promoters11. Cooperation between LDTFs and CBP/p300 facilitates Brd4 recruitment to its target promoters and enhancers11. Small-molecule competitive BET inhibitors such as JQ1 bind to the acetyl-lysine binding pocket of the BET bromodomains and displaces BET proteins from chromatin12. JQ1-mediated inhibition of Brd4 binding leads to the displacement of the Mediator complex and Pol II from enhancers, which in turn reduces eRNA production and associated gene expression14.The epigenomic reader Brd4 is a member of the bromodomain and extra-terminal domain (BET) family of nuclear proteins that also include Brd2 and Brd316. PPARγ and C/EBPα work in cooperation to activate many adipocyte-specific genes. The synchronized nature of adipogenesis in cell culture, wherein the majority of the cells in the confluent population differentiate from preadipocytes to mature adipocytes within 6–8 days, allows for a system conducive to studying gene expression during the process of differentiation17. Myogenesis is another model of synchronized cell differentiation. Myogenic differentiation protein (MyoD) and myogenic factor 5 (Myf5) are required for commitment to the muscle differentiation program, while myogenin plays a necessary role in establishing the terminal muscle phenotype18.Adipogenesis and myogenesis are two model systems that are well suited for studying cell differentiation. In adipogenesis, the induction of early adipogenic transcription factors (TFs), including CCAAT/enhancer binding protein-β (C/EBPβ), in turn induces the expression of two master adipogenic TFs, peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPαBrd4 KO mice, we provide in vivo evidence that Brd4 is essential for adipogenesis and myogenesis. During adipogenesis, Brd4 preferentially binds to active enhancers and controls the induction of cell-type-specific genes. Furthermore, we determine the sequential actions of LDTFs, enhancer epigenomic writers MLL3/MLL4 and CBP/p300, Brd4, transcription coactivator complex Mediator and general transcription factor TFIID, Pol II, and transcription elongation factor p-TEFb in enhancer activation and cell identity gene induction during differentiation. We further show that unlike its critical role in cell differentiation, Brd4 is largely dispensable for the maintenance of cell identity gene expression in differentiated cells.Brd4 is a prominent drug target for cancers but its role in normal cell differentiation and tissue development is largely unexplored. In this study, we use adipogenesis and myogenesis as model systems to explore the role of Brd4 in differentiation and development. Using tissue-specific Brd4 conditional knockout (KO) mouse strains, Brd4f/f and Brd4f/f #2, were used in this study. In the Brd4f/f strain, the exon 3 was flanked by two loxP sites  and muscle lineages20. The resulting Brd4f/f;Myf5-Cre mice survived until birth. E18.5 Brd4f/f;Myf5-Cre embryos were obtained at the expected Mendelian ratio but were unable to breathe and died immediately  or Cre , down-regulated , and unchanged  genes from D0 to D2 of differentiation  and during (D2) adipogenesis of ion Fig. . Among tion Fig. . During ion Fig. . These rBrd4 KO cells. To identify high-confidence Brd4 binding sites, we first removed false-positive signals obtained from Brd4 KO cells and then selected overlapping peaks from two biological replicates. In total, we identified 37,111, 13,676, and 27,351 Brd4 binding regions at D0, D2, and D7, respectively , during (D2), and after (D7) adipogenesis. To exclude false-positive genomic Brd4 binding sites due to off-target effect of the antibody, ChIP-Seq was also done in ely Fig. . Brd4 prely Fig. , but theely Fig. . Around ely Fig. .Fig. 3CePparg and Myod1 loci, which encode the master adipogenic TF PPARγ and myogenic TF MyoD, respectively  analysis revealed that at D0 before differentiation, Brd4 binds to genes associated with general biological functions. However, Brd4 moves to adipogenesis-related genes at D2 and D7 of differentiation Fig. . In C2C1ely Fig. . Togethe21. During (D2) and after (D7) adipogenesis, Brd4 binding regions were enriched with motifs of adipogenic TFs such as C/EBPα, C/EBPβ, and PPARγ as well as ATF4, which was recently identified as a novel TF that promotes adipogenesis22. ChIP-Seq analyses of C/EBPα, C/EBPβ, and PPARγ at D2 of adipogenesis confirmed the genomic co-occupancy of Brd4 with these adipogenic TFs , active promoter, and silent promoter  of Brd4 binding sites within active enhancers were occupied by enhancer epigenomic writers MLL4 and/or p300, indicating a substantial overlap Fig. . Among t25 and CBP/p300 facilitate Brd4 binding to active enhancers11, we hypothesized that MLL3/MLL4 are required for Brd4 recruitment to enhancers during differentiation. To test this hypothesis, we performed Brd4 ChIP-Seq in Mll3/Mll4 double KO cells at D2 of adipogenesis. Although deletion of Mll3/Mll4 did not affect Brd4 protein levels  of C/EBPβ+ MLL4+ active enhancers showed decreased C/EBPβ binding in Mll3/Mll4 KO cells, which consequently led to decreases in p300 and Brd4 binding  adipogenesis 27 on these enhancers during adipogenesis. We did not observe changes in C/EBPβ and MLL4 binding as well as H3K4me1 and H3K27ac levels on Brd4+ adipogenic enhancers in Brd4 KO cells. However, deletion of Brd4 markedly reduced MED1, TBP, Pol II, and CDK9 binding on Brd4+ adipogenic enhancers but not on enhancers near constitutively active genes Arid1a and Jak1 (n1 and n2)  on cell identity genes sis Fig. . We examn2) Fig. . Accordiers Fig. . These ders Fig. .Fig. 7BrPparg and Cebpa. We asked whether Brd4 is required to maintain adipocyte gene expression. To this end, we crossed Brd4f/f mice with Adipoq-Cre mice to generate adipocyte-specific Brd4 KO mice28. Deletion of Brd4 was successful in adipose tissues 13. Brd4 may regulate Mediator binding to enhancers through its physical interaction with Mediator complex27. On the other hand, since PPARγ physically associates with Mediator complex as well36, it is also possible that decreased Mediator binding in Brd4 KO cells is due to the decreased expression of PPARγ, a direct target of Brd4. Physical and functional association between Brd4 and CDK9, a component of p-TEFb, has been well documented37. Our data suggest that Brd4 is a molecular bridge between cell-type-specific enhancers and general transcription machinery. Taken together, we propose a model that adipocyte gene expression is induced by sequential actions of LDTFs C/EBPα, C/EBPβ, and PPARγ, enhancer epigenomic writers MLL3/MLL4 and CBP/p300, enhancer epigenomic reader Brd4, Mediator, and Pol II transcription machinery on enhancers  were purchased from Sigma. Anti-RbBP5 (A300–109A), anti-Brd4 (A301–985A100), and anti-MED1 (A300–793A) were from Bethyl Laboratories. Anti-C/EBPα (sc-61X), anti-C/EBPβ (sc-150X), anti-PPARγ (sc-7196X), and anti-p300 (sc-585X) were from Santa Cruz Biotechnology. Anti-H3K4me1 (ab8895), anti-H3K4me2 (ab7766), and anti-H3K27ac (ab4729) were from Abcam. Anti-Pol II (17-672) was from Millipore. For western blot analysis, all antibodies were diluted to 1 μg ml−1. Uncropped blots are available in the Supplementary Figure The lentiviral shRNA plasmids pLKO.1 targeting mouse Brd4 conditional KO mouse lines were used in this study. In the first Brd4 conditional KO mouse line (Brd4f/f), the first coding exon of Brd4 gene, exon 3, is flanked by loxP sites. Genotyping the Brd4 alleles, PCR was done using the following primers: 5′-GCCTAGATCAGTGCCTCCATTG-3′ and 5′-ACTGGAACTACATGGCAGCCTG-3′. PCR amplified 244 bp from the wild-type and 344 bp from the floxed allele. Brd4f/f mice were crossed with Myf5-Cre  or Adipoq-Cre (C57BL/6 background)28 to generate Brd4f/f;Myf5-Cre or Brd4f/f;Adipoq-Cre mice. For characterization of Brd4f/f;Adipoq-Cre mice, we used six 12-week-old male mice per genotype. Animals were not randomized and the researchers were not blinded during the experiment and when assessing the outcome. No animals were excluded from the analysis.Two Brd4 conditional KO mouse line (Brd4f/f #2), the heterozygous conditional Brd4 gene trap mice (Brd4tm1a(EUCOMM)Wtsi) were obtained from the KOMP2 program at Baylor College of Medicine and crossed with FLP1 mice (Jackson no. 003946) to delete the neomycin cassette. In the resulting Brd4f/f #2 mice, the exon 5 of Brd4 gene is flanked by loxP sites. Genotyping of the Brd4 alleles was done using the following primers: 5'-GGACATGGTGACAGAGTGG-3' and 5'-TCAAATGAATTCACTAGAACTAC-3'. PCR amplified 168 bp from the wild-type and 284 bp from the floxed allele. Brd4f/f #2 mice were crossed with Cre-ER (Jackson no. 008463) to generate Brd4f/f #2;Cre-ER mice.To establish the second All mouse experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of NIDDK, NIH.19.E18.5 embryos were isolated by Cesarean section, fixed in 4% paraformaldehyde, dehydrated in a methanol series, and embedded in paraffin for sectioning. Paraffin sections were stained with routine H&E or subjected to immunohistochemistry using anti-Myosin  and anti-UCP1  antibodiesBrd4f/f, Brd4f/f #2;Cre-ER and immortalized by infecting retroviruses expressing SV40T38. Adipogenesis of immortalized brown preadipocytes was induced with DMEM supplemented with 10% fetal bovine serum (FBS), 0.02 μM insulin, 1 nM T3, 0.5 mM IBMX, 2 μg ml−1 DEX, and 0.125 mM indomethacin for 2 days39. After this period, the culture medium was supplemented with FBS, insulin, and T3 only. 3T3-L1 cells were from Daniel Lane.Primary brown preadipocytes were isolated from interscapular BAT of newborn C2C12 myoblasts were purchased from ATCC and cultured in growth medium of DMEM containing 15% FBS. Myogenesis was induced by replacing growth medium to DMEM containing 2% horse serum when cells were ~70% confluent. Before changing the medium, cells were washed with plain DMEM twice.Brd4 exon 3 was done using SYBR green primers: forward 5′-CCCAGAGACCTCCAACCCTAA-3′ and reverse 5′-AACTGGTGTTTCCATAGTGTCTTGAG-3′. qRT-PCR of Brd4 exon 5 was done using the primers: forward 5′-TGACATCGTCTTAATGGCAGAAG-3′ and reverse 5′-CCTTTTGCCTGGACTATCATGAT-3′.Total RNA was extracted using TRIzol (Invitrogen) and reverse transcribed using ProtoScript II first-strand cDNA synthesis kit (NEB), following manufacturer’s instructions. qRT-PCR of 7 cells were washed with 20 ml cold PBS in culture dish twice and scraped off in 10 ml Farnham lysis buffer , then pelleted by centrifugation at 3000×g for 5 min at 4 °C. Cell pellet was resuspended in 10 ml lysis buffer and pelleted again to remove cytosolic proteins. Resulting nuclear pellet was sonicated in 2 ml TE buffer  to achieve DNA fragments of 200–500 bp. Detergents were added to digested chromatin fractions to make 1× RIPA buffer . After centrifugation, supernatant was collected in a new tube. For each ChIP, 8–10 μg antibodies were pre-incubated with 50 μl Dynabeads Protein A (Life Technologies) in 1 ml PBS overnight at 4 °C under gentle rotation. Next day, 1 ml chromatin from 1 × 107 cells was mixed with antibody-beads complex and incubated overnight at 4 °C with gentle rotation. Chromatin immunoprecipitates were washed twice with 1 ml RIPA buffer, twice with 1 ml RIPA containing 300 mM NaCl, twice with 1 ml LiCl buffer , and twice with 1 ml TE buffer. Samples were reverse-crosslinked in 100 μl elution buffer  overnight at 65 °C. DNA was purified by QIAquick PCR Purification Kit (QIAGEN) and quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific)24.For ChIP-Seq analysis, formaldehyde was added directly to cell culture medium to a final concentration of 2%. After 10 min of incubation at room temperature, glycine was added to a concentration of 125 mM to quench crosslinking reaction. Approximately 2 × 10For RNA-Seq, mRNAs were purified using Dynabeads mRNA Purification Kit (Invitrogen), and then they were used to synthesize double-stranded cDNAs using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). Sequencing libraries were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). All ChIP-Seq and RNA-Seq samples were sequenced on the Illumina HiSeq 2500.40. To eliminate non-specific binding of Brd4 antibody, we compared Brd4 ChIP-Seq data from Brd4 KO cells with that from two biological replicates of control (Pparγf/f) cells24, and kept only the identified Brd4 binding regions with enrichment level significantly higher in control cells than in the Brd4 KO cells, with an estimated false discovery rate (FDR) of <10−3. Then, we chose only the overlapping Brd4 binding regions from two biological replicates. For Brd4 ChIP-Seq data in Mll3−/−Mll4f/f cells, an FDR of <10−10 was used to find the high-confidence ChIP-enriched regions. Other published ChIP-Seq data sets were downloaded 41.To identify Brd4 binding regions, we used ‘SICER’ method with a window size of 50 bp and a gap size of 50 bphttp://cistrome.org/ap/root)42. The algorithm used in this motif tool is previously described in detail43. We selected the top 3000 Brd4 binding regions to screen enriched TF motifs at each time point.For motif analysis of Brd4 binding regions Fig. , we usedAll data sets described in the paper have been deposited in NCBI Gene Expression Omnibus under accession number GSE99101.Supplementary InformationPeer Review File


Here we study the evolution and functional diversification of chaperonin paralogs in the heterocystous, multi-seriate filament forming cyanobacterium Chlorogloeopsis fritschii PCC 6912. The genome of C. fritschii encodes two groES/groEL operons  and a monocistronic groEL gene (groEL2). A phylogenetic reconstruction reveals that the groEL2 duplication is as ancient as cyanobacteria, whereas the groESL1.2 duplication occurred at the ancestor of heterocystous cyanobacteria. A comparison of the groEL paralogs transcription levels under different growth conditions shows that they have adapted distinct transcriptional regulation. Our results reveal that groEL1 and groEL1.2 are upregulated during diazotrophic conditions and the localization of their promoter activity points towards a role in heterocyst differentiation. Furthermore, protein–protein interaction assays suggest that paralogs encoded in the two operons assemble into hybrid complexes. The monocistronic encoded GroEL2 is not forming oligomers nor does it interact with the co-chaperonins. Interaction between GroES1.2 and GroEL1.2 could not be documented, suggesting that the groESL1.2 operon does not encode a functional chaperonin complex. Functional complementation experiments in Escherichia coli show that only GroES1/GroEL1 and GroES1/GroEL1.2 can substitute the native operon. In summary, the evolutionary consequences of chaperonin duplication in cyanobacteria include the retention of groESL1 as a housekeeping gene, subfunctionalization of groESL1.2 and neofunctionalization of the monocistronic groEL2 paralog.Chaperonins promote protein folding and are known to play a role in the maintenance of cellular stability under stress conditions. The group I bacterial chaperonin complex comprises GroEL, that forms a barrel-like oligomer, and GroES that forms the lid. In most eubacteria the GroES/GroEL chaperonin is encoded by a single-copy bicistronic operon, whereas in cyanobacteria up to three  The recent genome sequencing of stigonematalean cyanobacteria revealed that species in that order encode a second copy of the groESL operon (Chaperonins are encoded in most eubacteria (∼70%) by a single bicistronic bacteria . Cyanobabacteria  and theybacteria . Under nbacteria . All cyaoEL gene . An exceL operon . These sL operon .groEL paralogs has been studied so far mostly in the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the filamentous, heterocystous cyanobacterium Anabaena sp. L31. In both species, the two groEL paralogs have been shown to be upregulated during heat stress  encode a single groESL operon and an additional monocistronic groEL . Cyanobacteria species that encode an additional groESL operon are mostly of the filamentous types included in Subsections III, IV, and V. Exceptions among the unicellular cyanobacteria (Subsection I) are Synechococcus sp. PCC 7335 and Synechococcus sp. PCC 7336 that encode two groESL operons. Within Subsection III, all three Pseudoanabaena strains encode two groESL operons, while Leptolyngbya sp. PCC 7375 encodes two groESL operons and two additional monocistronic groEL genes. Many heterocystous cyanobacteria that form linear or branching filaments (Subsections IV and V) encode an additional groESL operon .To study the evolution of groEL DNA sequences reveals two main clades that largely correspond to the monocistronic and operon encoded genes  form a sub-clade that is nested within the operon encoded groEL clade. In the following, we term the genes encoded in the ubiquitous groESL operon by groES1 and groEL1, while the additional operon components are termed groES1.2 and groEL1.2. The monocistronic groEL is termed groEL2. To further study the evolution of groES/groEL paralogs we reconstructed phylogenies that include all strains encoding more than two groEL paralogs and classified the genes into the three named paralogs . The tree topology suggests that the split between the operon encoded and monocistronic groEL2 is ancient  test; groESL1 and the monocistronic groEL2 is ancient and most probably occurred at the base of the cyanobacterial tree.A maximum-likelihood phylogenetic tree reconstructed from all cyanobacterial homologous ed genes A. Furth ancient B. To te ancient B and cogroEL duplicates by adding a constrained split between groEL1 and the remaining groEL duplicates . We note, however, that this topology includes also exceptional groEL paralogs that could not be classified into the three orthologous groups such as additional monocistronic groEL paralogs in Leptolyngbya sp. PCC 7375 and N. punctiforme PCC 73102. Testing for a constrained monophyly of groES1 and groES2 revealed that the constrained topology is not significantly different from the ML topology . To further characterize the evolutionary dynamics of the groES/groEL paralogs in stigonematalean cyanobacteria we constructed a phylogenetic tree that includes all paralogs encoded in the Nostocales (Subsection IV) and Stigonematales (Subsection V) genomes in our sample . The Nostocales–Stigonematales clade includes all heterocyst forming cyanobacteria and is considered monophyletic . Testing for a constrained topology where groEL2 is monophyletic yielded a topology that is not significantly different than the ML phylogeny . This result confirms that the duplication of groEL1 and groEL2 is ancient. A further test of the split between groEL1 and additional groEL duplicates reveals a topology that is not significantly different than the ML phylogeny . This indicates that the duplication of groESL1 operon occurred before the divergence of Stigonematales. Thus, the heterogeneous distribution of groESL1.2 across the Nostocales and Stigonematales is most probably due to a single duplication event and differential loss of groESL1.2 in the species that lack that operon rather than lateral gene transfer.We further tested for monophyly of the operon-encoded plicates C. The lphyletic . The grogroESL operon and the monocistronic groEL in cyanobacteria is known to depend on the presence of CIRCE and K-box elements at the 5′-UTR . The CIRCE element upstream of groESL1.2 is slightly diverged and is absent in Fischerella sp. PCC 73103 and Fischerella thermalis PCC 7521. The groEL2 is preceded in all genomes by a conserved CIRCE element but lacking a K-box element . To study differences in the transcriptional regulation of the groEL paralogs, we quantified their transcription level in C. fritschii PCC 6912 using qRT-PCR. In addition, we visualized the promoter activity of the three paralogs in real time using a green fluorescence protein (GFP) marker.Transcriptional regulation of the e 5′-UTR . ComparaP < 0.05, using paired t-test with Bonferroni correction). The groEL1 transcript is the most abundant and groEL2 transcript abundance exceeds that of groEL1.2 . We then compared the paralogs transcription level under high temperature by incubating C. fritschii at 50 °C. After 5 min of heat exposure (t5m) the transcription level of groEL1 increased 5-fold on average, while the transcription level of groEL1.2 and groEL2 increased more than 15-fold, on average, in comparison to their transcription level under standard growth conditions (t15m), the transcription level of groEL1 increased 8-fold and that of groEL1.2 increased 189-fold in comparison to the onset of the experiment (t0). The transcription level of groEL2 remained similar to t5m. The transcription level of all three paralogs increased further after 30 min of incubation at 50 °C, where the highest increase of about 2,000-fold was observed for groEL1.2. The transcription level of groEL2 increased 37-fold, whereas groEL1 transcription increased 78-fold in comparison to t0 . The three transcriptional fusion constructs were introduced independently into C. fritschii. The GFP fluorescence was observed in all transformed strains grown under standard conditions (t0), indicating that all promoters are active and GFP-fusions are functional. During high temperature stress conditions groESL1 promoter activity increased after 10 min (t10m), but decreased after 20 min (t20m) of incubation . Thus, promoter activity of the three paralogs as documented by GFP-fusion constructs supports the transcription level changes recorded using qRT-PCR.To further validate changes in the transcriptional regulation of the three paralogs under varying growth conditions, we created transcriptional fusions of each cubation A. ElevaC. fritschii PCC 6912 under 3-fold higher light intensity compared with standard conditions . The promoter activity of all paralogs, as documented by fluorescence intensity , validates the observed changes in transcription level under high light conditions.To compare the transcriptional regulation of the paralogs under high light conditions, we incubated nditions B. The tgroESL copies. Consequently, we compared the paralogs transcriptional regulation under diazotrophic conditions that induce heterocyst differentiation. Here we used the transcription level of glutamine synthetase (glnA) as a positive indicator for nitrogen deprivation . This was accompanied by an average increase of 26-fold in groEL1 transcript abundance after 3 h followed by a decrease after 8 h . The transcription level changes of groEL2 were similar to that of glnA, but with a maximum of 7-fold change after 3 h. The transcription level of groEL1.2 increased 18-fold after 3 h and continued to increase with a maximum of 47-fold change after 24 h in comparison to t0. The transcription dynamics of groEL1 suggest that it plays a role during the early stages of diazotrophy, while groEL1.2 seems to play a role in later stages of the adaptation to diazotrophic conditions. Consequently, we tested the promoter activity of groESL1 and groESL1.2 under diazotrophic conditions by time-lapse fluorescence microscopy  showed an increased activity after 96 h followed by further elevation in all cells during the time of incubation in diazotrophic conditions . The results of the interaction assays are summarized in a protein–protein interaction network , whereas any GroES1.2 protein interaction was only detected when it was C-terminally tagged. We could not detect an interaction between the different GroEL paralogs. Our results so far indicate that GroEL1 and GroEL1.2 form homogeneous barrel-like structures, while GroEL2 functions probably as a monomer. In contrast, the two small subunits, GroES1 and GroES1.2, were found to interact; hence, it is possible that they can form a heterogeneous lid structure. Moreover, we found GroEL1 and GroES1 to interact; this indicates that the  complex B. The aE. coli MGM100, in which the expression of the native groES/groEL operon is controlled by an arabinose-inducible promoter , and introduced them into E. coli MGM100. The ability of the introduced groES/groEL combination to complement the native E. coli groESL operon was scored positive if colonies were able to form upon induction with anhydrotetracycline. Using this approach we tested the complementation potential of the native C. fritschii operons as well as all GroES/GroEL combinations. In addition, because GroEL was shown to refold thermally denatured substrates independently of GroES in Anabaena sp. L31  possessed a duplicated groESL operon. Furthermore, the comparison of transcriptional regulation, chaperonin assembly and function among groESL1 and groESL1.2 paralogs in the stigonematalean cyanobacterium C. fritschii PCC 6912 demonstrates that they are functionally diverged. In those species where groEL diversification has been studied, frequently one groEL copy has maintained the housekeeping function while other paralogs have undergone sub- or neo-functionalization. For example, the genome of Sinorhizobium meliloti, an alphaproteobacterium that is often found in symbiosis with legumes, encodes a total of five groEL paralogs. Only one paralog (groEL1) serves the housekeeping function and is also essential for the symbiotic interaction with plants and the establishment of nodules in the root (groEL2) can substitute the housekeeping gene but its lower expression level is insufficient for the establishment of symbiosis in S. meliloti . Yet, the amino acid sequence similarity between the two chaperonins is high , hence, the mode of action is probably not different. This is supported by the ability of GroEL1.2 to complement a groESL deficient E. coli strain. Nevertheless, the lack of evidence for interaction between GroES1.2 and GroEL1.2 suggests that the groESL1.2 operon is dysfunctional, in a sense that its members do not interact, although transcribed together . We note that groESL1.2 evolves under purifying selection  .The search for GroES/GroEL homologs was performed with BLAST+  using thly 2014)  and Joinly 2014) . An e-va. 7.027b . Phyloge. 7.027b  for amin. 7.027b  for nucl. 7.027b . Testing. 7.027b . The cal. 7.027b . Codon a. 7.027b . PhylogeChlorogloeopsis fritschii PCC 6912 was obtained from the Pasteur Culture Collection (PCC) of cyanobacteria. Stock cultures were grown photoautotrophically in liquid BG11 medium supplemented with or without (BG11o) combined nitrogen at 37 °C and a light intensity of 24 µE m−2 s−1  15 and 50 µg ml−1, ampicillin (Amp) 100 µg ml−1, or spectinomycin (Sp) 50 µg ml−1 were used for selection.−2 s−1 . Transfol mating  with seli MGM100  for compC. fritschii PCC 6912 grown at 37 °C in BG11 medium with orbital shaking (100 rpm). A 30-ml of mid exponentially growing C. fritschii cells were harvested by centrifugation and DNA-Isolation was performed as described in  and high temperature (50 °C) growth conditions. Samples from diazotrophic conditions were harvested 3, 8, and 24 h after nitrogen deprivation. Upon harvesting samples were frozen in liquid nitrogen. Total RNA was isolated using Concert Plant RNA Reagent  according to the manufacturer’s instruction and treated with RNase-free DNaseI™ . One microgram of total RNA was used for single-strand cDNA synthesis with iScript™ . Quantitative RT-PCR was performed with primers listed in supplementary table S3, Supplementary Material online in a StepOne Real-Time PCR System (Biorad) with Power SYBR Green PCR master mix  and 50 ng cDNA as template. All samples were run in biological and technical triplicates. The transcript levels of groEL paralogous genes were normalized using housekeeping gene rnpB as a reference. Relative transcript levels were calculated using the ΔΔCt method  and high temperature conditions (50 °C), respectively. Samples from diazotrophic conditions were documented before and every 12 h after nitrogen deprivation over a period of 5 days. To fuse the promoter of groEL and groESL operons (groEP) to gfp:mut3.1, PstI and BamHI restriction sites were introduced to 5′ and 3′ ends of gfp-mut3.1 . To ensure aeration, 0.5 mm slices of solid media were removed from each side of the chamber. Twenty microliters of cells were then placed on top of the agarose slice and the chamber was sealed with a glass coverslip and visualized with an epifluorescence microscope .For time-lapse imaging, gene frame cover slips (Thermo Scientific) were attached to glass slides and the resulting chamber filled with 150 µl of BG11o medium containing agarose (0.5%) and supplemented with neomycin , IPTG (0.5 mM), Amp and Km. Blue colonies indicated positive interaction and the expression of the reporter gene was subsequently confirmed by β-galactosidase assay .To characterize the physical association between components of the E. coli MGM100 expressing groESL under the regulation of the arabinose (0.2%) inducible pBAD promoter (groESL1 and groESL1.2) and all chaperon subunit encoding genes  of C. fritschii PCC 6912 were amplified using the primers listed in supplementary table S3, Supplementary Material online. DNA fragments were cloned into pASK-IBA3plus  or pASK-IBA3sp, an aadA (SpR) cassette marker containing derivative of pASK-IBA3plus, using BsaI cutsites. Expression of the proteins was regulated by anhydrotetracycline treatment . For MGM100 co-expressing C. fritschii groEL and groES paralogs from different plasmids, both plasmids were co-transformed by electroporation. Transformed MGM100 was selected on LB plates containing Km, arabinose, Amp or Km, arabinose, Amp, Sp (for co-expression), and cells were grown in liquid media over night. Serially diluted cells were spotted onto selective solidified LB and grown at 30 °C for 18 hours. Growth of E. coli MGM100 on plates supplemented with glucose (and anhydrotetracycline) occurred only if the expressed groES/groEL from C. fritschii can complement the E. coli MGM100 strain.Complementation assays were performed with promoter . The twoSupplementary data are available at Genome Biology and Evolution online.Supplementary DataClick here for additional data file.