In multicellular organisms, epigenome dynamics are associated with transitions in the cell cycle, development, germline specification, gametogenesis and inheritance. Evolutionarily, regulatory space has increased in complex metazoans to accommodate these functions. In tunicates, the sister lineage to vertebrates, we examine epigenome adaptations to strong secondary genome compaction, sex chromosome evolution and cell cycle modes.Oikopleura dioica genome, we profiled 19 histone modifications, and RNA polymerase II, CTCF and p300 occupancies, to define chromatin states within two homogeneous tissues with distinct cell cycle modes: ovarian endocycling nurse nuclei and mitotically proliferating germ nuclei in testes. Nurse nuclei had active chromatin states similar to other metazoan epigenomes, with large domains of operon-associated transcription, a general lack of heterochromatin, and a possible role of Polycomb PRC2 in dosage compensation. Testis chromatin states reflected transcriptional activity linked to spermatogenesis and epigenetic marks that have been associated with establishment of transgenerational inheritance in other organisms. We also uncovered an unusual chromatin state specific to the Y-chromosome, which combined active and heterochromatic histone modifications on specific transposable elements classes, perhaps involved in regulating their activity.Across the 70 MB Compacted regulatory space in this tunicate genome is accompanied by reduced heterochromatin and chromatin state domain widths. Enhancers, promoters and protein-coding genes have conserved epigenomic features, with adaptations to the organization of a proportion of genes in operon units. We further identified features specific to sex chromosomes, cell cycle modes, germline identity and dosage compensation, and unusual combinations of histone PTMs with opposing consensus functions.The online version of this article (doi:10.1186/s13072-016-0110-4) contains supplementary material, which is available to authorized users. Oikopleura dioica, a member of the lineage that comprises the closest living relatives to vertebrates [Histone proteins, which package genomic DNA, provide multiple sites for covalent posttranslational modifications (PTMs) by evolutionarily conserved histone modifiers that form multimeric complexes and cooperate with nonhistone proteins , 2. Histtebrates .Oikopleura dioica is a semelparous pelagic tunicate  with a simple chordate body plan and short, 6-day life cycle [O. dioica has the smallest metazoan genome sequenced to date, organized in a haploid complement of 3 chromosomes. At 70 Mb, it is ~44-fold smaller than the human genome despite maintaining >18,000 protein-coding genes [trans-splicing of a short spliced-leader (SL) RNA occurs at the 5′ ends of 39% of protein-coding genes [O. dioica and the density of TEs is low, with most concentrated on the gene-poor Y-chromosome [O. dioica genome, but it has variety of LTR retrotransposons from the Ty3/gypsy group, divergent from those found in other organisms, as well as Dictyostelium intermediate repeat sequence 1 (DIRS1) and Penelope-like elements [env gene and are expressed in a variety of Oikopleura tissues including germline-associated cells [O. dioica has been assembled and includes ovary and testes samples [fe cycle . Severalfe cycle –24. O. dng genes  comparedng genes , 27. Intng genes , and trang genes . Transporomosome . Major celements . These aed cells . A compr samples . The ful samples .O. dioica genome that have potential regulatory function (introns and intergenic regions) have been reduced, often to the order of one nucleosome in size. How this compaction affects long-range enhancer-mediated gene regulation [O. dioica and nematodes, possibly correlating with limited cellular plasticity and lack of regeneration [O. dioica is an interesting model in which to investigate non-canonical Polycomb complexes and their functions in a rapidly evolving lineage.Regions of the gulation  and the gulation  is unknogulation . Polycomgulation . PRC1 isgulation , but itsneration . A numbeneration , 41. O. Oikopleura dioica is unusual among tunicates in that it has genetically determined separate sexes with heterogametic (XY) males and homogametic (XX) females. Organisms with heterogametic sex chromosomes have evolved dosage compensation mechanisms to equalize the abundance of transcripts produced by the single X-chromosome in males and the double X-chromosome in females [O. dioica, nor through what mechanism it might be achieved, if it does occur. females , 43. In  females . A commoO. dioica testis, at early day 6 (a few hours before germ-cell release), when it is a syncytium of mitotically proliferating, transcriptionally active, spermatogonia nuclei , where endocycling nurse nuclei share a common cytoplasm with meiotic nuclei arrested in prophase I [Here, we sampled ophase I , 45 , and four were specific to the testis . Specific chromatin states were associated with active promoters , transcription elongation  and silent regions , which included states specific to the Y-chromosome (state 11) and silent transcription factors (TFs) (state 1). State 2, which had no enrichment of any profiled modifications, covered 54% of the ovary and 40% of the testis genomes   Fig. c. State trans-spliced operon transcripts (since a stretch of 5′ sequence is removed from mRNAs and replaced by the SL RNA). Enrichment of H3K4me3, the hallmark of active promoters [p value = 2.044 × 10−10) but not at regions surrounding the 5′ ends of expressed downstream operon genes. This provides evidence that these are indeed co-transcribed from a single upstream promoter. Active promoter state 8  was enriched in active genes related to oogenesis and embryonic development  in these promoters may reflect the uncertainty of TSS annotations for a subset of romoters , was oveO. dioica, 15% of the ovary genome and 8% of the testis genome are transcribed [O. dioica. We identified 1106 genes in the ovary and 1389 genes in the testis, which have proximal promoter regions that are repressed but associated with RNAPII . Most of these genes encode proteins for RNA-processing functions, cell cycle and to a lesser extent, development, in both samples, and for nutrient response functions, particularly in the ovary  [Pausing of RNAPII is a pervasive feature of promoters in metazoans that facilitates integration of multiple cellular signals, poising genes for rapid expression and/or synchronous activation . In O. dnscribed . Serine- blooms) , 56, 57.O. dioica genome, despite its high compaction, reshuffling and loss of some regions of evolutionarily conserved synteny. More enhancers would likely be detected in early embryonic and larval samples when developmental regulatory networks are activated.We identified 8070 regions of H3K4me1 enrichment that did not overlap annotated gene start sites in the ovary and 12,272 in the testis. Of these, 1087 and 6829 overlapped regions of H3K27ac enrichment in the ovary and testis, respectively, and constituted candidate enhancers. We intersected these with regions associated with the co-activator p300, a tissue-specific enhancer-binding histone acetyltransferase , 59, andO. dioica testes deploy distinct epigenomic states (and possibly histone variants) in the control of important developmental regulatory loci.Repressed TF and zinc finger (ZF) genes in the ovary were associated with chromatin state 1  , and RA signaling is absent . dioica , 62. Sev. dioica  were alsDrosophila [Prior to H3K27me3 deposition, PRC2 can be recruited in a number of ways that depend on composition of the complex, its interacting partners, target genes and the cell cycle and developmental stage , 63, 64.osophila  and mammosophila , PRC exhO. dioica genome at a lower enrichment level than other marks  males and homogametic (XX) females. Autosomal and X-chromosome genes have similar expression levels in both ovary and testis . While we found regions of mutual exclusion of the repressive marks H3K27me3 and H3K9me3 in O. dioica Y-chromosome was enriched for repressive states characterized by H3K9me3, H3K9me2 and H4K20me3  . The TE orders DIRS (102 regions out of 519) and LTR (183 out of 1729) both had an unusually high frequency of this combination of heterochromatin and active marks, whereas Mariner, MITEs and repeats annotated as possible Env had lower than expected frequencies. We tested for the combination of H3K9me3 with each active mark separately and obtained similar results when considering H3K9me3 with H3K4me3   . TissueH3K79me2  and may O. dioica sperm chromatin does not undergo histone-to-protamine replacement that is typically preceded by massive H4 hyperacetylation in both human and Drosophila [O. dioica testis-specific variant H3t [O. dioica sperm, the association of generally activating H4ac with silent developmental genes in the testes could be viewed as a potentiating transgenerational mark. The lack of a transition to protamines, and extremely rapid embryonic and larval development, may have increased weighting on the intergenerational transmission of potentiated epigenetic states. Resolving individual lysine acetylations on H4 would be a step toward better understanding this unusual marking.Histone acetylations are generally associated with accessible chromatin and are related to transcriptional activation. We found, however, silent developmental genes marked by H4ac, particularly in the testis (state 15). In endocycling nurse nuclei, silent developmental genes were enriched in H3K79me3 (state 1) ntiation . Moreoventiation . The lonin HCNEs . We obseO. dioica male germ line that the somatic H3K36 methyltransferase SETD2 is repressed, leaving NSD-(mes-4)-like and PRDM9-like to deposit H3K36-methylation. Complementary patterns of H3K36me and H3K27me3 in testis indicate regulation of transcript levels in the germline analogous to mes-4 (NSD-like) catalyzed H3K36me in C. elegans, which antagonizes PRC on germline genes such that PRC is excluded from autosomes but remains on (and represses) the X-chromosome. The mutual antagonism between PRC2 and mes-4 (NSD-like) is thought to be important in the transgenerational inheritance of germline-specific transcriptional programs in C. elegans [O. dioica, and it would be of future interest to determine the terminal chromatin states of mature sperm in this regard. In C. elegans, this proposed mechanism operates in the absence of DNA methylation or histone replacement by protamines during spermatogenesis. Both of these latter mechanisms are features of mammalian spermatogenic programs, though protamine replacement is not complete and some histones remain. Interestingly, O. dioica is intermediate in that DNA methylation is present whereas histone replacement by protamines is absent [We found evidence of Polycomb interactions on autosomes and X-chromosomes in both sexes and propose roles of PRC2 in dosage compensation. We also observed in the  elegans , 85–87. s absent , offerinO. dioica offers an interesting model of more recent independent evolution of heteromorphic sex chromosomes. The O. dioica Y-chromosome contains a pseudoautosomal region, accumulated mobile elements, and a few male-specific genes, indicating that Y-chromosomal degeneration progressed rapidly [O. dioica Y-chromosome appears to have adapted to repress the activity of accumulated mobile DNA elements [Given the rarity of separate sexes among tunicates,  rapidly . Evoluti rapidly , but lit rapidly , 90. Pat rapidly . The comelements .C. elegans mobile elements [O. dioica has little heterochromatin, consistent with a scarcity of TE elements [O. dioica.Our finding of unusual chromatin states containing typically heterochromatic marks  combined with active transcription-related ones  might represent a transition state in the time course of heteromorphic chromosome evolution. To our knowledge, such patterns of H3K4me3 and H3K36me3 overlapping heterochromatin signatures have been observed only in mammalian imprinted loci  and C. eelements . In the elements . Howeverelements  and geneelements  that areelements . These eO. dioica genome, where intergenic regulatory regions are frequently on the order of one nucleosome. Indeed, we found that chromatin state domain widths in O. dioica were generally smaller than those in their chordate relatives, the vertebrates, and rarely exceeded 7 nucleosomes in size. Nonetheless, this is evidently compatible with fidelity of heritable transmission of epigenetic states in this species.The short life cycle and ability to rapidly regulate gamete output over 3 orders of magnitude , 56, 93 Our work provides the first comprehensive view of a protochordate epigenome. In the transcriptionally active endocycling, ovarian, nurse nuclei, histone modifications linked to RNAPII activity were conserved compared to those in other metazoans. We found evidence that RNAPII pausing is frequently involved in controlling gene transcription during oogenesis. We also identified candidate enhancers that have enhancer PTMs typical of other metazoan enhancer regions. Large heterochromatic domains of H3K9me3 were largely absent in the ovary.The epigenome of the male germ line reflected a shift from tissue-specific gene expression toward establishment of transgenerational inheritance and showed features related to the mitotic proliferation ongoing in this tissue. Our data support the involvement of a modified Polycomb complex in sex chromosome dosage compensation in homogametic females. We also identified a novel chromatin states that combines both euchromatic and heterochromatic histone PTMs that are associated with specific classes of TEs and the Y-chromosome.O. dioica have been epigenetically accompanied by reduced chromatin state domain widths and a general reduction of heterochromatin. It will be of interest to build on the foundation of the current study to determine how the three-dimensional genomic architecture of topologically associated domains (TADs) has been affected by these processes.Strong secondary genome compaction and disruption of evolutionarily conserved linear genome architecture in Oikopleura dioica were cultured at 15 °C [2, 4 mM MgCl2, 50 mM Tris/HCl pH 7.7, 0.5 mM PMSF and 1× protease inhibitor. Sampled testes were in the mitotically dividing pre-meiotic (spermatogonia) stage, using H3S28P immunostaining as a reference mark of meiotic versus mitotic events. For histone acetylation samples, 5 mM sodium butyrate was also added, and for histone phosphorylation samples, phosphatase inhibitor  was included. Samples were sedimented at 8000 rpm and snap-frozen in liquid nitrogen.at 15 °C . At lateO. dioica histone H3 and H4 sequences are identical [® Protein G (Invitrogen) and incubated with chromatin overnight (O/N). Immunoprecipitated DNA was purified by phenol–chloroform–isoamylalcohol extraction and EtOH precipitation followed by Min Elute PCR purification (Qiagen). Resulting DNA was amplified using Whole Genome Amplification kit 2 or 4 (Sigma) according to manufacturer’s instructions . With the exception of H3K4me2 and H3K9me2, histone PTM profiles were replicated at least twice in both ovary and testis samples. Amplified material was labeled and hybridized to O. dioica genomic microarrays (Nimblegen) [We performed native ChIP , with midentical , and we mblegen) .O. dioica p300 and CTCF. Antibodies were bound to 50 μl Dynabeads® Protein G and incubated with chromatin O/N. Immune complexes were washed twice with 800 μl RIPA, twice with 500 mM NaCl/RIPA and twice with LiCl buffer  and once with TE. Enriched DNA was recovered by 15-min elution at 65 °C in 1% SDS, 50 mM Tris–HCl pH 8, 1 mM EDTA and subjected to cross-link reversal for 5 h at 65 °C followed by RNase A and proteinase K treatments. DNA was then purified as for native ChIP. Immunoprecipitated DNA was amplified using the Whole Genome Amplification 4 kit (WGA4 Sigma), labeled and hybridized to Oikopleura genomic microarrays (Nimblegen).Animals were fixed in 1% formaldehyde in PBS for 12 min. Cross-linking was stopped by adding glycine to 0.13 M for 5 min followed by 3 washes with cold PBS. Fixed animals were kept in PBS containing 0.5 mM PMSF, 1× protease inhibitor and phosphatase inhibitor for dissection of gonads. Gonads were lysed in RIPA buffer , supplemented with fresh PMSF, protease and phosphatase inhibitor for 15 min. Chromatin was fragmented to a size range of 200–600 bp using a Bioruptor UCD-200 (Diagenode) for 8 × 30 s on/1 min off. ChIP was performed using 300 μl of centrifuged chromatin. We used custom antibodies  against Oikopleura dioica genomic DNA from day 6 immature ovaries and testes was isolated using proteinase K lysis followed by RNAse A treatment and phenol–chloroform extraction. Ethanol-precipitated DNA was diluted in 450 μl TE, randomly fragmented by sonication using the Bioruptor UCD-200 (Diagenode). Fragments ranged in size from 200 to 600 bp and were denatured at 95 °C for 5 min. Protein G Dynabeads (50 μl) were coupled to the monoclonal antibody against 5-methylcytidine (5mC) (Eurogentec #BI-MECY-1000). After three washes with IP buffer , DNA fragments together with 51 μl of 10 × IP buffer were added to the beads and incubated 16 h at 4 °C with constant rotation. Beads were collected with a magnetic rack and washed with 700 μl 1× IP buffer for 10 min with rotation. Washing was repeated twice, followed by elution in proteinase K buffer ) and incubation for 2 h at 55° C with shaking. Immunoprecipitated DNA was purified by phenol–chloroform–isoamylalcohol extraction and EtOH precipitation followed by Min Elute PCR purification (Qiagen). Resulting DNA was amplified using Whole Genome Amplification kit 4 (Sigma) according to manufacturer’s instructions .Ringo [2(Cy5/Cy3). We applied the NimbleGen normalization method, which adjusts for systematic dye and labeling biases by subtracting from individual log2 ratios, the Tukey’s biweight mean, computed across each sample’s log2 ratios. To reduce noise, we smoothened the normalized log2 ratios using a running median across a 150-bp window (approximate nucleosome size) with a minimum threshold of three nonzero probes. We used the resulting log2 ratios in all further analyses and browser visualizations.ChIP-chip data were preprocessed using the Bioconductor R package Ringo . We normO. dioica reference genome [trans-spliced with the spliced leader (SL) using an SL CAGE dataset [2 ratio at each probe position in a 1000-bp window centered on TSSs and TESs. Genes shorter than 1000-bp and with less than 1100-bp intergenic space were excluded, as were genes with no expression data. A total of 5444 gene models met our criteria for inclusion.We defined transcription start sites (TSSs) and transcription end sites (TESs) using gene models on the e genome . We catee genome . We furt dataset . We alsoRingo [y) as two underlying distributions: a null distribution from non-enriched regions and an alternative distribution from enriched regions. The null distribution is estimated for each sample individually using the empirical distribution of probe intensities: The mode (m0) is calculated and probe intensities y < m0 are reflected onto y > m0. A threshold y0 is then computed on the null distribution to define enrichment. We computed two enrichment thresholds using the 99 and 95% quantiles of the null distribution estimated for each sample individually. A region was called ChIP-enriched if a minimum of two probes exceeded the enrichment threshold. Regions were merged if the width between them was less than 50 bp. For samples with replicates, we defined final chers by intersecting the chers of each replicate: A region was only classed as ChIP-enriched if it was present in at least two replicates at the 95% threshold and present at the 99% threshold in at least one replicate. For H3K27me3, we also lowered the enrichment threshold to 75% in order to identify broader domains. We used the R package Ringo  to identO. dioica annotations [O. dioica homeodomain proteins were classified according to whether or not they had DNA-binding specificity PWM predictions using PreMoTF [http://stormo.wustl.edu/PreMoTF/). We calculated the specificity of each O. dioica gene across the developmental transcriptome using Shannon entropy, normalized to range between 0 and 1, following [The genome was segmented into 15 or 50 chromatin states using chromHMM . We creaotations  to defin PreMoTF  (http://ollowing . The speO. dioica GO term annotations [We used published otations . A subse


Caenorhabditis elegans, from embryogenesis to adulthood. Chromatin accessibility changes between developmental stages are highly reproducible, recapitulate histone modification changes, and reveal key regulatory aspects of the epigenomic landscape throughout organismal development. We find that over 5000 distal noncoding regions exhibit dynamic changes in chromatin accessibility between developmental stages and could thereby represent putative enhancers. When tested in vivo, several of these putative enhancers indeed drive novel cell-type- and temporal-specific patterns of expression. Finally, by integrating transcription factor binding motifs in a machine learning framework, we identify EOR-1 as a unique transcription factor that may regulate chromatin dynamics during development. Our study provides a unique resource for C. elegans, a system in which the prevalence and importance of enhancers remains poorly characterized, and demonstrates the power of using whole organism chromatin accessibility to identify novel regulatory regions in complex systems.Chromatin accessibility, a crucial component of genome regulation, has primarily been studied in homogeneous and simple systems, such as isolated cell populations or early-development models. Whether chromatin accessibility can be assessed in complex, dynamic systems in vivo with high sensitivity remains largely unexplored. In this study, we use ATAC-seq to identify chromatin accessibility changes in a whole animal, the model organism  Chromatin accessibility represents an essential level of genome regulation and plays a pivotal role in many biological and pathological processes, including development, tissue regeneration, aging, and cancer . HoweverThe primary limitation for studying chromatin accessibility in complex systems is that most assays lack the sensitivity and precision to detect regions active only in rare subpopulations of cells or require so many cells that precise temporal synchronization of samples is impractical. However, the Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) has been shown to assess native chromatin accessibility with high sensitivity and base pair resolution while requiring orders of magnitude less starting material than other assays . This apCaenorhabditis elegans is a particularly powerful model to study chromatin accessibility in a complex system and potentially identify novel regulatory regions. C. elegans has highly synchronous life stages, as well as consistent and well-characterized cellular composition throughout each stage of development . The low input requirements of ATAC-seq  and to generate three independent biological replicates that are tightly synchronized at three key life stages—early embryo, larval stage 3 (L3), and young adults—thereby limiting variation within stages . The insert size distribution of each C. elegans ATAC-seq library displays a stereotypical 147-bp periodicity that is consistent with the expected nucleosome occupancy of chromatin , indicative of ATAC-seq library quality  and emphasize single base pair resolution . The high correlation of ATAC-seq signal between each of the three biological replicates (Spearman's ρ > 0.837) demonstrates the high reproducibility of this approach (To sensitively measure high-resolution chromatin accessibility at different life stages in ChIP-seq ) allowed quality . We desisolution B, resultapproach C. The abapproach C. These approach B, anotheapproach . TogetheSupplemental Fig. S1C) and between L3 and young adult  . The larger number of differentially accessible peaks (both decreased and increased) observed during the transition from early embryo to L3 versus L3 to young adult could not simply be explained by differences in sequencing depth (see Methods) and is likely due to the massive changes in cell number and tissue composition that occur during this transition  , while the most enriched terms for genes with increased chromatin accessibility from embryo to L3 include larval development and locomotion . Similarly, we observe strong enrichments of GO terms reflecting the major phenotypic changes occurring between L3 and adult, including terms like larval development and reproduction .An example of a decrease in chromatin accessibility from embryo to L3 can be seen in the promoter region of the elopment . Conversly at L3 , 2000. CTogether, these results indicate that ATAC-seq in whole organisms can identify changes in DNA accessibility that represent key biological differences between stages, regardless of whether these changes are due to activation/repression of specific regions within a cell type or to changes in cell-type composition.Supplemental Fig. S3A–E; Supplemental Tables S8–S11; see Methods). ATAC-seq peaks from all three stages were significantly enriched in active and poised regulatory chromatin states , as defined by this ChromHMM model, and significantly depleted in heterochromatic states . ATAC-seq peaks were also enriched in H3K27me3-repressed regions, which is likely due to H3K27me3 marking inactive, yet accessible poised sites . Thus, ATAC-seq correctly identifies poised and active regulatory regions at both specific loci and genome-wide.Accessible chromatin encompasses several key features of the epigenome, including active and poised regulatory regions. To verify that our ATAC-seq data correctly identify regulatory regions throughout the epigenome, we used multiple histone modification ChIP-seq data sets from modENCODE  and Chroed sites . ATAC-seed sites B and genSupplemental Fig. S2A,B) and RNA-seq  were positively correlated with ATAC-seq signal near the TSS. These observations support the relationship between chromatin accessibility at the TSS and gene expression, despite the numerous other layers of gene regulation .To further assess the relevance of whole organism chromatin accessibility, we compared our ATAC-seq data to publicly available gene expression data  . Both GRSupplemental Fig. S3G). Conversely, the regions that gained accessibility during development were enriched for transitions from inactive chromatin states to active regulatory states  . Collectively these results show that ATAC-seq performed on an entire organism, even a complex multitissue adult, is sensitive enough to detect important global changes in chromatin structure that are consistent with predicted chromatin state transitions. Thus, ATAC-seq as a single assay constitutes an attractive alternative to performing multiple histone modification ChIP-seq experiments for identifying key regulatory regions, especially considering that ATAC-seq requires orders of magnitude less input than a single ChIP-seq.Beyond simply identifying regulatory regions important for individual life stages, chromatin accessibility dynamics should highlight regulatory regions critical for transitions from embryo to larval stages, and from larval stages to adulthood. We examined whether genomic regions that showed accessibility changes from one life stage to another were enriched for specific chromatin state transitions. Regions that lost chromatin accessibility from embryo to L3 or from L3 to young adult were enriched for transitions from active regulatory chromatin states  to repressed or heterochromatic states , as defined by the ChromHMM model and distinguished by the presence of H3K27ac (active enhancers) or H3K27me3 (repressed enhancers) . Furthermore, these distal noncoding ATAC-seq peaks were significantly more conserved than expected by chance , a defining feature of enhancers   . These aC. elegans, a small number of functional enhancers have been experimentally mapped, including four enhancers in the upstream regulatory region of hlh-1, the C. elegans MyoD ortholog that regulates muscle development . Given that hlh-1 is exclusively expressed in muscle  would enable us to capture distal regulatory dynamics of development. To experimentally test the functional enhancers predicted by our ATAC-seq data, we selected 13 distal ATAC-seq peaks  that exhibited the largest fold changes in accessibility between any two stages. We generated multiple transgenic C. elegans strains with these 13 putative enhancer regions upstream of a minimal promoter (pes-10) driving expression of green fluorescent protein (GFP) containing a nucleolar localization signal (NoLS) . To control for specificity, we also tested regions flanking 10 of the 13 putative enhancer regions . We created numerous (8–11) independent genetic strains for each validated regulatory site to ensure that no artifact of transgenesis  could be driving spurious GFP expression   . Thus, dgei-13, mlt-8, and nhr-25) are active in the head or tail hypodermis during development, while others are active specifically in the pharynx (C54G6.3) or along the flank of the worm (swip-10). In addition, the enhancers are located at a wide diversity of genomic positions relative to their putatively associated gene: upstream of the TSS (at distances varying from 1 to 9 kb), within introns, as well as downstream from the coding sequence. An interesting example is the nhr-25-associated enhancer, which is downstream from the 3′ UTR, more than 5 kb downstream from the TSS  (nhr-25 is a conserved nuclear receptor primarily expressed in the hypodermis (and somatic gonad) during larval development  indicates that ATAC-seq performed in whole worms is sensitive enough to identify regulatory regions active only in specific cell types, though we cannot rule out the possibility that these regions are also accessible but their activity repressed  in other cells.Importantly, the enhancers we experimentally identified display diverse spatiotemporal activity patterns. Three of the enhancer regions (putatively associated with the TSS) D. nhr-25elopment . The 3′ elopment E. The liCollectively, these findings demonstrate the power of using ATAC-seq dynamics as an unbiased approach to identify functional and conserved enhancers active in a small subset of cells. When applied to whole organisms or complex samples composed of diverse cellular populations, this approach could capture regulatory regions that may have been missed in studies of isolated cell populations.C. elegans transcription factor (TF) binding motifs . For example, the motif for BLMP-1, the C. elegans ortholog of human BLIMP-1/PRDM1, a TF that recruits chromatin-remodeling complexes in human B cells  and in ATAC-seq peaks that gained in accessibility in L3 versus embryo . Using this machine learning framework, we identified the motifs that are the most predictive of ATAC-seq dynamics; the top three most informative motifs in our model are the GAGA motif that we identified above and motifs for known chromatin regulators (BLMP-1 and ELT-3)  as welld ELT-3) C. Thus, Supplemental Fig. S7A) and was significantly enriched for regions that overlapped ATAC-seq peaks that gain accessibility in L3 compared to early embryo , indicating that EOR-1 is indeed bound to the GAGA motif and that EOR-1 may be involved in regulating chromatin accessibility. To examine if EOR-1 is found at closed chromatin, we quantified not only ATAC-seq signal but also ATAC-seq fragment size, as larger ATAC-seq fragments correlate with higher nucleosome occupancy and less accessible chromatin regions . In fact, the only factor with larger ATAC-seq insert sizes was the heterochromatin-associated protein HPL-2. The larger ATAC-seq insert sizes at EOR-1 binding sites are unlikely to be due to the binding of other TFs hindering the ability of transposons to interact with the genomic DNA because EOR-1 ChIP-seq peaks did not show unusual overlap with the other TFs assessed . These observations are consistent with the possibility that EOR-1 is uniquely present at regions with less accessible chromatin compared to other TFs.We next investigated whether EOR-1 may play a role in chromatin accessibility changes. An EOR-1 ChIP-seq data set at L3  revealed regions . EOR-1 CSupplemental Fig. S7F). As expected, canonical TFs are predominantly found at regions with short ATAC-seq insert sizes , whereas chromatin factors, especially HPL-2, are also present at regions with larger ATAC-seq fragment sizes (with many >147 bp). Strikingly, EOR-1 was found both at regions with short ATAC-seq insert sizes as well as at regions with larger ATAC-seq fragment sizes (>147 bp). Together, these results suggest that EOR-1 may, in some cases, bind to or at least be immediately adjacent to nucleosome-occupied DNA.To more closely investigate the chromatin accessibility landscape of EOR-1 peaks at the L3 stage, we generated aggregated histograms of the median ATAC-seq fragment sizes for the ChIP-seq peak summits of EOR-1, all canonical TFs, and the chromatin-associated factors HPL-1 and HDA-1  EOR-1 is capable of binding to both open and closed chromatin (depending on the loci or the tissue); for example, EOR-1 could bind open sites in one tissue but closed sites in another tissue A; (2) EOSupplemental Table S3) such as insulators and enhancers. This approach, developed here for C. elegans, should be readily applicable to other complex samples in vivo such as whole mammalian organs or tumor samples.Here, we show for the first time that sensitively measuring chromatin accessibility in a whole metazoan can detect dynamic changes throughout development and even identify novel functional enhancers active in only a small subset of the whole organism. Among the three developmental stages surveyed, we identified over 30,000 accessible sites which could serve as a catalog to facilitate the discovery of previously unknown distal regulatory loci  have previously been suggested to be promoters (despite their ChromHMM enhancer annotation)  . Thus, fnhr-25. This putative nhr-25 enhancer is located 5 kb downstream from the nhr-25 TSS and acts in an orientation-independent manner. Thus, this enhancer might be looping to the nhr-25 TSS to enhance transcription, although the limited spatial resolution of current Hi-C data in C. elegans , mouse (Nr5a1), and human (NR5A1) exhibit a consistent signature of three-dimensional chromatin architecture downstream from the 3′ UTR . While C. elegans, EOR-1. The EOR-1 motif we initially detected closely resembles a dimeric version of the canonical GAGA motif bound by Trl/GAGA-Associated Factor (GAF) in Drosophila. GAF is a multifaceted transcription factor that can associate with heterochromatin  (C. elegans (nhr-25 and swip-10) contain an EOR-1/GAGA motif. Collectively, these data suggest that EOR-1 might regulate chromatin accessibility at enhancers in C. elegans and potentially other species.In and RSC) . This isand RSC) , and a G elegans . Indeed,C. elegans as a paradigm, we have shown that ATAC-seq performed on complex, heterogeneous samples can reveal novel, spatiotemporally specific genetic regulators and that measuring chromatin accessibility across a developmental time course can identify important dynamic regions. We have highlighted important applications of this approach: discovering functional distal regulatory regions active in only a small subset of the total sample and identifying candidate regulators of genome-wide chromatin dynamics. This data set, which represents an initial atlas of genome-wide chromatin accessibility and candidate distal regulatory sites in C. elegans, should be a valuable resource for the community. The fact that a genome-wide chromatin accessibility assay performed in whole organisms can sensitively identify previously undiscovered functional enhancers in vivo raises the exciting possibility that distal regulation plays a more important role than previously believed in the nematode.Using Supplemental Methods.Brief methods can be found below, but in all cases more details can be found in the Three sets of completely independent biological replicates were prepared by harvesting and then flash-freezing tightly temporally synchronized samples at three different stages: early embryo (in utero), larval stage 3 (36 h post-egg lay), and young adult (57 h post-egg lay). Native nuclei were purified from frozen samples using mechanical homogenization as previously described . The purSupplemental Table S1). Prior to mapping, standard next-generation sequencing quality control steps, as well as ATAC-seq-specific quality control steps, were performed. Individual replicate ATAC-seq peaks were called using a custom pipeline which used MACS (v2.1)   to call S (v2.1) . The respes-10) driving expression of a C. elegans intron- and photo-stability-optimized GFP containing an N-terminal nucleolar localization signal. Putative regulatory regions were chosen by selecting ATAC-seq peaks that exhibited the largest differential accessibility between two stages and that were at least 1 kb from a transcription start site (as defined on the UCSC Genome Browser). Flanking negative control regions were chosen by selecting regions within 2 kb of the putative regulatory regions that were not in peaks of accessibility. Primers were designed to amplify each region as well as 50–500 bp flanking either side .For the enhancer screen, each putative regulatory region was cloned in the pL4051 plasmid (a gift from Andrew Fire) upstream of a minimal promoter . To quantify the consistency of GFP expression pattern, all lines (including the negative control lines) , negative control regions, and the no-insert controls were generated. Multiple independent lines were generated per construct . For eacl lines)  were scoC. elegans motif from cisBP (v1.02) (https://CRAN.R-project.org/package=gbm) had the best accuracy while still allowing for interpretation of which motifs were the most informative. Given the unbalanced classification problem, we used balanced accuracy as our primary metric of classification success .To predict changes in accessibility between early embryo and larval stage 3, the number of each mapped  (v1.02)  and eachhttps://github.com/brunetlab/CelegansATACseq, as well as in the Supplemental Material.All analysis source code is freely available at http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE89608.The raw data as well as stage-specific peaks for all three stages and the input control have been submitted to the NCBI Gene Expression Omnibus (GEO;


Lactobacillus plantarum is controlled by two gene clusters, and the global regulator catabolite control protein A (CcpA) may be involved in the regulation. To understand the mechanism, this study focused on the regulation relationships of CcpA toward target genes and the binding effects on the catabolite responsive element (cre). First, reverse transcription-PCR analysis of the transcriptional organization of the FOS-related gene clusters showed that they were organized in three independent polycistronic units. Diauxic growth, hierarchical utilization of carbohydrates and repression of FOS-related genes were observed in cultures containing FOS and glucose, suggesting carbon catabolite repression (CCR) control in FOS utilization. Knockout of ccpA gene eliminated these phenomena, indicating the principal role of this gene in CCR of FOS metabolism. Furthermore, six potential cre sites for CcpA binding were predicted in the regions of putative promoters of the two clusters. Direct binding was confirmed by electrophoretic mobility shift assays in vitro and chromatin immunoprecipitation in vivo. The results of the above studies suggest that CcpA is a vital regulator of FOS metabolism in L. plantarum and that CcpA-dependent CCR regulates FOS metabolism through the direct binding of CcpA toward the cre sites in the promoter regions of FOS-related clusters.Fructooligosaccharides (FOSs) metabolism in  Escherichia coli, has been termed carbon catabolite repression (CCR)  . CCR is on (CCR) .cre) with the assistance of seryl-phosphorylated histidine phosphocarrier protein (HPr-Ser∼P) , a protein of the LacI-GalR family . It is ar-Ser∼P) . The coneratures . The cre operons . The binsm genes . CCR is sm genes , stress sm genes , and metf of CCR .Lactobacillus paracasei 1195 was grown on media containing 1% FOS and 0.1% glucose, and predicted that the regulation is controlled by CCR via binding of CcpA to the cre site  were found to participate in the metabolism of FOS in Lactobacillus plantarum ST-III. A number of potential cre sites for CcpA binding have been predicted in the regions of putative promoters of the two clusters  are non-digestible food ingredients that can stimulate the growth and activity of beneficial microbes residing in the GIT . Despitecre site . Some crlus NCFM  and Bifi UCC2003 . In our clusters .L. plantarum is regulated by CcpA-dependent CCR, the present study investigated the growth of L. plantarum and its mutant strains in CDM medium containing FOS and limited glucose. Growth profiles, sugar consumption, and gene expression were monitored. The binding of CcpA to the cre sites was verified using electrophoretic mobility shift assays (EMSA) in vitro and chromatin immunoprecipitation assay (ChIP) in vivo. Our findings reveal the hierarchical nature of carbohydrate utilization in L. plantarum, and highlight the crucial role of CcpA in controlling FOS utilization.To determine whether FOS metabolism in Table 1 and Supplementary Table E. coli strain (DH5α and BL21), used as an intermediate cloning and expression host, was grown in Luria-Bertani (LB) medium at 37°C with aeration at 200 rpm/min. L. plantarum ST-III and its ccpA mutant strain were routinely cultured in de Man-Rogosa-Sharpe (MRS) broth  at 37°C without agitation. To measure the growth of the wild-type and ccpA mutant strains, cells were grown in chemically defined medium (CDM)  erythromycin for L. plantarum.The bacterial strains, plasmids and primers used in this study and their relevant features are listed in um (CDM)  supplemeL. plantarum ST-III or its ccpA mutant strain were transferred with 2% (v/v) inoculum into 500 mL of CDM containing 0.1% glucose and 0.4% FOS. The cultures were incubated without shaking for 16–18 h at 37°C. During the cells’ growth up to the stationary phase, the samples were withdrawn every 2 h to measure OD600 for growth analysis. To quantify sugars consumption and gene expression, culture samples were taken per hour (every 10 min in the diauxic growth period) during fermentation and centrifuged . The supernatants were collected for residual sugar detection, and the cell pellets were used for RNA isolation.Overnight cultures of g, 4°C, 15 min), the supernatant of each sample was filtered through a 0.45 μm nylon filter and then analyzed using a gradient elution procedure with H2O-250 mM NaOH as the mobile phase. The column was eluted at a flow rate of 0.5 mL/min and the injection volume of sample was 20 μL. Quantitative analyses were carried out using 5 mg/mL of solutions of the FOS standard set , glucose, fructose, and sucrose .For quantification of sugar consumption in the cell supernatants, high performance anion exchange chromatography (HPAEC) was carried out on an ICS5000 chromatograph  with a CarboPac PA20 anion exchange column  and a pulsed amperometric detector (PAD) as described previously . After cL. plantarum ST-III cells collected from various conditions was extracted using TRIzol reagent  according to the manufacturer’s instructions. RNA was subjected to RNase-free DNase I digestion and then purified using a PrimeScript RT reagent kit . The quantity and quality of total RNA were evaluated using a NanoDrop 2000C  and agarose gel electrophoresis. RNA preparations were stored at -80°C until use. Total RNA was reverse-transcribed to cDNA with a PrimeScript RT reagent kit  following the manufacturer’s instructions.Total RNA from -ΔΔCT method and the 16S rRNA gene was used as the normalized standard.For RT-qPCR analysis, the generated cDNA was mixed with 0.2 μM gene specific primers  was constructed for the collection of binding sites and those in the upstream regions of sacPTS1 and sacPTS26 clusters were searched. Scores of candidate sites were defined as the sum of positional nucleotide weights as previously described escribed  and valulox-based mutagenesis system was used for gene deletion of ccpA  with chromosomal DNA used as the template. The amplicons were respectively cloned in the XhoI and Eco53kI restriction sites of suicide vector pNZ5319, and the recombinant mutagenesis vector, pNZ5319-up-down, was electroporated into L. plantarum ST-III cells. Candidate double-crossover clones, were selected and confirmed by PCR. Then, the lox66-P32-cat-lox71 cassette was resolved to a single double-mutant lox72 site through the transient plasmid pNZ5348. A double-crossover ccpA mutant without any resistance gene (ΔccpA strain) was acquired and confirmed by DNA sequencing as described previously (The Cre- of ccpA . DNA fraeviously .6-tagged CcpA was performed using the pET-28a(+) vector as previously described  when OD600 reached 0.4 to 0.6 at 37°C. The culture was allowed to grow at 25°C for 8 h. Cells were collected by centrifugation at 6000 × g for 10 min, and resuspended in binding buffer , then 1 mM PMSF and 0.1 mg/mL lysozyme solution were added, and the cells were then lysed by sonication. The cell debris was removed by centrifugation at 10,000 × g for 10 min at 4°C before RNase and DNase were added. The soluble protein was purified by nickel ion affinity chromatography using a Chelating Sepharose Fast Flow column  according to the manufacturer’s instructions. The purified protein was desalted and concentrated by Amicon Ultra-0.5 centrifugal filter devices .The HissacK, Ppts1-sacA, Pagl4, and PsacR2) containing the putative cre sites  and were quantified with NanoDrop 2000C. A competition assay was performed by addition of 100-fold molar excess of unlabelled probe to the EMSA reaction mixture.Double-stranded 200-bp DNA fragments , 100 mM KCl, 2.5 mM MgCl2, 0.2 mM DTT, 2000 ng polydIdC and 10% glycerol. After incubation for 30 min at 30°C, the reaction system was loaded onto 2% agarose gels buffered with 0.5× TBE. The gels were scanned with ImageQuant LAS 4000 mini   was mutated and named creMUT. The main principle of the mutation was as follows: the defined base in the consensus motif was mutated to the other three bases, and the “W” that represents A or T was mutated to “S,” which represents G or C. Specific primers were then synthesized for PCR amplification, and the amplicons were purified and self-ligated with an Ezmax One-Step Cloning Kit  following the manufacturer’s instructions. The creMUT sequences were verified by DNA sequencing, and the probe preparation and EMSA analysis were the same as described above.Electrophoretic mobility shift assay was performed in a 20 μL reaction volume that contains 40 ng probe and different amounts of purified Hislthcare) . To veriL. plantarum ST-III were constructed using the pSIP409 vector as previously described (ccpA gene sequence was amplified by PCR using the Flag-ccpA-409F (containing a Flag-tag on the 5′ terminus) and Flag-ccpA-409R primers  to a final concentration of 50 ng/mL. The cells were fixed in 1% formaldehyde and quenched by the addition of glycine. Then the cells were washed twice with 5 mM Tris-HCl (pH 8.0) containing 25 mg/mL lysozyme and sheared to an average fragment size of 300 to 500 bp by sonication. After centrifugation, the 5 μL supernatant was retained for use as the input sample, and the rest of the supernatant was incubated with Flag antibodies. Normal Rabbit IgG was set up as a negative control. Immunoprecipitated and input samples were de-crosslinked by incubation in 1× IP elution buffer and 5 M NaCl and 20 mg/mL proteinase K for 65°C at 1.5 h. The purified ChIP products and genomic input DNA were analyzed by qPCR using specific primers .Previous sequence analysis suggested that the FOS-related gene clusters of ic units . To veriic units . The rescre sites to regulate its target genes. However, the cre sequence differs among Gram-positive bacteria . Using the generated PFM to search in the sacPTS1 and sacPTS26 clusters, six potential cre sites were found. In addition to the five cre sites found in the previous study, a new cre site was discovered in the Ppts1-sacA region. As a result, this region has three putative cre sites . The scores of the six candidate sites were all greater than 5, suggesting the possibility of CcpA binding  was monitored . A typical diauxic growth pattern with two distinct growth phases separated by a lag phase of about 2 h was observed. During the growth, residual sugars were detected in the supernatant at the same time. In the first 6 h, glucose and traces of fructose in the FOS mixture were consumed and the main components of FOS were kept constant. The diauxic lag is likely to have been caused by the depletion of glucose, as cessation of growth was observed at a similar time. After the diauxic lag phase, the cells resumed growing and entered a second growth phase using FOS as the carbon source. 1-Kestose was rapidly consumed and was undetectable after 10 h of fermentation. Nystose and fructosyl-nystose gradually decreased to an undetectable level at 14 and 16 h, respectively. To examine the kinetics of transcription of the FOS-related genes during the diauxic growth, RT-qPCR was performed to analyze the changes in the expression levels of these genes. RNA samples were collected at different sampling times and CT values obtained at 8 h of incubation were chosen as the control values to calculate the fold changes in gene expression over time. Each gene of the two clusters was analyzed using specific primer pairs , which is consistent with the result of the RT-PCR experiments. Then sacK, sacA, and pts26 genes were chosen as the representatives for gene expression in the subsequent studies. As shown in Figure 4C, the expression levels of sacK were very low during the first growth phase when glucose was utilized as the preferred carbon source. On entering into the lag phase, gradually higher expression was observed. The maximum transcription levels were observed at 9 and 10 h when 1-kestose and nystose were actively hydrolyzed. The expression profiles of sacA and pts26 were similar. Expression was repressed during the first growth phase, then, after glucose depletion, the repression began to release and the transcription levels remained almost constant during the second growth phase. After 13 h, the transcription levels of all three genes were sharply decreased and this period was associated with the depletion of FOS in the medium. These observations are consistent with the results of previous transcriptome experiments in which glucose repressed the transcription of FOS-induced genes (The growth of ed genes .ccpA strain was constructed using the Cre-lox-based mutagenesis system (ccpA strain on glucose and FOS was compared with its parent strain. The ΔccpA strain exhibited a significantly reduced growth rate compared with the wild-type strain in glucose (a doubling time of 98.4 ± 1.0 min versus 76.2 ± 6 min) and FOS (a doubling time of 102.6 ± 1.5 min versus 81.9 ± 7.2 min). These results are in accordance with findings that CcpA inactivation significantly affected the growth rate of other microorganisms . The components in the FOS mixture were used simultaneously with glucose during the whole growth process, although glucose was consumed at a faster speed. All of the sugars were depleted at the end of fermentation.To determine whether the inhibition of FOS utilization by glucose is regulated by catabolic repression via CcpA, a Δs system . The grorganisms . The delccpA strains was also measured by RT-qPCR in the early logarithmic phase of the growth. The expression levels of the sacK, sacA, and pts26 genes in the wild-type and ΔccpA strains were measured with glucose, FOS and the mixture of glucose and FOS as the carbon sources respectively. The CT values of the wild-type strain grown on glucose were chosen as the control for the three genes to calculate the fold changes between the different conditions. As expected, all three genes were significantly down-regulated for the wild-type strains grown on glucose and in the mixture of glucose and FOS, compared with the wild-type grown on FOS (Table 2). In contrast, after ccpA inactivation, repression of these genes by glucose was drastically relieved in the ΔccpA strain compared with the wild-type. For example, the expression levels of the sacK, sacA, and sacPTS26 operons in the ΔccpA strain were respectively 2.9-, 3-, and 3.9-fold higher than the corresponding values for the wild-type strain when the cells were grown in a mixture of glucose and FOS. These results confirm the dominant role of CcpA in the CCR of FOS metabolism in L. plantarum ST-III. Notably, in the ΔccpA strain, sacA and pts26 showed 1.5- to 1.8-fold increases in the presence of FOS  versus glucose alone. This result implies that in addition to the CcpA-dependent CCR, these genes may be induced or derepressed by FOS.Gene expression of the FOS-related clusters in the wild-type and Δcre sites in vitro, His6-tagged CcpA was expressed heterologously in E. coli, and the purified protein was used to perform an EMSA , with increasing amounts of His6-tagged CcpA (0 to 1.2 μg), the intensities of the bands representing the shifted CcpA-DNA complex strengthened. In contrast, when labeled and unlabelled probes were used for a specific competitive assay (lane 5), no shift was detected for the labeled probe, demonstrating the binding specificity of CcpA to these DNA fragments.To determine whether CcpA can bind to the six putative  an EMSA . As showcre sites within the regions, EMSA experiments were performed to examine whether mutation of cre , indicating that the CcpA did not bind to the three promoter regions that included creMUT. As the Ppts1-sacA region exists at three putative cre sites, the region was mutated three times and EMSA was conducted. The binding affinities of His6-tagged CcpA protein to the Ppts1-sacA region were weakened after one (cresacA1) or two sites (cresacA1 and cresacA1-sacpts1) were mutated (data not shown). When the Ppts1-sacA region was mutated at third time (cresacA2), the binding affinity of His6-tagged CcpA protein to the Ppts1-sacA region vanished. These results indicate that all three cre sites can bind to the CcpA protein.Next, to verify that CcpA combined directly with the ragments . Then DN in EMSA . The resin vivo. The CcpA protein was Flag-tagged at its N terminus, and then the successful expression of 409-Flag-ccpA in L. plantarum was confirmed via western blot analysis . The cross-linked DNA fragments were analyzed by RT-qPCR. As shown in Figure 6B, it is clear that the four regions were remarkably enriched by CcpA protein compared to the negative control sample, suggesting that CcpA interacts specifically with those promoter regions in vivo. Together, these findings suggest that CcpA protein can specifically bind to the four promoter regions, and six cre sites exist in these regions with high-affinity binding for CcpA.Chromatin immunoprecipitation was then performed to test for the binding of CcpA to the promoter regions Lactobacillus plantarum is a normal inhabitant of the human GIT and previous reports have shown that it can utilize FOS as efficiently as glucose, with two gene clusters participating in FOS metabolism  and “WTGNNARCGNWWWCAW” in Bacillus subtilis .Regulation of the transcription of CcpA-regulated genes involves the binding of CcpA to subtilis  and L. plantarum , respectlantarum . In thisequences , but theccpA strain, demonstrating that other regulators participate in FOS metabolism of L. plantarum. These results confirm our previous prediction that L. plantarum have a double effect of global and local regulation of FOS metabolism. The local regulators SacR1 and SacR2 may also be involved in metabolic regulation in L. plantarum (In the absence of a functional CcpA, other regulatory processes could be studied when CcpA-mediated repression is abolished. In our study, FOS in the medium resulted in activation of the FOS-related genes in the Δlantarum . In theslantarum . AlthougL. plantarum grown on FOS in the presence of limiting glucose demonstrated that FOS utilization is subject to CCR. Inactivation of the ccpA gene eliminated these phenomena, proving the dominant role of CcpA in CCR for FOS metabolism. The binding of CcpA to the cre sites in the promoter regions was verified both in vivo and in vitro, which suggests that CcpA regulates CCR through direct regulation of the transcription of FOS-related clusters. As CCR is part of the regulatory network, further analysis of CCR may lead to deeper insights into a complex regulatory network. Our evolving understanding of the mechanistic interactions of probiotics and prebiotics will provide the molecular basis for the design of effective probiotic–prebiotic combinations to maximize host benefits.The diauxic growth, hierarchical use of sugars and repression of FOS-related genes in ccpA strain. HY executed the target genes structure analysis and confirmed CcpA binding to the putative cre sites. HT designed the research.CC wrote the manuscript and the statistical analysis. YL analyzed the growth and expression of related genes in wild-type and mutated strains. LW executed the construction of the expression vector and ΔThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation. The characteristics of cells are principally determined by patterns of gene expression. During the course of development, the generation of various cell types consisting of our body are driven by the dynamic alteration of gene expression patterns. Human pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs)  and induThe changes of gene expression patterns during myogenic differentiation have been well characterized. Several myogenic transcription factors (TFs) are identified as markers for specific stages in differentiation , 10. TheIn this review, we discuss the current methods for differentiating skeletal muscle cells from hPSCs through enforced epigenetic and transcriptional manipulation that can directly activate the myogenic gene expression program.The first attempts to direct myogenic differentiation with forced expression of TFs have been performed using mouse ESCs (mESCs). Darabi et al. demonstrated that overexpressing Pax3 or Pax7 during the embryoid body (EB) formation of mESCs induces efficient myogenic differentiation –21. WhenIn these experiments, the introduction of TFs was performed with lentiviral or adenoviral vectors, and their overexpression was not directly carried out in hESCs or hiPSCs but rather in mesodermal and mesenchymal-like cells derived from hESCs/hiPSCs. Indeed, the efficiency of myogenic differentiation is low when adenoviruses encoding MYOD1 are directly transduced to hESCs . Albini The functional activities of TFs require their physical access to the genome, which is tightly enclosed in chromatin structure consisting of DNA and histone complexes. Especially, the chromatin structure around the TF-binding sites plays critical roles in the regulation of gene expression –25. The Pluripotent ESCs/iPSCs possess unique chromatin signatures to be prepared for differentiation. In ESCs/iPSCs, lineage-affiliated genes are transcriptionally poised by “bivalent” histone modifications, consisting of H3 Lys-4 trimethylation (H3K4me3) and H3 Lys-27 trimethylation (H3K27me3) –32. H3K4In hESCs, more than 1500 genes are categorized as bivalent “H3K4/K27me3-modified genes” , 31, 37,Histone methylation is dynamically regulated by two kinds of enzymes, histone methyltransferases and demethylases, which add and remove the histone lysine methylation, respectively. The addition of H3K27me3 is mediated by the Polycomb repressive complex containing the histone methyltransferase—EZH2—as the enzymatic subunit –40. On tManipulating JMJD3 expression has the potential to change the epigenetic status of hPSCs toward differentiation. Indeed, Akiyama et al. have revealed that the forced expression of JMJD3 results in genome-wide demethylation of H3K27me3 in hESCs . Furtherβ-catenin signaling is responsible for meso/endodermal differentiation [The forced expression of JMJD3c enables the hESCs to upregulate developmental genes that are accompanied by H3K27me3 demethylation . In thisntiation , 53, ectJMJD3c overexpression also activates the PAX3 and PAX7 genes, but not MYF5 or MYOD1 , indicatThe chromatin structure established in H3K27me3-deficient ES cells provides a suitable state for MYOD1-mediated myogenic differentiation. Akiyama et al. have shown that JMJD3c overexpression followed by MYOD1 overexpression significantly upregulates markers for skeletal muscle differentiation—MYOG, MEF2C, CKM, and SIX1 . The myoIn previous studies, the overexpression of TFs was performed by viral or transposon vectors such as lentivirus, adenovirus, and piggyBac transposons. These vectors can effectively induce the expression of exogenous genes in hPSCs, but they have considerable limitations in terms of therapeutic applications: for example, possible insertional mutagenesis may occur due to random integration of the vectors into the host genome.Synthetic mRNAs (synRNAs) encoding developmental regulator genes is one of the most promising approaches for directing the differentiation of hPSCs. This approach eliminates the risk of genomic DNA integration and insertional mutagenesis and is, thus, considered suitable for therapeutic applications. It has been shown that the transfection of synRNAs encoding reprogramming TFs into fibroblast cells can efficiently generate hiPSCs . FurtherAkiyama et al. have demonstrated that transfection of JMJD3c-synRNAs prior to MYOD1-synRNAs dramatically increases the efficiency of myogenic differentiation of hPSCs . The majA recent study showed that the SWI/SNF chromatin remodeling component BAF60C also promotes MYOD-mediated myogenic conversion in hESCs . There aIn this review, we have provided an overview of the current status of skeletal muscle generation from hPSCs using epigenetic and transcriptional manipulation . Direct The authors declare that there is no conflict of interest regarding the publication of this paper.