Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ One crucial component of this regulatory system is the σ54 protein, which directs RNA polymerase to conserved DNA sequences located in the − 12 and − 24-bp regions of σ54 promoter elements2. Enhancer binding proteins (EBPs), which are transcriptional activators, are also crucial for the normal function of the σ54 regulatory system. Namely, EBPs are ATPases that use the energy from ATP hydrolysis to help σ54-RNA polymerase form an open promoter complex and initiate transcription5. Bacteria typically have one gene for σ54, but often have multiple genes for EBPs; each EBP works with σ54 to regulate a subset of σ54 promoters, which the EBP identifies via specific tandem repeat sequences or enhancer elements7. Interestingly, the tandem repeat binding sites of EBP dimers are typically located 80- to 150-bp upstream of the − 12 and − 24-bp regions of σ54 promoters; hence, it seems likely that many σ54 promoters have intrinsically curved DNA sequences or binding sequences for DNA bending proteins, as EBP dimers directly contact σ54-RNA polymerase10.The σ7. Typically, the N-terminal signaling domain modulates the ATPase activity of the EBP in response to an intracellular or extracellular signal. In some cases, the N-terminal domain binds directly to a signaling molecule. However, the N-terminal domain of most EBPs is modified  by a signal transduction partner such as a histidine protein kinase that detects the signal11. Because σ54-RNA polymerase requires the energy from EBP-catalyzed ATP hydrolysis to initiate transcription and the EBP’s ATPase activity is controlled by signal input, the σ54 system is able to tightly control transcription of its target genes.EBPs generally contain three domains: an N-terminal signaling domain, a central ATPase domain that is responsible for ATP hydrolysis and transcriptional activation, and a C-terminal DNA-binding domain (DBD) that recognizes a specific DNA sequence54 was viewed as specialized regulatory system that was mainly dedicated to transcription of genes involved in nitrogen assimilation or nitrogen fixation13. In recent years however, it has become clear that the σ54 system is important for transcription of many types of bacterial genes. For example, the σ54 system in Bacillus cereus, Pseudomonas aeruginosa and Escherichia coli modulates transcription of genes involved amino acid metabolism, the response to reactive nitrogen species and the phage shock response, respectively16. In Caulobacter crescentus, Pseudomonas aeruginosa, Pseudomonas putida and Vibrio cholerae the σ54 system regulates genes that are important for flagellar biosynthesis and motility21, and in Pseudomonas aeruginosa the σ54 system is implicated in transcription of genes involved in quorum sensing, biofilm formation and virulence24.Historically, σ54 system in the soil bacterium Myxococcus xanthus has been studied extensively and has some rare properties. Namely, M. xanthus is one of the rare bacterial species in which the σ54 system has been linked to growth in nutrient rich conditions. For example, inactivation of the nla4 or nla18 EBP gene severely impairs M. xanthus growth in nutrient rich media27. Presumably, the relatively slow growth of the nla4 mutant and nla18 mutant is due at least in part to changes in the basal levels of the intracellular starvation signal (p)ppGpp and the resulting changes in gene expression.The σM. xanthus also has an unusually large repertoire of 53 EBP genes28. All of the EBPs were characterized a number of years ago and many of the EBPs were implicated in motility30 and in starvation-induced biofilm formation36, which yields spore-filled aerial structures called fruiting bodies. Six of the EBPs that begin functioning in the early to middle stages of biofilm development, which is also known as fruiting body development, form a regulatory cascade30. This EBP cascade is reminiscent of the sigma factor cascade that controls the sequential stages of spore development in Bacillus subtilis37, as pairs of EBPs functioning at one stage of development directly activate transcription of an EBP gene important for the next developmental stage.39. The consensus Nla28 binding site [CT(C/G)CG(C/G)AG consensus half site], which was generated from these studies, was subsequently used to search the M. xanthus genome sequence for Nla28 target promoters/genes located outside the EBP cascade39. A number of these putative Nla28 target promoters were identified in natural product gene clusters. This was an intriguing finding, as Volz et al. previously showed that two M. xanthus EBPs (HsfA and MXAN4899) are capable of binding to fragments of natural product gene promoters40. Furthermore, it was previously suggested that the σ54 system might be a key regulator of polyketide (PK) and non-ribosomal peptide (NRP) natural product genes in M. xanthus, and in bacteria in general, based on bioinformatics38.Nla28 is one of the early-functioning developmental EBPs that participates in the transcriptional cascade. A putative tandem repeat promoter binding site for Nla28 dimers was identified and analyzed using bioinformatics and experimentation54 system modulates expression of M. xanthus natural product genes. We focused on three potential σ54 promoter targets of Nla28 that are located in M. xanthus natural product gene clusters38. The activities of the three natural product promoters increase during growth, with peak activities occurring during the transition into stationary phase. The activities of the promoters also increase during starvation-induced development. These findings are consistent previous studies linking expression of bacterial natural product genes to changes in nutritional status45. Our in vivo and in vitro mutational analyses indicate that three natural product promoters are indeed σ54 promoter elements and direct targets of Nla28, as predicted via bioinformatics. An examination of the characterized and uncharacterized σ54 promoters in M. xanthus natural product gene clusters yielded a striking result; the vast majority are predicted to be intragenic not intergenic promoters. These results are consistent with previous experimental findings, which placed many characterized M. xanthus σ54 promoters in intragenic regions39.Here, we explored the idea that the σM. xanthus strains were grown at 32 °C in CTTYE broth  or on CTTYE plates containing 1.5% agar. Fifty μg/ml of kanamycin or 10 μg/ml of tetracycline were added to CTTYE broth and CTTYE agar plates as needed. CTT soft agar (CTTSA), which is used to plate electroporated M. xanthus cells, contains 1% Casitone, 10 mM Tris (pH 8.0), 1.0 mM KH2PO4 (pH 7.6), 8.0 mM MgSO4, and 0.7% agar. Submerged culture development of M. xanthus strains occurred in 24-well polystyrene plates containing 100 μl of MC7 buffer . Unless otherwise stated, E. coli strains were grown in Luria–Bertani (LB) broth  supplemented with 100 μg/ml of ampicillin.Bacterial strains and plasmids used in the study are listed in Table M. xanthus strains were grown by inoculating cells into flasks containing CTTYE broth and incubating the cultures at 32 °C with vigorous swirling. Development was induced as previously described25. Briefly, M. xanthus cells were grown in CTTYE broth until the cultures reached a density of approximately 5 × 108 cells/ml, the cells were pelleted, the supernatant was removed, and the cells were resuspended in MC7 buffer to a density of 5 × 109 cells/ml. Forty μl aliquots of the cell suspensions were placed into polystyrene plate wells containing 100 μl of MC7 buffer and the polystyrene plates were transferred to a 32 °C incubator for 24 h.M. xanthus strain DK162246 was extracted using ZYMO Research gDNA extraction kit. Oligonucleotides used in PCR reactions were synthesized by Integrated DNA Technologies (IDT) and are listed in Table Chromosomal DNA from wild-type 54 promoter elements were generated using the Quick Lightning Mutagenesis Kit from Agilent Technologies and the manufacturer’s protocol. Briefly, promoter fragments containing the putative σ54-RNA polymerase binding site in the − 12 and − 24-bp regions and the upstream Nla28 tandem repeat binding site were cloned into pCR 2.1 TOPO vector (Invitrogen). Mutations in the − 12-bp region, the − 24-bp region, the spacer between the − 12 and − 24-bp regions were generated using primers carrying the appropriate nucleotide changes  were synthesized by IDT; the first putative Nla28 half binding site in each promoter was changed to AAAAAAAA. The mutant promoter fragments were then subcloned into the promoterless lacZ expression vector pREG172747, introduced into M. xanthus strains and analyzed as described below.Site-directed mutations in putative σes Table , plasmidlacZ expression vector pREG1727 to create lacZ transcriptional fusions47. The plasmids were introduced into strain DK1622 or a derivative of strain DK1622 carrying an insertion in the nla28 gene, and cells carrying a plasmid integrated at the Mx8 phage attachment site in the chromosome were identified via PCR. The in vivo activities of wild-type and mutant promoters were determined by measuring the specific activities of β-galactosidase in cells developing in submerged cultures for 1, 2, 6, 12 or 24 h, or growing in CTTYE broth for various amounts of time49. Mean wild-type and mutant promoter activities were compared using a two-way analysis of variance (ANOVA) and Tukey’s multiple comparisons post hoc tests, considering two independent variables in each group. The significance level was set at p < 0.05 or lower. GraphPad Prism software v8.4  was used for all analyses.Wild-type and mutant MXAN1286, MXAN1579, MXAN1603 and MXAN3778 promoter fragments were cloned into the promoterless nla28 gene corresponding to the Nla28 DNA binding domain (Nla28-DBD)30 was PCR amplified using gene-specific primers (Table E. coli strain BL21 (DE3) using chemical transformation. Cells containing the Nla28-DBD expression plasmids were grown in rich LB broth to a density of 2 × 108 cells/ml. Protein expression was induced by the addition of 0.3 mM IPTG to the culture and the subsequent incubation of the culture for 12 h at 15 °C. Cells were pelleted via centrifugation and resuspended in 25 ml column buffer  per liter of culture. The resuspended cells were lysed by a combination of freeze-thawing and sonication, and pelleted by centrifugation. The crude extract (supernatant) containing Nla28-DBD was diluted by adding 125 ml of cold column buffer to every 25 ml aliquot of crude extract. For purification of Nla28-DBD from the diluted crude extract, 15 ml of amylose resin was placed in a 2.5 × 10 cm column and the amylose resin was washed with 250 ml cold column buffer. Diluted crude extract containing Nla28-DBD was loaded onto amylose columns at a flow rate of 5 ml/min and washed with 600 ml cold column buffer at a flow rate of 10 ml/min. Nla28-DBD was eluted using 100 ml cold column buffer containing 10 mM maltose; the flow rate was 5 ml/min and 20 fractions containing 5 ml were collected. The presence of eluted Nla28-DBD was detected by UV absorbance at 280 nm. Nla28-DBD- containing fractions were pooled and incubated with 1 mg of Factor Xa at 4 °C overnight to cleave the MBP tag. Nla28-DBD was separated from MBP and concentrated to about 1 mg/ml using Amicon Ultra centrifugal filter units (EMD Millipore). SDS-PAGE and Bradford assays were used to determinate the purity and concentration of Nla28-DBD.A fragment of the rs Table , and the1286, Cy5-P1579 and Cy5-P3778) were generated via PCR; each promoter fragment contained a putative wild-type Nla28 binding site. Three mutant derivatives of these 5′ Cy5-labelled promoter fragments  were synthesized; the first putative Nla28 half binding site in each promoter was changed to AAAAAAAA. All PCR-generated and synthesized promoter fragments were gel-purified and used in subsequent EMSAs. In EMSA reactions, 2 μM purified Nla28-DBD was incubated with 1 ng of 5′ Cy5-labelled wild-type promoter fragment  or 5′ Cy5-labelled mutant promoter fragment  in EMSA buffer  for 30 min at 30 °C. The samples were then analyzed using PAGE under non-denaturing conditions and imaged using a Bio-Rad imager.Purified Nla28-DBD was expected to bind to wild-type MXAN1286, MXAN1579 and MXAN3778 promoter fragments carrying a putative Nla28 tandem repeat binding site. Using the 5′ Cy5-labelled oligonucleotides shown in Table 38, the algorithm developed by Studholme et al.50 was used to examine whether the σ54 system might be a common regulator of bacterial natural product genes. Namely, 180 annotated PK and NRP gene clusters from 58 bacterial species were analyzed for sequences that closely match the σ54 promoter consensus in the − 12-bp region and in the − 24-bp region . The results, which uncovered 124 clusters with at least one σ54 promoter based on consensus matching, supported the idea that the σ54 system might be a general regulator of bacterial natural product genes.In a previous studyM. xanthus) uses the σ54 system for transcription of PK and NRP gene clusters, as predicted in the bioinformatics analysis of Stevens et al.38. We focused on the putative σ54 promoters upstream of MXAN1286, MXAN1579, MXAN1603 and MXAN3778, as these genes were linked to natural product biosynthetic genes/clusters. Namely, the MXAN1286 gene is embedded in a putative NRP gene cluster and flanked by genes that are likely to be involved in NRP biosynthesis. MXAN1579 and MXAN1603 are predicted to be part of another M. xanthus NRP gene cluster. MXAN1603 is a putative non-ribosomal peptide synthetase gene. MXAN1579 is embedded in the cluster and flanked by genes such as MXAN1603 that are likely to be involved in NRP biosynthesis. MXAN3778 is adjacent to a putative hybrid non-ribosomal peptide synthetase/type I polyketide synthase gene and possibly part of the same operon38.The goal of the work presented here was to examine whether a major producer of bacterial natural products ] and the distance from putative Nla28 binding sites2. The mutations that we generated are the following: the GC dinucleotide in the − 12-bp region was replaced with a TT, the GG (GA in MXAN3778) dinucleotide in the − 24-bp region was replaced with a TT, or 1 bp in the spacer between the − 12 and − 24-bp regions was deleted. Subsequently, wild-type and mutant promoter fragments were fused to the promoterless lacZ gene in plasmid pREG172747 and the lacZ transcriptional fusion plasmids were introduced into wild-type M. xanthus strain DK1622 (the plasmids integrated at the Mx8 phage attachment site in the chromosome).To confirm that the natural product loci have bona fide σon Table . HoweverlacZ expression. As shown in Fig. 54-RNA polymerase binding substantially impacted the activities of the MXAN1286, MXAN1579 and MXAN3778 promoters in growing and developing cells, supporting the prediction that the three natural product loci use σ54 promoter elements for transcription.Wild-type and mutant promoter activities during growth in CTTYE broth and fruiting body development in MC7 starvation buffer were inferred from the levels of 54-RNA polymerase binding site in the MXAN1603 promoter region only had a modest impact on growth-related and developmental activities. Our interpretation of this result is that the fragment of the MXAN1603 promoter region contains an unidentified promoter element that is responsible for the majority of the observed development and growth-related activities and that the putative σ54-type promoter element only makes a minor contribution to the observed activities.In contrast to the other promoters, the MXAN1603 promoter only showed a slight increase (about 1.3-fold) in activity during growth in CTTYE Figure A and dur54 promoters, as EBP-mediated ATP hydrolysis opens the σ54-RNA polymerase promoter complex so that transcription can initiate5. Since the σ54 promoters in the MXAN1286, MXAN1579, and MXAN3778 loci were identified as potential targets of Nla28, we determined whether the activities of wild-type promoter fragments are reduced as predicted in a mutant containing an inactivated nla28 gene25 . MXAN1286, MXAN1579, and MXAN3778 promoter activities in wild-type and nla28 mutant cells grown in CTTYE broth are shown in Fig. nla28 abolished the growth phase regulation of all three promoters and caused about 3.1- to fourfold reduction in peak promoter activities at the highest cell densities. Inactivation of nla28 also abolished the developmental activities of the promoters, as the promoters did not show the typical increases in activities when nla28 cells were placed in MC7 starvation buffer  Nla28 half binding site in each promoter fragment was converted to all A nucleotides. Wild-type and mutant promoters were introduced into wild-type strain DK1622 and promoter activities during growth in CTTYE broth and development in in MC7 starvation buffer were determined CG(C/G)AG], in the σsee Fig. . To exam54 promoter elements of the MXAN1286, MXAN1579 and MXAN3778 natural product loci and that the tandem repeats that we identified in the σ54 promoter elements are Nla28 binding sites.Electrophoretic mobility shift assays (EMSAs) were used to confirm that the MXAN1286, MXAN1579, and MXAN3778 natural product promoters are targets of the Nla28 EBP. In particular, we used EMSAs to determine whether the purified DNA binding domain of Nla28 (Nla28-DBD) is capable of binding a fragment of the MXAN1286 promoter, MXAN1579 promoter, and MXAN3778 promoter. Each promoter fragment, which corresponded to DNA upstream of − 12 and − 24-bp regions, contained a putative binding site for a Nla28 dimer. As shown in Fig. M. xanthus EBPs (HsfA and MXAN4899) are capable of binding to fragments of natural product gene promoters40. With this information and the preliminary data from Nla28 studies in mind, Stevens et al. asked if the σ54 system might be a common regulator natural product genes38. Namely, a bioinformatics analysis was used to search for putative σ54 promoters in the sequences of 180 PK or NRP gene clusters from 58 bacterial species. The results, which revealed that about 70% of natural product gene clusters have at least one putative σ54 promoter, suggested that the σ54 system might indeed be a common regulator of natural product genes.For decades, bacterial-derived PK and NRP natural products have been a crucial source of therapeutic agents such as antibiotics and yet little information about the regulation of these genes has been uncovered. In a notable study in 2012, Volz et al. showed that two M. xanthus is an excellent system to study natural product gene regulation, as this bacterium is a major producer of PKs and NRPs and over 80 putative σ54 promoters were identified in the PK and NRP gene clusters of strain DK162238  contain bona fide σ54 promoter elements and that the σ54 promoters are targets of the EBP Nla28. Our results also indicate that the σ54 promoters are crucial for both growth-related and developmental activities. We propose that the fourth natural product promoter region that we characterized, MXAN1603, also contains a σ54 promoter. However, it seems likely that the MXAN1603 promoter region contains an unidentified promoter element that is responsible for the majority of the observed developmental and growth-related activities and the σ54-type promoter only makes a minor contribution to these activities.Our in vivo and in vitro data indicate that three of the 55. Nla28 begins modulating gene expression in the early stages of starvation-induced fruiting body development39, which led to the suggestion that the Nla28/Nla28S signal transduction system might be a general regulator of starvation-induced or stress-responsive genes55. The findings presented here are consistent with this idea. In particular, the MXAN1286, MXAN1579, and MXAN3778 σ54 promoters are induced in the early-middle stages of fruiting body development and the developmental activities of these promoters are dependent on Nla28 ppGpp accumulates when M. xanthus transitions from exponential growth to stationary phase, since this seems to be the case in other bacterial species58. In addition, bacterial natural product gene expression was previously linked to (p)ppGpp accumulation59 and stringent response-associated genes such as relA and spoT have been linked to synthesis of bacterial natural products60.The above results point to nutrient depletion/starvation as the potential trigger for Nla28/Nla28S-mediated activation of the MXAN1286, MXAN1579 and MXAN3778 promoters. We suggest that the stringent response and accumulation of (p)ppGpp might also be a key inducing factor, as accumulation of this starvation signal is known to initiate 54 promoters that we characterized are located within the coding sequences of genes (intragenic) and not in intergenic regions , the σ54 promoter is located in the coding sequence of an upstream gene . The question that arises is, why would a σ54 promoter be located so far upstream of the single gene or operon whose transcription it aims to regulate? One possibility is that the natural product σ54 promoter is positioned at a distant location to produce a relatively long 5′ untranslated region (UTR) in the mRNA and that the 5′ UTR provides an additional layer of regulation for natural product gene expression. We should note that a substantial number of the internal σ54 promoters are predicted to yield relatively long 5′ UTRs as well. Why would many natural product genes need multiple levels of regulation? Natural product synthesis is often energy expensive and many natural products are toxic at high levels; hence, tight and signal-responsive regulation of natural product gene expression might be important in many cases.A second and relatively large group of putative natural product σ54-mediated regulation of natural product genes and other types of bacterial genes will require researchers to go beyond predicting where σ54 promoters are located. Indeed, we argue that the work of confirming putative σ54 promoters and analyzing the mRNAs under σ54 control is crucial, as the results will help address a number of long-held assumptions about bacterial promoters and bacterial gene regulation in general.Of course, addressing questions about the role and mechanism of σSupplementary Information


In human, UGT2B17 gene expression is detected in hepatic tissues as well as in steroid target tissues.2 Nonetheless, shown elegantly in the study by Allain et al.1 is that in lymphoid cell models and in CLL patients occurs a lineage-inappropriate UGT2B17 overexpression, predominantly mediated from the alternative UGT2B17_n2 transcript that comprises the additional exon 1c, extending the 5′-untranslated region. Lower UGT2B17 expression from the alternative UGT2B17_n3 and n4 transcripts, comprising the alternative 1b exon, was also reported. Of note, all novel transcripts identified encode a functional UGT2B17 protein.1 Since UGT2B17 represents a main conjugating enzyme for fludarabine glucuronidation and consequently UGT2B17 misexpression could affect primary response to first-line treatment with fludarabine in CLL patients, we fully agree with Allain et al.1 that the molecular machinery underlying high UGT2B17 expression in lymphoid cells warrants further clarification. Herein, we present evidence that both UGT2B17 exons 1b and 1c were evolutionarily derived from endogenous-retrovirus (ERV) sequences and that UGT2B17 overexpression in B cells of CLL patients is driven by the in cis aberrant activation of long terminal repeats (LTRs) of the ERV1 family.We read with great interest the article by Allain et al.3 The typical genomic structure of a retrovirus consists of a gag, pro, pol, and env genes flanked by two LTRs that naturally comprise core transcription regulatory elements and transcription factor (TF) binding sites.3 The majority of ERVs in human genome has undergone recombination events between the 5′- and 3′-LTRs, resulting in solitary LTRs.4 Interestingly, many solitary LTRs have preserved their ancestral promoter function and, when positioned in the sense orientation of an adjacent host gene, could drive ectopic transcription initiation.4 Accordingly, because epigenetic silencing represents the predominant path to ERV transcription inactivation, alterations of the epigenetic landscape occurring during cellular transformation events could likely lead to ERV derepression with a significant impact on host gene transcriptional networks, a process designated as onco-exaptation.5ERVs represent heritable provirus insertions into the host genome DNA, remnants of exogenous infectious retroviruses.6 of the genomic segment comprising UGT2B17 exon 1c and its non-canonical proximal promoter sequence reveals that these genetic elements are comprised within a Harlequin-int LTR/ERV1 sequence, of sense orientation to UGT2B17 gene , which, among 74 cell types examined, is functional specifically in the lymphoid cell lines -regulated genes was documented, subsequently pointing to NF-κB as a key regulatory “hub point” that targets the non-canonical promoter and drives UGT2B17 misexpression in CLL cells.TF chromatin immunoprecipitation and sequencing (ChIP-seq) data in 91 cell types from the ENCODE Project10 Subsequently, it would be tempting to deduce that the efficacy of UGT2B17 expression as a prognostic marker of high-risk CLL2 is mechanistically linked to the retroviral origin of its ectopic promoters. Most importantly, since the LTR-driven misexpression of UGT2B17 may impact the effectiveness of fludarabine-based interventions, quantitative PCR-based assays of LTR-driven UGT2B17 ectopic transcription in cancerous B cells would help tailor fludarabine dosage to better stratified CLL patients.It has been shown that the positive selection of novel hypomethylation motifs, which could subsequently allow the epigenetic derepression of ERV transcription, entails co-evolution of genetic sub-clonal complexity in CLL.


The rapid renewal of intestinal epithelium is mediated by a pool of stem cells, located at the bottom of crypts, giving rise to highly proliferative progenitor cells, which in turn differentiate during their migration along the villus. The equilibrium between renewal and differentiation is critical for establishment and maintenance of tissue homeostasis, and is regulated by signaling pathways  and specific transcription factors . Such regulation controls intestinal cell identities by modulating the cellular transcriptome. Recently, chromatin modification and dynamics have been identified as major actors linking signaling pathways and transcriptional regulation in the control of intestinal homeostasis. In this review, we synthesize the many facets of chromatin dynamics involved in controlling intestinal cell fate, such as stemness maintenance, progenitor identity, lineage choice and commitment, and terminal differentiation. In addition, we present recent data underlying the fundamental role of chromatin dynamics in intestinal cell plasticity. Indeed, this plasticity, which includes dedifferentiation processes or the response to environmental cues (like microbiota’s presence or food ingestion), is central for the organ’s physiology. Finally, we discuss the role of chromatin dynamics in the appearance and treatment of diseases caused by deficiencies in the aforementioned mechanisms, such as gastrointestinal cancer, inflammatory bowel disease or irritable bowel syndrome. The intestinal epithelium is entirely renewed every 3 to 5 days. This renewal is mediated by Intestinal Stem Cells (ISCs), which reside at the bottom of proliferative compartments, called “crypts” , 2. The in vivo [The fine regulation of this renewal allows the intestinal epithelium to reach a crucial equilibrium, called “homeostasis”, which is required for essential functions of the tissue, such as being a barrier, or an immune or metabolic regulator. This homeostasis, which has been extensively studied during the last decades, is controlled by signaling pathways , such asin vivo . Althougin vivo –17. Shivin vivo , 18. Morversus erasers), allowing dynamic regulation of these marks. Some modifications can directly affect chromatin structure, others function as signaling events. Their presence or absence is recognized by proteins (readers) containing specific domains, such as bromodomains, PHD domains, chromodomains, etc. …, which mediate regulation of DNA-linked processes, like transcription.Chromatin is a highly dynamic structure involving the association between DNA and proteins (histones and non-histones). Wrapping 146 bp of DNA around a histone octamer (2 H2A/H2B dimers and 1 H3/H4 tetramer) gives rise to a nucleosome, the basic unit of chromatin . ChromatThe role of chromatin modification in cell fate has been extensively studied in embryonic stem cell maintenance and differentiation (reviewed in –23). RecThe dynamics of several chromatin modifications during specification and differentiation of ISCs, in association with transcriptional changes has recently been investigated . The autCdkn2a/b (INK4 locus). Another prominent change is the decreased presence of histone variant H2A.Z at the promoters of 421 of these genes  is important for the proliferation of intestinal cells . Zhao anSI, LPH) and for goblet cells  in cellulo and in vivo. H2A.Z.1 inhibits binding of the transcription factor CDX2 at the promoter of its target genes  effects of each H2A.Z isoform on transcription , 38–40, +-dependent proteins [Histone acetylation consists of adding an acetyl group to lysine residues on histones. By neutralizing the histone’s positive charge, acetylation decreases the interaction between DNA and histones , leadingproteins . HATs arproteins . HDACs aproteins , 43.in vivo of ISC markers (Lgr5 and Olfm4) and loss of ability to form 3D intestinal organoids [Many studies have addressed the role of histone acetylation in intestinal homeostasis by investigating the involvement of HDACs and HATs. Tou and colleagues showed that HDAC1 and HDAC2 are the most highly expressed HDACs in adult intestinal epithelium . Moreoverganoids . This dorganoids , and thirganoids  or β-hydrganoids ). Altogerganoids , 48.in cellulo and in vivo. SAHA treatment upregulates enterocyte-specific genes, such as Sucrase-Isomaltase, in Caco-2/15 cells (an in vitro model of enterocyte differentiation) and in mice [Hdac1 and Hdac2 KO animals [In accordance with this hypothesis, pharmacological HDAC inhibition leads to increased expression of differentiation marker genes  in mice . This re in mice . The HDA animals , indicatAltogether, these results show the essential role of HDAC in intestinal epithelium, especially for the maintenance of cells in an undifferentiated state Fig. .Fig. 3RoThe analysis of acetylation writers, HATs, showed that these enzymes are important for intestinal homeostasis by functioning as transcriptional coactivators for specific transcription factors. The two HATs, p300 and CBP, are highly expressed all along the adult intestinal epithelium, suggesting their involvement in establishing or maintaining the identity of many cell types  Fig. 3C. Anotherin cellulo. PCAF recruitment leads to H3K9 acetylation, RNA Pol II binding and Secretin expression [HATs are also important for the activity of the transcription factor NeuroD. NeuroD is crucial for enteroendocrine cell differentiation, especially by promoting the expression of Secretin , 52. Neupression . These rSurvivin and c-Myc) in cellulo [via histone acetylation at these promoters but also by promoting acetylation of β-Catenin itself, which causes its stabilization and increases its transactivation abilities [in vivo [Another study also revealed the role p300 and CBP play in modulating two TCF4/β-Catenin targets  impairing the interaction between p300 and MYB [+) number and of Lgr5 expression [Plt6 mice also shows that CBP is unable to compensate for the p300 functional deficiency. The intestinal specific KO of the CREB encoding gene leads to decreased proliferative cell number, loss of stemness and increased enteroendocrine and Goblet cell numbers [Finally, another study suggested a role for p300 in mediating activity of two transcription factors in the intestine, MYB and CREB . The aut and MYB . This mupression , a phenopression , 62. The numbers . Howeverin cellulo models. In vivo analyses of HAT functions by deleting HAT-encoding genes in intestinal epithelium, are necessary to confirm and clarify their roles in homeostasis.Altogether, these results demonstrate how HATs, by acting as transcriptional coactivators Fig. , controlOne of the most studied chromatin marks involved in cell fate control is H3K27me3, and Polycomb Repressive Complex 2 (PRC2) functions as its specific writer. H3K27me3 is associated with transcriptional repression, by playing a scaffold role for repressor complexes (such as the PRC1 polycomb complex), or by inhibiting recruitment of activator complexes or deposition of the active mark H3K27ac .HNF1α/CDX2), PRC2 is involved in repressing, in a CDX2/HNF1α-dependent manner, differentiation marker genes expression . The authors hypothesize that PRC2 could be essential for preserving progenitor identity by maintaining the proliferation abilities of these cells   Fig. .in vivo by monitoring loss of KI67 staining in the intestine of mouse KO for Eed (another subunit of this complex) [Cdkn2a cell cycle inhibitor locus (Ink4). This result probably explains the loss of H3K27me3, observed at this locus during enterocyte differentiation in Kazakevych et al.’s study [Eed also induces a loss of ISCs, revealed by diminishing Lgr5 staining, observed by RNA in situ hybridization about 6 weeks after induction of the Eed KO, and by loss of the ability to form organoids [in vivo study [AhCre;Eedloxp/loxp), full recombination requires many successive induction events, and recombination efficiency is lower compared to that observed in Koppens’s work. Lgr5 expression may thus be compensated by wild-type crypts that are probably overactivated [PRC2’s essential role in progenitor cells proliferation was confirmed complex) , 66. The’s study  , but also by triggering their choice towards the absorptive lineage (inhibition of Math1 expression)  Fig. .ATP-dependent chromatin remodeling complexes contain at least one ATPase subunit, which catalyzes the remodeling reaction, and other subunits acting as cofactors to regulate targeting on specific chromatin regions and to modulate activity. Among the different families of remodeling complexes, the SWI/SNF , INO80/SWR and NURD families have been shown to regulate important processes for intestinal cell fate determination.Arid1a, a subunit involved in DNA interaction, leads to loss of ISCs and thus to decreased tissue renewal. Indeed, ARID1a promotes expression of the gene encoding transcription factor SOX9, essential for ISC maintenance [Brg1 gene, encoding the ATPase subunit, also causes a drastic decrease in expression of ISC marker genes  [Brg1 KO likewise leads to a huge increase in secretory cell number, in a Math1–dependent manner [Arid1a KO diminishes secretory cell number via an uncharacterized mechanism [The SWI/SNF complexes contain BRG1 or BRM ATPases . This fantenance . Deletio Ascl2…) . These rt manner . In contechanism . Interesin cellulo expression of β-Catenin- and CDX2-target genes  [BRG1-containing SWI/SNF complex can also act as an effector of transcription factors to regulate gene expression. Indeed, BRG1 interacts with β-Catenin  and CDX2or CDX2) , 73. AltThe Tip60/p400 complex, belonging to the INO80/SWR family is a major regulator of the Wnt pathway and controls proliferation of normal and colorectal cancer cells . MaintenThe Mi-2/NuRD (Nucleosome Remodeling and Deacetylase) complex is a chromatin complex associated with transcriptional repression; it contains subunits with ATP-dependent chromatin remodeling activity (CHD3 or CHD4), or HDAC activity (HDAC1 or HDAC2). The MBD2 subunit allows recruitment of the complex to the DNA through binding to methylated DNA , whereasLect2 gene and inhibits its expression. Lect2 being an endogenous inhibitor of the Wnt pathway [Axin2, CyclinD2…) in a context of high Wnt activity (Apc−/− mice) [In intestinal cells, MBD2/NuRD binds to the DNA-methylated promoter of the  pathway , MBD2/Nu/− mice) .Lgr5, CyclinD1 or c-jun itself, when the JNK pathway is inactive. Activation of the JNK pathway leads to c-jun phosphorylation, loss of MBD3/NuRD binding and thus expression of c-jun target genes. This mechanism is essential for controlling proliferation in intestinal crypts but has no effect on secretory differentiation [Notch1 expression [In addition, Aguilera and collaborators found that MBD3 allows interaction of NuRD with the transcription factor c-jun in intestinal cells . Throughntiation . Finallypression  and to eAltogether, these results  is a DNA mark known to repress transcription when it occurs at promoters enriched in CpG (CpG islands). It is very stable and can be transmitted through somatic cell divisions. However, in specific context, this mark can be dynamic in order to regulate biological processes . For exade novo DNA methylation [DNA methylation is deposited by DNMT (DNA methyltransferase) enzymes, with DNMT1 as the methyltransferase responsible for maintaining DNA methylation during cell division, and DNMT3 proteins mediating hylation . Removalhylation .Lph, Alpi or Krt20, and an increase at enhancers of ISC genes, such as Olfm4. The intestine-specific knock-out of Dnmt1 leads to a slight increase of crypt size and Olfm4 expression, suggesting that DNA methylation could participate in controlling stemness features. The Dnmt1 KO also leads to overexpression of Dnmt3b, and not Dnmt3a, in the mouse crypts [Dnmt1 and Dnmt3b leads to a huge decrease in mice survival, which is not observed with both single KOs. This mortality is associated with the loss of proliferative cells (Ki67+ cells) and of tissue renewal [Dnmt3b overexpression compensates for the loss of Dnmt1 to preserve progenitor proliferation and tissue maintenance.Two studies showed that DNA methylation is mostly static in intestinal epithelium . The only DNA methylation differences observed during ISC differentiation are the loss of this mark at enhancers of a few genes more highly expressed in differentiated cells , 83. Anoe crypts . The dou renewal . These rAltogether, these results show that DNMT1 and DNA methylation are essential for restriction of stemness in the lower part of the crypt and suggest that these actors are important for progenitor proliferation Fig. . The anaIn addition to its essential role in the setup of intestinal cell identity, as discussed in the previous section, chromatin is also involved in the control of tissue plasticity. The term “plasticity” describes, in this review, all changes in cell identity which are not directly linked to tissue homeostasis, but induced following environmental modifications or stresses that trigger an adaptive response.The choice between absorptive and secretory lineages takes place in progenitor cells. It is controlled by the Notch pathway thanks to a phenomenon called “lateral inhibition”. Indeed, in the Transient Amplifying (TA) compartment , a cell with an active Notch pathway, which corresponds to an absorptive progenitor, inhibits the Notch pathway in its neighboring cells. These cells then express the transcription factor MATH1 to acquire features of secretory lineage progenitors. This phenomenon is coupled with proliferation arrest in these secretory progenitors. In contrast, absorptive progenitors continue to divide and give rise to enterocytes –89. ThisMath1-KO mice [Myc, Mcm2…) in Ent-pro, and the overexpression of genes essential for the secretory lineage setup in Sec-pro  [Kim and collaborators collected absorptive progenitors (Ent-pro) from -KO mice , thus wi-KO mice . They co-KO mice . The diseurog3…) .MATH1 expression on cell transcriptome, in cells which do not express Math1 initially and which have a chromatin landscape different than Ent-pro or Sec-pro cells .However, this binary choice between absorptive or secretory fate can be reversed, if needed, in case of alteration in one lineage . Indeed,Dll1+ cells (secretory progenitor cells) are able to replace Lgr5+ ISCs when these cells are lost, and to give rise to all epithelial cell types [Another phenomenon of intestinal plasticity is dedifferentiation of intestinal cells to acquire previously lost ISC features. This process is essential for maintaining the epithelium integrity and thus organism survival , 94. Thell types . Mechanill types .Jadhav and colleagues  analyzedLgr5+ ISC loss [The role of chromatin modifiers during the dedifferentiation process was addressed in only one study. Chiacchiera and colleagues demonstrated that PRC2, essential for stemness and progenitor identity in homeostatic conditions, is essential for the dedifferentiation abilities of progenitors after ISC loss .Altogether, these results show that chromatin dynamics are involved in the tissue response to stress (here ISC loss), and that the pre-existing chromatin landscape within each cell type could orientate the choice of the cell type to start dedifferentiation Fig. .Fig. 4ChChromatin is undoubtedly essential for the intestine’s adaptation to changes in the outer compartment. Indeed, it was shown that chromatin regulation is required for the epithelium response to commensal microbiota colonization Fig. . AnalysiIntestinal epithelium’s major role is to serve as a front line barrier between the organism and its external environment. This tissue must therefore permanently adapt to environmental cues, such as bacterial colonization. Many studies analyze the host-bacteria relationship, with an emphasis on immune response. However, important studies also show that interaction between commensal microbiota and intestinal epithelium leads to important modifications in the intestinal cell transcriptome , including genes other than immunity genes , 104. HoChromatin modifications, thanks to their highly dynamic properties, are obvious candidates for mediating response to bacterial colonization. Despite extensive transcriptome modification, little or no global chromatin changes in the presence or absence of microbiota was observed by analyzing DNA accessibility by DHS-seq , 105. ThOnly one analysis demonstrates the causal role of chromatin in transcriptome regulation in response to microbiota colonization . The autIn this review, we highlight important data uncovering the role of chromatin dynamics in the setup and control of intestinal epithelial homeostasis. Transcriptional programs are central elements in cell identity, and important modifications in gene expression are required for ISC differentiation. By their prominent role in transcription control, chromatin modifications and modifiers are major regulators of these processes, as are transcription factors and signaling pathways Figs.  and 3. IOne of the remaining issues is how the dynamics of these marks and/or modifiers is regulated with respect to signaling pathways. Even though regulation of H2A.Z dynamics is controlled, at least in part, by the Wnt signaling pathway  As described in this review, all data so far point to a function for chromatin modification in intestine homeostasis, via its role in controlling gene regulation. However, chromatin modifications also regulate other processes requiring access to the DNA double helix, such as DNA repair or replication, two processes known to be critical for intestine homeostasis and physiopathology. For example, the control of DNA replication licensing  is esseRecent data also indicate that chromatin modifications may be the basis of important epigenetic information in the gut. For example, promoters of the 628 genes comprising the adult epithelium signature  are marked by the active mark H3K4me3 in ISCs as well as enterocytes . This caEpigenetic mechanisms are also known to be important for the organism’s response to the environment; in the intestinal epithelium, they could play a major role in the physiopathology of the organ, in particular for metabolic diseases, such as obesity. Indeed, more than 80% of obese people regain weight after a restriction diet, in a mechanism known as “Yo-yo dieting” . This adTo what extent such epigenetic mechanisms relying on chromatin modifications are involved in the intestine physiopathology is clearly an open question, whose answer may have important consequences for the therapeutic strategies to fight against metabolic diseases.Chromatin dynamics is regulated by enzymes which, through their action on chromatin and thus on epigenetic information, may have long term consequences on gene expression. As a result, chromatin modifiers are promising therapeutic targets for many diseases, in particular - but not only - in cancer. Much effort has been made to identify molecules affecting their activities, called “epigenetic drugs”, and many of them are currently used or tested in clinical trials. Given the prominent role of chromatin modifiers in intestine homeostasis, whether such molecules have interesting therapeutic potential in intestine diseases is clearly worth investigating.Lgr5. However, tumor growth resumed after the treatment, and the authors hypothesized that this reappearance was due to dedifferentiation of tumor cells, as already observed in healthy intestine, leading to recovery of tumoral growth. An innovative strategy for the fight against gut cancer, but also in others organs, would be to prevent the dedifferentiation process in addition to CSC ablation [For example, understanding how chromatin structure affects cellular plasticity may provide important insights for cancer treatment. Indeed, cancer stem cells (CSCs) are often defined as “fuel” for the tumor, allowing tumor growth and the appearance of differentiated tumor cells . Thus, Cablation . Dediffeablation .Muc2 Knock-Out leads to spontaneous appearance of chronic colitis [Epigenetic drugs might also be considered for their therapeutic potential against immune-linked disease, such as Intestinal Bowel Disease (IBD), Crohn’s Disease or Ulcerative Colitis. IBD describes idiopathic diseases involving extensive inflammation of intestinal mucosa, due to a strong immune response, which leads to destruction of intestinal structure and function. In addition, to its direct role in controlling immune response (reviewed in ), chroma colitis . How chr colitis . Thanks Finally, epigenetic drugs could also be valuable in metabolic diseases associated with digestive enzymes misregulation, such as lactose intolerance. Indeed, by targeting chromatin modifiers, epigenetic drugs could restore normal expression of digestive enzymes. They could be particularly valuable for treating Irritable Bowel Syndrome (IBS), which is associated to lactose intolerance and other intestinal dysfunctions  and for which the only available treatment, probiotics, has alone a relatively low efficiency .In this review, we have shown that chromatin dynamics plays an essential role in the homeostasis of the intestinal epithelium, by controlling the establishment and maintenance of cell identities on one hand, but also by playing an essential role in the plasticity of these cells in response to various stresses on the other hand. Finally, these fundamental roles of chromatin modifications could make them major targets in the fight against intestinal diseases such as cancers, IBD and IBS.


Mesenchymal stem cells (MSCs) present in multiple tissues can self-renew and differentiate into multiple lineages including the bone, cartilage, muscle, cardiac tissue, and connective tissue. Key events, including cell proliferation, lineage commitment, and MSC differentiation, are ensured by precise gene expression regulation. ATP-dependent chromatin alteration is one form of epigenetic modifications that can regulate the transcriptional level of specific genes by utilizing the energy from ATP hydrolysis to reorganize chromatin structure. ATP-dependent chromatin remodeling complexes consist of a variety of subunits that together perform multiple functions in self-renewal and lineage specification. This review highlights the important role of ATP-dependent chromatin remodeling complexes and their different subunits in modulating MSC fate determination and discusses the proposed mechanisms by which ATP-dependent chromatin remodelers function. Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are multipotent stromal cells that can differentiate into a variety of mesoderm cell types, including osteoblasts, chondrocytes, myocytes, and adipocytes –3. MSCs Specific gene expression programs, which depend largely on the organization of the associated chromatin, define a variety of cellular processes like differentiation, proliferation, and stemness . ATP-depATP-dependent chromatin remodeling complexes mainly consist of an ATPase and multiple subunits. The ATPase subunit hydrolyzes ATP, while the associated subunits regulate ATPase catalytic activity and genome binding. Therefore, different combinations of ATPase and associated subunits result in various chromatin remodeling complexes with different functions , 18. AllThe interaction of ATP-dependent chromatin remodeling complexes and histone acetyltransferases (HATs) in gene transcription has been demonstrated. For example, the yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex cooperates with the SWI/SNF complex via the cell wall integrity pathway for mandatory nucleosome displacement, which is essential for full gene expression . It has −/−Lsh mouse embryos, which lack DNA methylation from transposons, centromeric repeats, and several gene promoters, can reestablish DNA methylation and silence the misregulated genes with LSH reexpression [ATP-dependent chromatin remodeling complex can also cooperate with DNA methylation in various cellular processes. Several ATP-dependent chromatin remodeling enzymes, including the mammalian SNF2 family and ATPases ATRX and LSH (HELLS), are involved in DNA methylation at the fifth carbon of cytosine (5mC), which is an abundant epigenetic modification in vertebrate genomes –28. The pression . The intβ-actin are shared by the complexes [The switch/sucrose nonfermentable (SWI/SNF) complex  is composed of at least 15 different subunits that invariably include a core ATPase of either Brm (Brahma) or Brg1 (Brahma-related gene 1) that can provide the necessary energy to the complex for nucleosome remodeling activity . BRM andomplexes . The BAFomplexes .SChLAP1 and SMARCB1/SNF5. SChLAP1 is an aberrantly expressed lncRNA identified in prostate cancer tissues [SChLAP1 and SNF5 in human prostate cells, while SChLAP1 overexpression resulted in decreased SWI/SNF occupancy genome-wide [Targeting of SWI/SNF to genomic sites is partly modulated via interactions of its associated subunits with transcription factors, histone modifications, and noncoding RNAs (ncRNAs), which were recently described. One example is the interaction between long ncRNA (lncRNA)  tissues , while S tissues . Direct ome-wide .BRG1 overexpression induces BRG1 occupancy at the NANOG promoter, thereby increasing chromatin compaction and recruiting HDACs [in vitro and in vivo [SWI/SNF is critical for stem cell self-renewal and cell differentiation. BRG1, BAF47, and BAF155 depletion can impair the survival of totipotent cells and cause peri-implantation embryonic lethality in mice –38. BRG1ng HDACs . Further in vivo .ex vivo osteoblast cultures and in skeletal tissues of mouse embryos [β; thus, BRG1 can induce OC expression regulated by RNA polymerase II [in vivo enhanced by p38 to form a complex that is transcriptionally active [β-LAP∗, which can bind to the Ric-8B promoter. This leads to Ric-8B expression downregulation in differentiating osteoblasts [Baf180/Baf200/Brd7 largely compromised the osteogenesis and osteolineage gene expression, while Baf180 loss was found to impair MSC ossification in vivo [Several studies have indicated that SWI/SNF is required for osteogenic induction . Brg1 ex embryos . This exerase II . Brg1 any active . In addieoblasts . Several in vivo . By comp in vivo . FGF rec in vivo .EΒPα, C/EΒPβ, C/EBPδ, and PPΑRγ2 to induce uncommitted fibroblasts into adipocytes [γ2 and C/EBPβ [γ promoter [γ and LPL [γ2 and GLUT4 [ATP-dependent chromatin remodeling is also essential in promoter activation during adipogenic differentiation of MSCs. BRG1 and hBRM can cooperate with C/ipocytes , 53. In d C/EBPβ . CARM1 opromoter –57. In M and LPL . Moreove and LPL . Further and LPL . miRNA37nd GLUT4 . This inα and RB family proteins [α target genes while stimulating β-oxidation of fat in hepatocytes [α, which inhibit cell cycle reentry and enhance differentiation, respectively [SWI/SNF is also important for hepatocyte differentiation. During early liver development, BRM or BRG1 can decrease the expression of tryptophan oxygenase, a gene specific to the late stage . During proteins . BAF60A atocytes . Moreoveatocytes . On the ectively .Brg1 deletion in the developing heart results in dysregulated cardiac gene expression and severe cardiac morphogenesis anomalies. By mediating remodeling of promoter chromatin and BRG1 recruitment, BAF250A regulates the expression of Mef2c, Nkx2-5, and Bmp10 during the differentiation of cardiac progenitor cells into beating cardiomyocytes [BRG1 is essential for regulating gene expression and the differentiation of cardiomyocytes . In a momyocytes . In addimyocytes . BAF250Amyocytes . Moreovemyocytes . BAF60c myocytes . Furthermyocytes , 73.Baf45a and/or Baf53a mediated by short hairpin RNA leads to decreased proliferation, while Baf45a overexpression improves neural progenitor cell mitosis. The SWI/SNF complex specific to neural progenitors regulates Notch and Shh signaling to promote proliferation and maintain the cells in the transition state from progenitors to postmitotic neurons [MSCs could also differentiate into astrocytes or neurons when cultured with retinoic acid and neurotrophic factors derived from the brain . Studies neurons . Meanwhi neurons . Downreg neurons .Snf2h, which is critical for early embryonic development, is ubiquitous in various tissue types, while the Snf2l expression is restricted to the brain and postnatal reproductive tissues [Snf2h loss leads to lethality, while mice with a Snf2l deficiency can still survive [ISWI complexes contain one of two conserved ATPase SMARCA5 (SNF2H) or SMARCA1 (SNF2L) along with two to four associated subunits . The exp tissues , 81. The survive . Further survive .Snf2h deletion compromises the proliferation of granule neuron progenitors and Purkinje cells with increased cell death, which leads to defects in postnatal neural maturation [Foxg1 by binding to its promoter region. Therefore, SNF2L is required to maintain the balance between proliferation and differentiation of neural progenitors during brain development. This can be confirmed by the increased proliferation and self-renewal of neural progenitors in conditional Snf2l mutants accompanied by increased FOXG1 expression [ISWI complexes containing either SNF2H or SNF2L are critical for ectoderm-derived lineage development , 84, 85.pression .Egr1, a gene specific to thymocyte maturation [Bptf mutants were not able to differentiate any ectoderm, endoderm, or mesoderm tissue types, suggesting the important role of BPTF in germ layer formation. In addition, Bptf mutants failed to form distal ventral endoderm, and the expression of SMAD-responsive genes depended upon BPTF, suggesting that NURF functions as a transcription cofactor for SMAD [Snf2l mutant mice responded differently under gonadotropin induction, and thus, they yielded significantly fewer eggs and exhibited fewer secondary follicles compared to control WT mice. The study also indicated that Fgl2 transcription, which can encode a prothrombinase for mouse reproduction to mediate folliculogenesis, is regulated by Snf2l [ISWI is also essential in mesoderm-derived lineage differentiation. The nucleosome remodeling factor (NURF) complex, which includes SNF2L-containing ISWI, plays an important role in erythropoiesis . On the turation . Bptf mufor SMAD . SNF2L ifor SMAD , 89. SnfDrosophila ISWI or BPTF leads to repressed histone H1 levels and a general male X chromosome decondensation [Many genes are heterochromatinized upon differentiation, and thus, regularly spaced nucleosomes are needed for higher order compaction. The ISWI-containing chromatin remodeling complex ACF1 is required for nucleosome assembly. In the meantime, centromeric chromatin is assembled by RSF1, while heterochromatin formation is regulated by NoRC; thus, rDNA repeats can be silenced –92. Defiensation , 94. Theensation .Taken together, ISWI complexes have been shown to have specific roles in cell proliferation, differentiation, or maturation . SNF2H-cNine chromodomain helicase DNA-binding (CHD) proteins (CHD1-9), which can either function alone or cooperate with other proteins to form the complexes, constitute a CHD subfamily. Among them, different CHD complexes have distinct roles in early development and cell lineage differentiation.CHD1 has been shown to be required for maintaining the self-renewal ability and pluripotency of embryonic stem cells , 97. CHDChd8 in neural progenitor cells, the neural developmental genes containing those ASD-related genes were downregulated [In the developing brain, the NuRD complex, which contains CHD4, is required for synapse formation . This coegulated .Cdkn1a, which is a cell cycle inhibitor gene. Therefore, the disruption phenotype in the FOG2-NuRD interaction can be rescued through Cdkn1a deletion. Furthermore, CHD7 is involved in transcription activity in various heart development processes. Chd7 mutant mice exhibited CHARGE syndrome in cardiac aspects [CHD complexes also play an important role in heart development. A NuRD complex containing CHD3 or CHD4 is involved in the proliferation of cardiomyocytes by interacting with the transcription factor FOG2 . Once th aspects , 109 whi aspects .In a well-established MSC model with the induction of osteoblast lineage differentiation, CHD1 is essential for osteogenesis by regulating the transcriptional program of osteoblast differentiation, specifically at later stages. Moreover, CHD1 depletion was shown to reduce the induction of lineage-specific genes in adipocyte differentiation, indicating that CHD1 has a more general role in regulating transcriptional programs related to MSC differentiation . CHD7 isOverall, CHD complexes function to regulate transcription or suppression of different genes and induce various lineage differentiations in MSCs . This prThe ATPase subunits of INO80/SWR are another subfamily of ATP-dependent chromatin remodeling complexes that exhibit a conserved insertion in the ATPase/helicase domain. This is required for the interaction between RVB1/RVB2 helicase and these complexes . The INOIno80 mutation in spermatogonia before meiosis led to reduced synapse formation and double-strand break defects [Hamlet (ZNHIT1), a SRCAP subunit, is required for muscle differentiation [Hamlet is phosphorylated at the promoter region of Myog, a muscle-specific transcription factor gene. H2A.Z is then recruited to phosphorylated P18Hamlet/SRCAP, forming the chromatin structure necessary for Myog transcription.MSCs transfected with siRNAs targeting INO80 resulted in an impaired mineral deposition in osteogenic induction conditions, and the implanted mice with INO80-silencing MSCs also exhibited decreased bone formation. This suggests the essential role of the INO80 complex in MSC osteogenic differentiation and its potential application in tissue engineering in the clinic and osteoporosis treatment  Table 1Table 1.  defects , 124. P4 defects , 126. In defects . Moreoventiation . P18HamlOver the past years, MSCs have become the focus of intense interest. Thus, they have been investigated for their capacities for self-renewal and lineage specification. The application of MSCs has been considered as a solution for the poor ability of adult tissue regeneration and a potential treatment for human diseases. Gene expression programs work at the chromatin level, so the organization of chromatin is essential in both normal and malignant development and tissue regeneration. We propose that the efficiency of differentiation of MSCs into a variety of cell types will be enhanced by modifying the composition of ATP-dependent chromatin remodeling complexes. As mentioned above, ATP-dependent chromatin remodeling complexes catalyze critical functions in self-renewal and multilineage differentiation of MSCs. In addition, the role of ATP-dependent chromatin remodeling in embryonic stem cells in diverse tissue types also raises the possibility that it may have similar functions in MSCs. The diversity of combinations of multiple subunits has specific functions in chromatin remodelers. For example, a specific combination plays an important role in ATP-dependent chromatin remodeling in differentiated cells, while another combination is crucial for some tissue progenitors. Moreover, ATP-dependent chromatin remodelers can regulate specific transcription in various cell types or with different transcriptional programs in the same cell type depending on the collaboration of these chromatin remodelers with histone-modifying complexes that can induce the binding of histone marks to regulatory sites. The insights of the ATP-dependent chromatin remodeling complexes and their roles in MSC fate determination will provide potential strategies for regeneration and cell-based tissue-engineering therapy.


Core histone variants, such as H2A.X and H3.3, serve specialized roles in chromatin processes that depend on the genomic distributions and amino acid sequence differences of the variant proteins. Modifications of these variants alter interactions with other chromatin components and thus the protein’s functions. These inferences add to the growing arsenal of evidence against the older generic view of those linker histones as redundant repressors. Furthermore, certain modifications of specific H1 variants can confer distinct roles. On the one hand, it has been reported that the phosphorylation of H1 results in its release from chromatin and the subsequent transcription of HIV-1 genes. On the other hand, recent evidence indicates that phosphorylated H1 may in fact be associated with active promoters. This conflict suggests that different H1 isoforms and modified versions of these variants are not redundant when together but may play distinct functional roles. Here, we provide the first genome-wide evidence that when phosphorylated, the H1.4 variant remains associated with active promoters and may even play a role in transcription activation. Using novel, highly specific antibodies, we generated the first genome-wide view of the H1.4 isoform phosphorylated at serine 187 (pS187-H1.4) in estradiol-inducible MCF7 cells. We observe that pS187-H1.4 is enriched primarily at the transcription start sites (TSSs) of genes activated by estradiol treatment and depleted from those that are repressed. We also show that pS187-H1.4 associates with ‘early estrogen response’ genes and stably interacts with RNAPII. Based on the observations presented here, we propose that phosphorylation at S187 by CDK9 represents an early event required for gene activation. This event may also be involved in the release of promoter-proximal polymerases to begin elongation by interacting directly with the polymerase or other parts of the transcription machinery. Although we focused on estrogen-responsive genes, taking into account previous evidence of H1.4′s enrichment of promoters of pluripotency genes, and its involvement with rDNA activation, we propose that H1.4 phosphorylation for gene activation may be a more global observation. Histone H1 and the linker histones are members of a family of lysine-rich proteins that have classically been identified as structural components, playing a role in the formation of higher order chromatin structures associated with inaccessibility to transcriptional machinery . HoweverThe H1 family is evolutionarily diverse, and present in non-allelic amino acid sequence-variant forms in many metazoans. Humans express eleven H1 variants; seven variants are ‘somatic’, whereas the rest are selectively expressed in germline tissues. Five of the seven somatic variants—H1.1, H1.2, H1.3, H1.4 and H1.5—are replication-dependent and are expressed predominantly during the S-phase of the cell cycle. These variants contain a more conserved amino acid sequence as compared to the replication-independent variants, namely H1.0 and H1.X. N-terminal domain (NTD) and a longer C-terminal domain (CTD). The GD sequence is highly conserved amongst species, but there are extensive differences in the NTD and the CTD [Metazoan H1 variants share a common tripartite structure containing a central globular domain (GD) that is flanked by a shorter  the CTD ,9. Fluor the CTD .L)/arginine (R)) were the predominant sites of phosphorylation. These sites are also phosphorylated during mitosis, but during that time, threonine-containing CDK sites (TPXZ) and some non-CDK motifs are also phosphorylated [Like core histones, H1 CTDs and NTDs undergo several types of posttranslational modifications, including methylation, acetylation and phosphorylation. Based on older chromatography reports and more recent mass spectrometry data, phosphorylation is observed to be the most abundant modification, progressively increasing during the cell cycle and transiently peaking at mitosis ,12,13. Torylated ,14.Several studies have suggested that the phosphorylation event changes how H1 interacts with the chromatin. While some studies suggest that H1 phosphorylation results in its complete depletion from the chromatin ,15, otheEarlier studies from our lab, conducted on HeLa S3 cells, identified the predominant interphase phosphorylation sites in H1.2 and H1.4 as H1.2-S173, H1.4-S172 and H1.4-S187 . The accHere, we provide the first genome-wide view of pS187-H1.4 dynamics using an inducible system—specifically, estradiol-responsive MCF7 cells. Using an affinity purified version of the same pS187-H1.4 antibody, we show that pS187-H1.4 is strongly associated with the promoters of active genes. Further, we show that these ‘active’ signals are quenched by a known CDK9 inhibitor, Flavopiridol. We combined pS187-H1.4 ChIP-sequencing data in conjunction with publicly available data for RNA Polymerase II (RNAP II), and Global Run-On Sequencing (GRO-seq) data derived via a similar system, to further analyze their correlation. We showed that pS187-H1.4 stably binds RNAPII and co-localizes with active RNAP II peaks, suggesting a functional interaction between the two proteins. In addition, these signals were corroborated by a GRO-seq expression dataset to show that the active pS187-H1.4 signals were seen at the promoters of genes undergoing transcriptional elongation.Taken together, these data provide evidence for a more nuanced role of H1 and put forth the possibility of a new layer of regulation within the accepted model of transcriptional activation.We previously generated a collection of unique, highly specific antisera using synthetic phospho-peptides and recombinant proteins and demonstrated their ability to recognize phosphorylation at single sites ,19. ThesAlthough these antisera are highly specific, their efficiency for high throughput studies was limited. Therefore, for this study, we purified pS187-H1.4 and pan-H1.4 antisera using a modified affinity chromatography approach to generate a highly efficient set of H1 antibodies. The purified pS187 H1.4 antibody was re-tested for specificity in accordance with ENCODE guidelines, as shown in http://homer.ucsd.edu) [We used the purified pS187-H1.4 and pan-H1.4 antibodies to perform chromatin immunoprecipitation (ChIP) on MCF7 cells to reveal the genome-wide distribution of all versions of the H1.4 protein (pan-H1.4) and the specifically phosphorylated version (pS187-H1.4). Peaks generated from the sequencing data were then associated with their nearest genes. We noted an enrichment of pS187-H1.4 ChIP peaks around the promoter regions A. The ascsd.edu) . In stricsd.edu) , providiIn order to further characterize the status of the promoters associated with pS187-H1.4 peaks, we used H3K4me3 ChIP-Seq data from a separate study conducted on MCF7 cells . The H3KThis alignment also revealed that pS187-H1.4 peaks align with the ‘dip’ between paired H3K4me3 peaks  AccessiC , which is usually found to be proximal to the promoters of genes that are transcriptionally ready or actively transcribed. We used MCF7 ChIP- seq data from a different study  AccessiA. This cIn order to confirm the physical interaction of the pS187H1.4 with RNA pol II in our estradiol inducible MCF7 system, we performed native-ChIP, which does not rely on formaldehyde cross-linking, thereby providing an accurate and unbiased view of protein–protein interactions. Further, to ensure that the interaction between the RNAPII and the pS187H1.4 is within a very close range (~1–2 nucleosomes), we used micrococcal nuclease digestion to obtain a majority of mononuclesomes C. FollowTaken together, these data show that the pS187-H1.4 peaks have a distinct pattern of distribution and strong association with ‘active’ promoters. In order to better understand the genome-wide dynamics of pS187-H1.4 at the promoters, we sought to further explore the pS187-H1.4 binding in a transcription-activating estradiol-inducible system.To ask whether pS187-H1.4 binds to promoters as they are activated, we used the purified pS187-H1.4 and pan-H1.4 antibodies to perform chromatin immunoprecipitation (ChIP) on MCF7 cells treated with 20 nM 17β-Estradiol (E2) for 30 min. This treatment is sufficient to ‘activate’ the early estrogen-regulated network of genes, following 72 h of hormone starvation .The estradiol response can vary in different cell lines to the point where some genes may be oppositely regulated . For exaWe conducted ChIP-seq in both untreated and E2-treated MCF7 cells with pS187-H1.4 and pan-H1.4 antisera, and processed the data as described in the methods section. We then used the peaks from pan-H1.4 and pS187-H1.4 before and after E2 treatment to generate metagenome profiles to visualize their distribution across the genome with respect to a normalized gene . We aligWe focused our attention on promoter regions specifically in further analyses. First, we plotted the average pS187-H1.4 signal before and after E2 treatment, focused on the 3000 bp regions upstream and downstream of genome-wide TSSs A. This pIn comparisons between ChIP in E2-treated and untreated MCF7 cells, we found 727 pS187-H1.4 differential peaks with at least a twofold increase in normalized mapped reads. We visualized this increase using an aggregate plot averaging the pS187-H1.4 signal across all differential peaks, to reveal a strong preferential enrichment at the promoter , which when used at 10 nM and for a short treatment, in this case, 1 h, is specific to CDK9 ,39,40. Ahttp://great.stanford.edu) to reveal the correlation of the nearest genes 1 Kb upstream and downstream of the E2-induced pS187-H1.4 peaks for previously established functional annotations. The top ten most enriched annotations from the MSigDB perturbation category are listed with their enrichment p-values in While the ‘metagenome’ profile and aggregate plots detect patterns of enrichment across the genome, they average the architecture of protein binding across all genes, and thus may obscure critical differences at the level of individual, or subsets of individual, loci. We used the GREAT platform (FLOT1) gene from the list of genes associated with the 2X pS187-H.14 differential peaks. Here, we see that the FLOT1 promoter is clustered very near the promoter of neighboring gene, IER3. Both promoters show a basal level of pS187-H1.4 signal in untreated cells; after E2 stimulation, pS187-H1.4 ChIP signals were markedly increased. In addition to this increase in pS187-H1.4 signal after estradiol treatment, we also observed a clear overlap of the pS187-H1.4 signal with that of H3K4me3, as was seen genome-wide . Following hormone depletion, the cells were induced with 20 nM beta-estradiol (E2) dissolved in ethanol for 30 min before harvesting. In the case of drug treatments, the cells were pre-treated with Flavopiridol  for 1 h before E2 induction to selectively inhibit CDK9.MCF7 cells, a kind gift from the Katzenellenbogen lab (University of Illinois at Urban Champaign) were grown in RPMI 1640 media supplemented with 5% fetal bovine serum (FBS) and 1% Penicillin-streptomycin, and were subcultured with a 0.25% trypsin-EDTA treatment followed by seeding at 1.5 × 10siRNA treatments were conducted using Lipofectamine RNAiMAX  and Luciferase control, H1.2/H1.4 and H1.4 siRNA according to manufacturer’s protocol. The cells were seeded the day prior to transfection to achieve 50–60% confluency at the time of treatment. siRNA and lipofectamine were diluted in Opti-MEM , mixed, incubated for 5 min, and the complexes were then added directly to the cells now growing in phenol-red free CD-FBS Media to allow estradiol depletion along with siRNA knockdown simultaneously. After 72 h, the cells were placed in E2/ethanol for 30 min before harvesting.6 cells were then incubated with primary antibody overnight at 4 °C. The antibody–chromatin complexes were then incubated with 50μL BSA-blocked Dynabeads  for 4 h at 4 °C and then collected using a magnetic separator. Following sequential ChIP wash-buffer and TE buffer washes, the beads were eluted using 200 μL freshly prepared elution buffer. 200 mM final concentration NaCl was added to these eluates and incubated at 65 °C overnight to reverse crosslink the immunocomplexes. Following RNAse A and Proteinase K digestion, the DNA was then purified using phenol/chloroform extraction and then precipitated using glycogen as a carrier. The precipitated DNA was then dissolved in Tris-EDTA buffer and used for qRT-PCR or Illumina sequencing.Chromatin immunoprecipitation was performed using protocols described before  with minMCF7 cells (±E2) were collected following trypsinization. The nuclei were isolated for chromatin extraction. Briefly, the cells were washed with 0.1% tween-20 in 1XPBS, 0.1%NP40 in Tris-MgCl2 (TM2) buffer, and TM2 buffer to extract the nuclei. The nuclei were then subjected to micrococcal nuclease (2 units) treatment for 6 min before using 0.5X PBS for overnight chromatin extraction. pS187-H1.4 antibody was then added to this chromatin extract and allowed to bind overnight. Sepharose beads were used to elute the chromatin–antibody complex. This mixture was then boiled with Laemmeli buffer and then run on a 4–20% polyacrylamide gel for western blot.For expression analyses, RNA was extracted from cells using the trizol extraction method  and furt−4. Genes annotated at 100 bp from differential pS187H1.4 peaks were submitted for GO analysis to DAVID and GREAT [ChIP products were used to prepare libraries using a kit from KAPA Biosystems- Roche . Manufacturer protocols were followed for sheared DNA sizes 500–700 Kb. The quality control of libraries was conducted using a bioanalyzer and then sequenced using the Illumina-Hiseq 4000 by the W. M. Keck Center for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Center . Sequence data were mapped with Bowtie2  to the Und GREAT ,46. MetaTwo biological replicates of pS187-H1.4 and pan-H1.4 ChIP-sequencing were performed. The IP efficiency of replicate 1 was better than replicate 2; however, the peak enrichment patterns agreed closely, and the correlation coefficient scores calculated between the replicates were >0.94. Browser views demonstrated close to identical trends . Data frPrevious attempts to understand the functions of histone H1 have involved mapping the distribution of all H1 variants together across the genome, under the assumption that all of the variants were interchangeable and redundant in function. However, like core histones, H1 variants also undergo distinct post-translational modifications that impart different functionalities to these proteins. Among the known modifications, phosphorylation is the most abundant ,47, but In the present study, we have addressed this gap in knowledge by employing a unique antibody designed to detect a specific phosphorylated version of variant H1.4, pS187-H1.4. Early studies show that the sequence of human H1.4 shares a 93.5 sequence identity with its mouse ortholog ,49; thisAlthough there have been a number of studies looking into histone PTMs, there is currently no genome-wide study describing the predominant interphase phosphorylation sites. This study provides the first genome-wide evidence of the functions of pS187-H1.4, in the context of estradiol (E2) regulation. Earlier reports have described the estrogen response as being carried out through estrogen receptor isoforms (ERα or ERβ) dimerizing with 17β-estradiol and binding to specific motifs in the DNA to elicit a transcriptional response ,53,54. IIn a previous study by Liao and Mizzen , CDK9 waThe association of pS187-H1.4 with features across the genome, and particularly promoters, strengthens the hypothesis that H1.4 is important for the activation of genes via the RNAPII regulatory pathway. However, it is noteworthy to observe that a strong enrichment of pS187-H1.4 is observed in CpG Islands, which are known to mainly contain sites of active transcription . The regTogether, this suggests that pS187H1.4 may be able to interact with multiple different transcriptional regulators, all with the final outcome of transcription activation. This suggests that, globally, pS187H1.4 may be a versatile factor with the ability to interact with various transcription regulatory components. Although this study was focused on the estradiol-based response system as a model, we observed a basal level of pS187-H1.4 proximal to the promoters. Together, these data suggest that pS187H1.4 may play a more global role in the regulation of transcription.Since H1 has been largely regarded as a general repressor, the progress toward understanding the function and the mechanisms of action of individual H1 family members has been slow. Further, the paucity of appropriate ChIP-grade antibodies has also been a limiting factor. However, our novel, highly specific antibodies have allowed us to uncover a potential role of pS187-H1.4 in transcriptional activation for the first time, opening up a new area of study and suggesting that the roles of other specific H1 variants and their various modified forms should be further studied.In order to further elucidate the effect of H1.4 phosphorylation on gene activation, the mechanism of action must be tested. In particular, the extent and nature of the downstream effects must be assessed in cases where the phosphorylation event is prevented. Identifying the mechanism of CDK9 recruitment to this H1.4 will also yield an insight into the nature of its activity with regard to RNAPII regulation. However, these studies raise important issues regarding the functions of H1.4 variants that suggest their broad significance in gene regulatory mechanisms, and which we are now well poised to address.


Coxiella burnetii. Annotation of transcriptional regulatory elements will aid researchers in identifying genetic mutations of immunological consequence. Here we report the first genome-wide survey of regulatory elements in any sheep immune cell, utilizing alveolar macrophages. We assayed histone modifications and CTCF enrichment by chromatin immunoprecipitation with deep sequencing (ChIP-seq) in two sheep to determine cis-regulatory DNA elements and chromatin domain boundaries that control immunity-related gene expression. Histone modifications included H3K4me3 (denoting active promoters), H3K27ac (active enhancers), H3K4me1 , and H3K27me3 (broad silencers). In total, we identified 248,674 reproducible regulatory elements, which allowed assignment of putative biological function in macrophages to 12% of the sheep genome. Data exceeded the FAANG and ENCODE standards of 20 million and 45 million useable fragments for narrow and broad marks, respectively. Active elements showed consensus with RNA-seq data and were predictive of gene expression in alveolar macrophages from the publicly available Sheep Gene Expression Atlas. Silencer elements were not enriched for expressed genes, but rather for repressed developmental genes. CTCF enrichment enabled identification of 11,000 chromatin domains with mean size of 258 kb. To our knowledge, this is the first report to use immunoprecipitated CTCF to determine putative topological domains in sheep immune cells. Furthermore, these data will empower phenotype-associated mutation discovery since most causal variants are within regulatory elements.Alveolar macrophages function in innate and adaptive immunity, wound healing, and homeostasis in the lungs dependent on tissue-specific gene expression under epigenetic regulation. The functional diversity of tissue resident macrophages, despite their common myeloid lineage, highlights the need to study tissue-specific regulatory elements that control gene expression. Increasing evidence supports the hypothesis that subtle genetic changes alter sheep macrophage response to important production pathogens and zoonoses, for example, viruses like small ruminant lentiviruses and bacteria like  Cells were isolated from collected lavage fluid by centrifugation (400 × g for 10 min) and washed with DPBS at room temperature. Erythrocytes within the pellet were lysed by suspension in sterile water for 30 s. The harvested cells were confirmed to be morphologically consistent with macrophages on cytological evaluation as others have reported  and slowly frozen to −80°C in isopropyl alcohol baths  for short term storage.Animals were cared for and handled according to protocols approved by the Institutional Animal Care and Use Committee at Washington State University under Animal Subject Approval Form 4618. Sheep were humanely euthanized with intravenous sodium pentobarbital and lungs were removed firstly during routine postmortem examination by a veterinarian. No gross lesions were detected in the sheep. Alveolar macrophages were collected from the lungs of 2, 1-year-old, clinically healthy, crossbred  ewes using methods modified from those previously described . Brieflyreported . Cells w1). Cells and buffers were maintained on ice during all steps. Nuclei were isolated from approximately 5 × 107 unfixed, thawed cells firstly by incubation on ice in hypotonic buffer . Sodium butyrate 5 mM was included to inhibit histone deacetylases during processing. Next, 0.2% IGEPAL CA-630 detergent  was added to the suspension with gentle Dounce homogenization using a tight pestle. The nuclei suspension was then carefully layered onto 8 mL of buffer containing 1.2 M sucrose and centrifuged at 4,000 × g for 20 min at 4°C. Detergent layers were removed carefully from the nuclei pellet, then the pellet was resuspended in micrococcal nuclease digestion buffer with protease inhibitors. The pellet was briefly vortexed and then 60 Kunitz units of micrococcal nuclease  was added for 12 min incubation at 37°C to digest the chromatin into mono- and di-nucleosomes. Addition of 20 mM EGTA quenched the digestion reaction and soluble chromatin fragments were recovered in the supernatant by probe-free, cup horn sonication for 2 × 30 s on ice at high power (260 watts). A sample of purified digested chromatin was checked for adequate fragmentation on an agarose gel and on a fragment bioanalyzer  to ensure oligonucleosome fragment lengths within 100–450 base pairs. Average chromatin fragment size was approximately 150 bp in both biological replicates. Chromatin concentration was then measured by fluorescence quantification using the Qubit dsDNA HS kit (Thermo Fisher Scientific).Native chromatin isolation and immunoprecipitation was modified from methods published previously for tissues . AdditioSupplementary Methods 1Input nucleosomal DNA for each ewe were used as negative controls (no addition of antibody or magnetic beads). Chromatin for immunoprecipitation was pre-cleared by incubation with protein G coupled magnetic beads . Antibodies to the following targets were used for each chromatin immunoprecipitation: five microliters of anti-H3K4me3, anti-H3K27ac, anti-H3K27me3, anti-H3K4me1, and 10 microliters of anti-CTCF  following the manufacturer’s protocol with 15 PCR cycles to minimize duplication bias and size selection of 250–600 bp to include the bulk of immunoprecipitated fragments ligated to adapters. Preparation for multiplexing was accomplished by utilizing indexing adapters included in the kit. ChIP library size was assessed by Fragment Analyzer  with the High Sensitivity NGS Fragment Analysis Kit , and library concentration was determined by StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with the KAPA Library Quantification Kit . Each library was diluted to 4 nM with RSB , followed by denaturation with 0.1 M NaOH, and 20 pM was clustered in a high-output flow cell using HiSeq Cluster Kit v4 on a cBot (Illumina). After cluster generation, the flow cell was loaded onto HiSeq 2500 for sequencing using HiSeq SBS kit v4 (Illumina). DNA was sequenced with a read length of 50 bp from a single end generating between 41.96 million and 80.15 million filter-passed reads for each library. These were derived from a total of 644,923,132 reads for the experiment that passed initial sequencing quality filters (97% pass-rate) .PRJEB40528 (ERP124181). Optional parameters used for all bioinformatics tools and detailed bioinformatics protocol are included in Sheep ChIP-seq sequencing files generated for this article are publicly available in the ENA database and FAANG data portal under project accession 2. Reads were filtered for quality and unique mapping with SAMtools v1.9 . Reads were quality checked with FastQC software  with attols v1.9 . Peaks fols v1.9 . Effectius peaks . These rOvis aries Refseq Annotation Release 103  see . Correlacis-acting regulatory elements include 71,933 regions marked by H3K4me3 classified as promoters and 68,818 marked by H3K27ac grouped as enhancers or highly active regions. Regions enriched for H3K4me1, considered primed and active enhancers were discovered at 31,800 genomic locations that included both broad and narrow regions of signal enrichment. Silencers were regions with signal enrichment for H3K27me3 found at 53,879 broad regions that cover long stretches of DNA. Lastly, 22,244 very narrow regions marked by CTCF were identified that denoted genomic locations of insulators indicative of chromatin domain boundaries. Consensus regulatory elements were used for further analysis since there was acceptable agreement amongst animal replicates  and annotated with the nearest Refseq gene. The majority of H3K4me3 enriched regions were located within genes (intron and exon) or near the 5′ end of genes within 2 kb of TSS annotated as promoter regions . Twenty-More than half of H3K27ac enhancer regions were annotated within introns . Half ofPOL3D, ACTB  were associated with an H3K4me3 enriched regulatory element. Regulatory elements with H3K4me3 signal were identified with gene-rich regions of the genome, 77% were within 20 kb of the nearest gene. Regions with ChIP enrichment for active promoters and enhancers, H3K4me3, H3K27ac, and H3K4me1 were at constitutively expressed housekeeping genes including 3D, ACTB , and GAPke PPARG  and at e RNA-seq . Several RNA-seq . Lineageq . The top third H3K4me3 enriched regions corresponded to genes from the RNA-seq data with an average expression of 77 TPM (r = 0.12).Promoter regions enriched for H3K4me3 were then filtered for those within only 2 kb of annotated genes, as these were most likely to be a correct match between regulatory element and TSS. Approximately 73% of genes with active H3K4me3 enrichment within 2 kb of the TSS were also expressed in RNA-seq data from sheep alveolar macrophages in the Sheep Gene Expression Atlas. Analysis of regions with H3K4me3 and H3K27ac enrichment showed 78% were associated with gene expression from RNA-seq regardless of distance from that annotated gene. Maximum signal enrichment of H3K4me3 at promoter peaks within 2 kb of TSS was positively correlated with gene expression TPM when compared to RNA-seq data (f 77 TPM . Within P < 0.05), greater than two fold overrepresentation for biological processes like viral protein processing, positive regulation of antigen receptor-mediated signaling pathway, type I interferon-mediated signaling, regulation of autophagy, and mitotic spindle assembly checkpoint, among others. Antigen processing and presentation via MHC class II was overrepresented at 1.76-fold  than known motifs (P = 10–30) within bivalent promoter sequences. A de novo motif with similarity to the binding motif for yeast protein STB1 was found in 38% of regions. Known motif analysis revealed bivalent promoter sequences were enriched for “CCCGC” and “CGCGCG” sequences and the motif for the Drosophila GAGA factor protein.Further analysis of promoters revealed a small subset of 3,641 regions with signal enrichment for both H3K4me3 and the repressive mark H3K27me3. Annotation with the nearest gene revealed 45% of these regions were within 2 kb of a TSS. These 1,630 regions were considered bivalent promoters. Regions were associated with 1,166 protein-coding genes. Gene ontology analysis was largely enriched for genes involved in molecular functions for transcriptional regulation, transcription factor activity, DNA and RNA binding, and RNA polymerase II regulation . BiologiTotal enhancer regions with either H3K4me1, H3K27ac, or both were more numerous than H3K4me3 promoter regions. We found that genes have multiple enhancer regions. On average five significant regions enriched for enhancer signal were associated with each unique gene. In fact, we found that 69% of active enhancer regions marked with H3K27ac were found in clusters of two to seven (average of 3.2) significant regions around the same gene. Multiple regions meant that enhancers are further from the genes they control. H3K27ac regions were a mean of 1.9 kb further from their genes than H3K4me3 regions. Our data revealed that 56% of genes may be controlled by multiple active distal enhancers  in macrophages.de novo motifs within H3K27ac regions was a 15-bp sequence with 0.89 match score similarity to the known binding motif for the protein peroxisome proliferator activated receptor gamma (PPARG). PPARG is a transcription factor specific to macrophages within the lung microenvironment. The de novo PPARG motif was discovered in approximately 10% of active enhancers. Primed enhancers denoted by H3K4me1 were generally enriched for known lineage specific and pioneering factor motifs. Approximately half of H3K4me1 regions contained the known motif for the transcription factor protein PU.1. Primed enhancers in sheep additionally contained binding motifs for the transcription factor proteins CCAT enhancer binding protein beta  and CEBPC, SpiB, and SpiC, and a de novo motif for NFKB1 reflecting presence of immunity related binding sites , to the proteins Zfp281 and NFKB1. Several additional C2H2 zinc finger protein binding sites were discovered with borderline significance (P = 10–42) for transcription factor proteins ZKSCAN1, ZNF467, and ZNF165. Silencer regions were found near homeobox transcription factor genes (HOX family) involved in embryologic development. The closest genes to H3K27me3 regions in the sheep genome included 46% of all homeobox genes know in humans.Silencer regulatory elements denoted by H3K27me3 signal were generally not near genes on a genome-wide basis, since 69% of significant regions were in intergenic regions (see de novo binding motifs in sheep and top known motifs (P = 10–1482) and amongst closest matches to de novo motifs (P = 10–4338) (Insulator element CTCF peaks were scanned with HOMER to determine the top n motifs . CTCF an10–4338) . Signifi10–4338) . These rP = 8.9 × 10–8) indicating a pattern to their location rather than pure noise. Like H3K4me3 regions, hyperChIPable regions were found within the first intron of genes or at the TSS (39%)  of the genome. However, they were broad at 8 kb, longer than regions found in any individual target. Although the majority of hyperChIPable regions were found within intergenic regions (59%), there was moderate but significant enrichment at promoters (39%) see .cis-acting regulatory elements for housekeeping genes, genes associated with macrophage differentiation, and tissue-specific alveolar macrophage genes. We found bivalent regulatory elements at the promoters of few genes and annotated GO processes that varied from the processes found in more typical promoters. We also identified significant regions bound by CTCF with N-ChIP that revealed insulators and allowed the first preliminary estimates of chromatin domains in sheep alveolar macrophages. Our collective data assigned a putative biological regulatory function in macrophages to nearly 12% of the sheep genome.We identified repressive and active regulatory elements and validated regions by comparison to public RNA-seq data on alveolar macrophages from the Sheep Gene Expression Atlas . These dAlveolar macrophages served as a biologically interesting tissue given their myriad local functions and importance to zoonotic intracellular pathogens. We were able to identify GO overrepresentation in regulatory element associated genes involved in pathways reflective of tissue-resident macrophage main functions in homeostasis such as protein catabolism, autophagy, and nitrogen metabolism . Both inWe found evidence in ChIP-seq enriched active regions (H3K4me3 and H3K27ac) of increased guanosine and cytosine content, as others have shown in various cell types that promotes open chromatin . HeterocTLR4 promoters and immediate early genes that help to induce rapid expression after exposure to foreign and injurious stimuli like LPS . The de novo motifs were found in approximately half of the sheep insulator regions, meaning half do not contain recognizable motifs or may have bindings sites adjacent to immunoprecipitated regions since CTCF binds chromatin in large protein complexes. However, from We defined the first We found complex patterns of overlapping histone modifications across the regulatory element landscape. The “histone code” precisely titrates gene expression at multiple levels and is better assayed by analysis of multiple ChIP-seq targets together as we saw in sheep . This “cSome promoter regions in sheep macrophages were found to paradoxically have both H3K4me3 active marks and H3K27me3 repressive marks. These bivalent promoters signify unique genes that have highly variable and responsive gene expression . GO reveIn contrast, overlap of H3K27me3 and H3K27ac histone modifications are antagonistic to one another and not found in the same regions . AccordiLastly, because we produced data for multiple marks, we were able to elucidate putative hyperChIPable regions in the sheep genome. These regions were found in all immunoprecipitated datasets and were not specific for any one target or antibody . HyperChin vivo conditions rather than from cell culture conditions to identify the regulatory elements that are uniquely used by the immune system. We found modest, positive correlation that was highly statistically significant (P = 10–130), between signal enrichment of H3K4me3 in promoters and gene expression in alveolar macrophages determined in the Sheep Gene Expression Atlas , IL-6, and TLR4 until pathogens are detected  under accession numbers SAMEA7423844 and SAMEA7423845 and samples under SAMEA7423964 and SAMEA7423965. Raw DNA sequencing data in .fastq.gz format is available on the FAANG data portal and within European Nucleotide Archive (ENA) project accession PRJEB40528 (https://www.ebi.ac.uk/ena/browser/view/PRJEB40528). Analysis BED files of the peak calls in each animal, the consensus regions for each mark, bivalent promoters regions and hyperChIPable regions are available on the Open Science Framework website upon request and will be made public, https://osf.io/8rq4c/?view_only=1937b862ea874a14b85fff2043a9b522. Publicly available datasets from the Sheep Gene Expression Atlas (Roslin Sheep Atlas) were analyzed in this study. This data can be found here: University of Edinburgh DataShare portal at http://dx.doi.org/10.7488/ds/2112 (GCA_002742125.1 (https://www.ncbi.nlm.nih.gov/assembly/GCF_002742125.1/)  .The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Washington State University.Experimental protocols were designed by AM, MM, MH, BM, and SW. Cell collection was completed by AM, MM, and MH. Experiments, data generation, and initial data analysis were completed by AM. The bioinformatics pipeline was designed by AM and DH. Final analysis was completed by AM, MM, MH, DH, BM, and SW. AM, MM, BM, and SW acquired funding. AM wrote the first draft of the manuscript and created visualizations. All authors contributed to editing the manuscript and visualizations. All authors read and approved the final manuscript.The 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.