Early animal development is characterized by intense reorganization of the embryonic genome, including large-scale changes in chromatin structure and in the DNA and histone modifications that help shape this structure. Particularly profound shifts in the chromatin landscape are associated with the maternal-to-zygotic transition, when the zygotic genome is first transcribed and maternally loaded transcripts are degraded. The accessibility of the early zebrafish embryo facilitates the interrogation of chromatin during this critical window of development, making it an important model for early chromatin regulation. Here, we review our current understanding of chromatin dynamics during early zebrafish development, highlighting new advances as well as similarities and differences between early chromatin regulation in zebrafish and other species. This focused review highlights current advances in our understanding of chromatin dynamics during early zebrafish embryogenesis. We provide an emerging picture of chromatin changes during the awakening of the zebrafish zygotic genome, integrate this knowledge with new data gained from other species, and highlight research directions where the strengths of the zebrafish model provide high potential for new advances.Animal genomes undergo a period of intense reorganization during early development as zygotic transcription initiates and embryos transition from a totipotent to a lineage-committed state. This reorganization is reflected by dramatic changes in chromatin structure and in the DNA and histone modifications that help drive this structure. During the last two decades, zebrafish have become an important and established model for studying chromatin in the context of vertebrate development. Two recent comprehensive reviews detail the many seminal insights into chromatin regulation that have been obtained using zebrafish3. Early cleavage stages exhibit several unique features, including a high ratio of cytoplasmic to nuclear volume, rapid cell divisions in which the entire genome is replicated in less than 15 minutes, and sac-like vesicles called karyomeres that transiently encase individual or groups of chromosomes near the end of mitosis7. Zebrafish display a conserved fate map following blastula stages and undergo further cell fate restriction during gastrulation8.Zebrafish offer a powerful system for studying chromatin transitions associated with early development. External fertilization means that embryos are accessible for observation, manipulation, and molecular interrogation from the 1-cell stage onward without surgical intervention. Following fertilization, zebrafish embryos develop quickly over the next 24 hours, moving through a stereotypical program defined by cleavage, blastula, gastrula, and segmentation periods. During this time, embryos can be accurately staged on the basis of morphological characteristics as well as time post fertilization11. The timing of this transition varies between organisms. Activation occurs as early as the 1- to 2-cell stage in mouse, between the 4- and 16-cell stages in most other mammals, and as late as cell divisions 6 to 8 inDrosophila and zebrafish9. In addition to dramatic transcriptional changes, MZT coincides with other important changes, including lengthening of the cell cycle, emergence of cell cycle check points, and the capacity for cells to undergo apoptosis11.The maternal-to-zygotic transition (MZT) represents a critical milestone in early animal embryogenesis. During this transition, developmental control shifts from maternally provided proteins and RNAs to zygotic transcripts4. However, a minor wave of zygotic genome activation (ZGA) precedes this stage, and the earliest zebrafish transcripts emerge from the miR-430 microRNA gene cluster at the 64-cell stage (2 hpf)17. MicroRNAs from this cluster in turn play a key role in the degradation of maternally loaded transcripts during MZT18.The transition from a fully quiescent to active genome during MZT offers a unique and exciting opportunity to tease apart the relationship between chromatin and transcription. In zebrafish, the bulk of zygotic transcription starts after the 10th cell division at the 1000-cell stage (3 hours post fertilization [hpf])24. In zebrafish, transcription factors Pou5f3, Nanog, and SoxB1 proteins are critical for ZGA, binding to thousands of putative regulatory elements during this period27. These transcription factors appear to be involved in nucleosome displacement through a two-step process. Before ZGA, they provide non-specific competition with histones on strong nucleosome footprints and then at ZGA they act synergistically to maintain open chromatin at regions with high nucleosome affinity29. Binding is associated with increased accessibility at MZT30, and recent work suggests that accessibility precedes and is predictive of future transcription31.Initiation of zygotic transcription is generally mediated by a small number of pioneer factors, although the specific factors involved differ between species27. Injection of translation-blocking morpholinos for either Pou5f3 or SoxB1 prevents ZGA14, suggesting that translational regulation of these RNAs likely contributes to the control of ZGA.During egg production, many mRNAs and proteins required for early development are maternally deposited. The extent to which early transcription factors are maternally loaded at the protein level is currently unclear. However, mRNAs encoding key transcription factors, including Pou5f3, Nanog, and SoxB1 proteins, are detected in embryos prior to the 64-cell stage, indicating that they are maternally loaded11. Of particular note, recent studies in Xenopus suggest that the concentration of histones in the early embryo may be critical for ZGA36. This also appears to be the case in zebrafish, as injection of core histones into the early zebrafish embryo is sufficient to delay ZGA whereas histone depletion accelerates ZGA36. The concentration of histones on DNA does not change during the period leading up to ZGA, but the non-DNA-bound core histone concentration is decreased by early cleavage divisions36. This results in a high nucleus-to-cytoplasmic ratio of histones at ZGA. This observation has led to a model in which reduced concentrations of unbound histones allow key transcription factors to successfully compete for DNA binding, thereby initiating ZGA37. One challenge to this model is that zebrafish embryos injected with mRNA encoding the cell cycle regulator Chk1 arrest development between the 4- and 16-cell stages and maintain a low overall nuclear-to-cytoplasmic ratio yet these embryos are still able to activate a subset of zygotic genes38. Although further investigation is needed, short genes seem to be less affected by the nuclear-to-cytoplasmic ratio during embryogenesis compared with long genes39. This raises the possibility that ZGA is differentially regulated at different subclasses of sequences.The exact sequence of events that leads to ZGA is not well understood and remains an area of intense investigation. In addition to translational regulation of transcription factors, depletion of repressors, accumulation of activators, and local changes in chromatin accessibility have been implicated in promoting ZGA6.Intriguingly, in zebrafish, early replication of “first wave” zygotic genes precedes initiation of their transcription. This finding raises the possibility that DNA replication is controlled by the same factors that poise these genes for transcription or that the pre-MZT replication timing program itself helps to prime early ZGAThe relatively large number of cell divisions occurring between fertilization and ZGA has made zebrafish an appealing model for profiling the chromatin changes during this critical period. Genome-wide changes in DNA methylation, histone modifications, and chromatin structure have all been profiled at high temporal resolution during early zebrafish embryogenesis . As in o44. Most CpGs in vertebrate genomes are methylated; the primary exception consists of non-methylated islands that generally overlap with promoters and other cis regulatory elements48. 5mC is essential for viability in vertebrates, and global loss of 5mC results in lethality in mice, frogs, and zebrafish60. Vertebrate 5mC is established byde novo DNA methyltransferases of the Dnmt3 family and maintained by the maintenance DNA methyltransferase Dnmt1 and its cofactor Uhrf161. In addition to encoding Dnmt1 and Uhrf1, the zebrafish genome encodes six Dnmt3 orthologs, which exhibit differential expression during development69. Zebrafish also harbor orthologs of the 5mC dioxygenases Tet1, Tet2, and Tet3, which can promote active 5mC removal through the iterative oxidation of 5mC73.5-methylcytosine (5mC) is the most common DNA modification in vertebrate genomes. It is associated with transcriptional repression and predominates at CpG dinucleotides83. In contrast, zebrafish and other non-mammalian vertebrates do not appear to undergo similar large-scale demethylation91. Instead, at least in zebrafish, the developing embryo adopts the 5mC landscape of the paternally inherited genome through gradual refashioning of the maternal methylome88. Surprisingly, the paternal genome is not required for this process46. The specificde novo methyltransferases involved in establishing 5mC on the maternal methylome during this window have yet to be identified. Regions of methylation loss most likely undergo passive demethylation as Tet enzymes and oxidative derivatives of 5mC are undetectable during early zebrafish development95.The dynamics of DNA methylation are considerably different in zebrafish and mammals. The mammalian methylome is erased and re-established during preimplantation development and primordial germ cell formation92. As in mammals, Tet enzymes are important for demethylation of enhancer chromatin at later stages of zebrafish development97. Very recent work suggests that 5mC patterns are also broadly stable in the zebrafish germline, although there is one curious exception91. There appears to be female-specific germline amplification and demethylation of an 11.5-kb repeat region encoding 45S ribosomal RNA91. The failure to undergo large-scale erasure and re-establishment of 5mC in the zebrafish embryo or the germline may explain the transgenerational accumulation of 5mC at transgenes in zebrafish99. These observations also raise the possibility that inherited 5mC drives epiallelic regulation of endogenous zebrafish genes in some contexts.The absence of Tets in zebrafish during early embryogenesis is in contrast to mammals, where Tets play a critical role in shaping the early embryonic methylome102. Unlike mammalian sperm, where the bulk of histones are replaced with protamine, zebrafish sperm rely entirely on histones to package their DNA104. In contrast to 5mC, most histone modifications undergo erasure and re-establishment during early zebrafish development, although reprogrammed histone modifications do not necessarily match the pattern of either gamete109.Modification of the N-terminal tails of histones plays an instrumental role in shaping genomes into regions that are restrictive or permissive for transcriptionH3K27ac. One of the earliest histone modifications detected in the developing zebrafish embryo is histone H3 lysine 27 acetylation (H3K27ac), a modification associated with transcriptional activation27. Deposition of H3K27ac is associated with initial access to promoters for early transcribed genes, and the highest H3K27ac enrichment occurs at Pou5f3-, SoxB1-, and Nanog-primed loci at ZGA31. Maternal depletion of the histone acetyltransferasesep300b,crebbpa, andcrebbpb reduces detectable zygotic transcripts at the dome stage (4.3 hpf). Conversely, premature increases in the histone acetyltransferases Brd4 and P300 are sufficient to prematurely activate zygotic transcription38. Together, these findings suggest a fundamental requirement for H3K27ac in promoting transcription during ZGA. Mechanistically, it is likely that increased histone acetylation during MZT helps relieve the repressive activity of histones, thereby promoting zygotic transcription38.H3K4me3/H3K27me3. Trimethylation of histone H3 lysine 4 (H3K4me3) and trimethylation of histone H3 lysine 27 (H3K27me3) have important antagonistic roles in transcriptional regulation. H3K4me3 is typically associated with activation whereas H3K27me3 is associated with repression. A third class of bivalently marked genes harbor both modifications and exist in a state that is poised for transcription113. In zebrafish, H3K4me3 and H3K27me3 are present in sperm but are erased in the early embryo109. Early chromatin microarray experiments revealed colocalized enrichment of H3K4me3 and H3K27me3 at promoters and transcriptional start sites at ZGA and these findings have recently been confirmed using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq)109. Only a subset of genes marked by H3K4me3 or H3K4me3/H3K27me3 are actively transcribed at ZGA, and many bivalently marked genes remain poised for later expression. In addition, a large class of promoters is marked only by H3K4me3 in sperm and the pre-ZGA embryo. Some of these become bivalent after ZGA. Other genes are newly marked exclusively with H3K4me3 at ZGA109. Large-scale resetting of H3K4me3 and H3K27me3 is also observed in early mammalian embryos117.118. However, although maternal/zygotic zebrafish mutants lacking this enzyme exhibit global depletion of H3K27me3, they show only limited changes in early gene expression, develop normally through gastrulation, and form a normal body plan121.Somewhat surprisingly, H3K27me3-mediated repression may have only limited roles in the zebrafish embryo. Mouse mutants for the H3K27 methyltransferase Ezh2 die during embryogenesis between 7.5 and 10.5 days post coitumH3K9me3. Trimethylation of histone H3 lysine 9 (H3K9me3) is enriched at repetitive sequences, including transposons, pericentromeric satellite sequences, telomeres, and some gene clusters122. H3K9me3 is an essential component of highly condensed constitutive heterochromatin, which silences expression from repetitive sequences122. H3K9me3 is broadly depleted in early mouse, worm, fly, and zebrafish embryos, and large-scale establishment follows ZGA125. In zebrafish, the establishment of H3K9me3 appears to rely on miR430-mediated degradation of maternally loaded Smarca2, an ATP-dependent chromatin remodeling protein typically associated with the BAF complex108. Other mechanisms have been proposed in flies and worms126. The lack of H3K9me3-marked heterochromatin in early embryos is somewhat surprising, as H3K9me3 is thought to promote genome stability127. The mechanisms that allow early embryos to maintain genome integrity in the absence of H3K9me3-marked constitutive heterochromatin are not known.129. Chromatin structure can be visualized on multiple levels that reflect distinct aspects of DNA packaging. Structure in the zebrafish embryo has been recently assessed at the level of local chromatin accessibility by assay for transposase-accessible chromatin using sequencing (ATAC-seq), at the level of chromatin interactions by high-throughput chromatin conformation capture (Hi-C), and at the level of cellular ultrastructure by transmission electron microscopy (TEM).Beyond the direct modification of histones and DNA, profound structural changes are observed as the embryonic genome passes through early developmentAccessibility. Fine-scale structural organization of chromatin is achieved through nucleosome positioning, which can promote or constrain transcription factor access to DNA. In zebrafish, prior to the major wave of ZGA, nucleosomes already appear positioned downstream of zygotic transcriptional start sites. During ZGA, these nucleosomes become organized into regular, well-positioned arrays near gene promoters130. Accessibility, as assessed by ATAC-seq, is detected at a small subset of sequences, including the miR-430 cluster at the 64-cell stage, but far more regions of accessibility emerge by the 1000-cell stage. At the onset of the major wave of ZGA, the majority of accessible regions are in promoters whereas by the dome stage accessibility at distal regulatory regions predominates31.Compartments and topologically associated domains. Hi-C–based mapping of chromatin interactions in somatic cells has revealed two levels of organization. First, chromatin can be segregated into distinct compartments; the A compartments contain transcriptionally permissive chromatin, and B compartments contain repressed chromatin131. Compartments can be further subdivided into genomic regions known as topologically associated domains (TADs), which are thought to serve as regulatory scaffolds133. At 24 hpf, zebrafish have A/B compartments and TADs with genomic features similar to those observed in mammals, including enrichment of binding sites for the regulatory factor CTCF at TAD borders138. In most studied organisms, there is an absence of both compartments and TADs in early embryogenesis and these structures emerge during the major wave of ZGA140. In contrast, Hi-C in zebrafish reveals evidence of compartments and TADs at the 128-cell stage (2.25 hpf) prior to the major wave of ZGA. These structural features are lost during major ZGA and then re-established as embryonic development progresses134. The mechanisms that drive these very early chromatin interactions are not clear, nor are the reasons that TADs dissolve at ZGA. There is also a possibility that formation of karyomeres in the early embryo impacts TAD organization, although this has not been explored experimentally. Along with CTCF, the cohesin complex has been implicated in TAD formation. Intriguingly, there is global shift in the genomic distribution of the cohesion complex component Rad21 during the window in which TADs are remodeled in zebrafish, raising the possibility that there is a causative relationship between these events143.Ultrastructure. TEM provides an additional approach for visualization of condensed chromatin ultrastructure. By this approach, condensed chromatin regions appear as electron-dense aggregates within cell nuclei. Consistent with the lack of histone modifications associated with heterochromatin, early zebrafish embryos also lack condensed chromatin ultrastructure. Just prior to the major wave of ZGA, at the 512-cell stage, embryonic nuclei are completely devoid of the electron-dense aggregates, and aggregates similar to those classically observed in somatic cells emerge in all embryonic nuclei between 3.7 and 6 hpf108. This time line of compaction correlates with H3K9me3 establishment in the embryo108. The clear presence of A and B compartments in zebrafish embryos by Hi-C at the 128-cell stage is curious in light of the lack of ultrastructure visualized by TEM at the 512-cell stage. Future analysis will be required to determine whether differences reflect minor discrepancies in timing between analyses or rather suggest that early Hi-C interactions are insufficient to drive chromatin segregation at the ultrastructure level.144. At ZGA, genes marked by placeholders become either active and marked by H3K4me3 and H3K27ac or silenced and marked by H3K4me3/H3K27me3. The accumulation of these modifications suggests that these specialized nucleosomes poise gene promoters in an unmethylated state that can be readily activated at ZGA or during later development105. The presence of placeholders also suggests a clear mechanism by which methylation of the maternal methylome is remodeled during embryogenesis in the absence of information from the paternal methylome105.In addition to foundational work profiling chromatin changes during embryogenesis, a number of important conceptual advances have emerged from recent studies in zebrafish. Among these is the concept of the placeholder nucleosomes, which enable programming of DNA methylation on the maternal genome during early embryogenesis and prime promoters for later expression. Placeholder nucleosomes contain the histone H2A variant H2A.Z and H3 histones that are monomethylated on lysine 4 (H3K4me1). In both sperm and cleavage-stage zebrafish embryos, placeholder nucleosomes occupy virtually all regions lacking DNA methylation, and perturbation of these placeholders causes expansion of 5mC domains108. Recent evidence also suggests that resolution of primed promoters into active or silenced states relies predominately on maternal factors, although the specific factors involved have not been identified27. Around one third of zebrafish embryonic transcripts are exclusively maternal, whereas most additional transcripts are expressed both maternally and zygotically16. These RNA pools include transcripts for many candidate chromatin regulators. To date, one challenge to understanding the function of maternally loaded gene products has been the need to generate maternal/zygotic mutant embryos. For genes that are also required later in development, generation of maternal zygotic mutants often requires the labor-intensive process of germ cell transplantation, which can present an obstacle to rapid progress145. New technologies for germline-specific CRIPSR/CAS9-mediated mutation or degradation of maternally loaded gene products in the embryo are expected to aid in probing the functions of these maternally loaded factors151.Another emerging theme arising from work in zebrafish is the importance of maternally loaded RNA and protein in shaping the early chromatin landscape. Depletion of maternally loaded histones is implicated in activation of zygotic transcription, while clearance of the maternal factor Smarca2 is required for H3K9me3 establishment and condensed chromatin ultrastructure155. Haploid embryos have been elegantly used to demonstrate that the female methylome can reset without any input from male chromosomes and to show that decreased DNA content leads to delayed ZGA46. The recent discovery of the Ly6/uPAR protein Bouncer as necessary for species-specific fertilization in fish also opens up the opportunity to make hybrid embryos from medaka sperm and zebrafish eggs156. A detailed assessment of ZGA or the early chromatin environment has yet to be undertaken in these embryos, but they offer a unique system in which to probe DNA intrinsic versus extrinsic factors that contribute to the shifting landscape during early embryogenesis.The ability to readily manipulate the chromosomal content of the early embryo provides a unique tool for probing early chromatin regulation. Haploid zebrafish embryos are relatively normal during the first day of development, and triploid zebrafish are viable to adulthood the clarity and accessibility of the zebrafish embryo provide an exciting opportunity to visualize zygotic transcription and associated chromatin dynamics in intact embryos. Visualization of newly synthesized transcripts in the early embryo can be achieved by 5-ethynyl uridine (EU) labeling bulk RNA followed by click chemistry or through targeted visualization of highly expressed RNAs using fluorophore-conjugated morpholinos38. At the same time, CRISPR-dCAS9-GFP complexes can be used to visualize specific DNA loci within embryonic nuclei38. With these approaches, two foci of miR-430 expression, corresponding to the two chromosomal miR-430 gene clusters, can be visualized in a small fraction of embryonic nuclei beginning at the 64-cell stage, and all nuclei show two miR-430 foci by the 512-cell stage38. In addition to containing miR-430 transcripts, foci contain activated RNA polymerase II and RNA transcripts derived from gene clusters encoding zinc finger transcription factors17. Signal from these transcriptional hubs appears to be specific to the early embryo as they dissolve after the major wave of ZGA6.In addition to the ability to manipulate chromosome content,159. By this approach, H3K27ac is observed in two nuclear foci corresponding to the miR-430 gene clusters in 64- to 1000-cell stage embryos. Intriguingly, H3K27ac still appears at these foci when zygotic transcription is inhibited, suggesting that the establishment of H3K27ac at these foci precedes activation of transcription157.Injection of fluorescently labeled modification-specific antigen-binding fragments (Fabs) provides an additional new tool, allowing monitoring of chromatin changes in live zebrafish embryos160.Additional new technologies for visualizing chromatin and transcription in the early zebrafish embryo continue to emerge. Light-sheet microscopy tracking transcription factors, including Sox19b, was recently used to demonstrate that the chromatin-bound fraction of transcription factors increases over early embryonic cell divisions. Furthermore, new preprinted work visualizing transcriptional activity by three-color stimulated emission depletion (STED) super-resolution and live-cell microscopy suggests that, after transcription initiates, regions of active euchromatin form RNA-enriched microenvironments that exclude inactive euchromatinThe past few years have led to an explosion in articles exploiting the zebrafish system to understand chromatin dynamics and their relationship to transcription during the initial activation of the zygotic genome. Recent high-resolution profiling of DNA, histone modification, and structural changes occurring during this period offer a critical foundation for understanding how these many signals are integrated in the early embryo, while new technologies that are well suited to the zebrafish model offer the opportunity for continued advances.


Pioneering studies have showed that ZNF143 binds directly to promoters and contributes to chromatin interactions connecting promoters to distal regulatory elements, such as enhancers. Further, it has proved that ZNF143 is involved in CCCTC-binding factor (CTCF) in establishing the conserved chromatin loops by cooperating with cohesin and other partners. These results indicate that ZNF143 is a key loop formation factor. In addition, we report ZNF143 is dynamically bound to chromatin during the cell cycle demonstrated that it is a potential mitotic bookmarking factor. It may be associated with CTCF for mitosis-to-G1 phase transition and chromatin loop re-establishment in early G1 phase. In the future, researchers could further clarify the fine mechanism of ZNF143 in mediating chromatin loops with the help of CUT&RUN (CUT&Tag) and Cut-C technology. Thus, in this review, we summarize the research progress of TF ZNF143 in detail and also predict the potential functions of ZNF143 in cell fate and identity based on our recent discoveries.ZNF143, a human homolog of the transcriptional activator Staf, is a C2H2-type protein consisting of seven zinc finger domains. As a transcription factor (TF), ZNF143 is sequence specifically binding to chromatin and activates the expression of protein-coding and non-coding genes on a genome scale. Although it is ubiquitous expressed, its expression in cancer cells and tissues is usually higher than that in normal cells and tissues. Therefore, abnormal expression of ZNF143 is related to cancer cell survival, proliferation, differentiation, migration, and invasion, suggesting that new small molecules can be designed by targeting ZNF143 as it may be a good potential biomarker and therapeutic target for related cancers. However, the mechanism on how ZNF143 regulates its targeting gene remains unclear. Recently, with the development of chromatin conformation capture (3C) and its derivatives, and high Xenopus oocytes and named it Staf (selenocysteine tRNA gene transcription activating factor). In the same year, -Staf from mouse mammary gland, which is consistent with human ZNF143. ZNF143 is a member of the Kruppel family and is a widely expressed transcriptional activation factor that regulates gene expression associated with cell cycle and DNA replication  encompasses seven tandemly repeated zinc fingers of the C2H2 type, is highly basic, while the regions A  encodes four repeated motifs and C  are acidic  and the element of repeated motifs (activation domain) are especially well conserved among these three proteins . It is rTF ZNF143 regulates gene expression associated with cell cycle. Many studies utilize knockdown or overexpression methods to evaluate the effect of ZNF143 on cancer cell progression. For example, znf143a, a novel paralog of znf143, encodes a strong transcriptional activator protein and performs a similar role in the normal development of zebrafish embryos but expressed at a different level during early development. In mouse embryonic stem (ES) cells, ZNF143 regulates Nanog by regulating the binding of Oct4, and ZNF143 is also critical for maintaining human ES cell identity  elements in vitro and in transfected cells. ZNF143 plays an important role in the transcription of neuronal nitric-oxide synthase (nNOS) exon 1, the mutation of the binding site of ZNF143 leads to a significant reduction in the activity of this exon  (ZNF143 acts as a transcription-activated factor under the joint action of RNA polymerase III . The snRnt (DSE) . By compnt (DSE) . U6 snRNnt (DSE) . Schaub To maintain stable ZNF143 expression at normal levels, the transcription feedback regulation mechanism is the simplest and most direct means. ZNF143 selectively adjusts reverse expression by using a low affinity binding site (TSS2) located downstream of the TSS. When ZNF143 expression is higher than normal, transcripts containing longer 5′-UTR (few translation products) are produced by TSS2 transcription. In addition, when ZNF143 levels are lower than normal, the canonical TSS1 binding site is used to express transcripts containing shorter 5′-UTR (many translation products). This transcriptional auto-regulatory mechanism regulates ZNF143 expression by the conversion of the TSS switch, which plays an important role in cell proliferation and growth . Given tin silico) and biochemical methods. They speculated that at least 2500 ZNF143-binding sites are distributed in 2000 promoter regions throughout the mammalian genome. Further research has found that the presence of ZNF143-binding site alone can initiate the expression of a luciferase reporter gene, suggesting that ZNF143 itself exhibits the ability to recruit the transcription machinery  and cleavage under targets and tagmentation (CUT&Tag), will be viewed as a cost-effective and versatile alternative to ChIP because of low backgrounds, which requiring only ∼1/10th the sequencing depth as ChIP (CTCF, cohesion, and ZNF143 are three major regulators involved in the establishment and maintenance of long-range chromatin interactions. In mammalian cells, TAD-free analysis indicates that the blocking effects of CTCF, cohesin, and ZNF143 depend on the distance between loci because each protein may participate at different scales of chromatin organization . CTCF an as ChIP . Based oDuring mitosis, transcription is globally shut down, chromatin condenses, the nuclear envelope is disassembled, and most TFs are stripped off the mitotic chromosomes. How do the new daughter cells faithfully re-establish the cell-type specific transcription program? Recent discoveries that a select set of TFs remain associated with mitotic chromosomes suggest a phenomenon termed mitotic bookmarking . For exaAs a key TF, the role of ZNF143 in cancer progression through transcriptional regulation of genes related to DNA replication and cell cycle . FurtherBY, GaY, and YL drafted the manuscript. CZ, QW, and GuY critically revised the 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.


The two factors interact via their key activation domains; the low glucose inhibitory domain (LID) of ChREBPα and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in  Glucose and lipid metabolism are tightly connected and coordinately regulated in mammals to maintain whole-body energy homeostasis. In the liver, excess dietary carbohydrates are converted to fatty acids through de novo lipogenesis (DNL) destined for long-term storage as triglycerides in adipose tissue. Dysregulation of lipogenesis contributes to non-alcoholic fatty liver disease (NAFLD), which is associated with increased risk of metabolic syndrome and type 2 diabetes ,2. SeverN-acetylglucosamine (O-GlcNAc) [Lpk), acetyl-CoA carboxylase (Acc), fatty acid synthase (Fasn) and stearoyl-CoA desaturase-1 (Scd1) [de novo synthesis of fatty acids. The discovery of the O-GlcNAc modification of LXR [While showing high sequence homology, the two LXR subtypes differ in their distribution (reviewed in Reference ): LXRα i-GlcNAc) ,16,17,18-GlcNAc) ,20, whic1 (Scd1) ,22. Thisn of LXR , proposin of LXR . Howevern of LXR ,26.Chrebpα transcription start site (TSS), lacks most of LID (the first 177 amino acids), has escaped glucose regulation and acts constitutively independent of glucose concentration [ChREBP is a glucose-activated TF that belongs to the basic helix-loop-helix leucine zipper (bHLH/Zip) family . ChREBP ntration . In addintration ,39.Pklr (Lpk) and Mlxipl (Chrebpβ) and less ChREBP recruitment to the Lpk promoter [Lpk in the livers of mice fed an oleic acid-enriched diet [Chrebpα expression or directly by modulating ChREBPα activity.We previously reported that LXR deficiency leads to reduced ChREBP activity, resulting in reduced hepatic expression of ChREBP-specific target genes promoter . Moreovepromoter . Recentlhed diet . It is, In the current study, we demonstrate that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. Moreover, LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction, where LXRα ligand binding restricts the activation of glycolytic and lipogenic target genes. We speculate that this novel function of LXRα as a ligand-driven molecular switch for ChREBP, has been overlooked due to LXR’s role in promoting DNL.Formaldehyde (F1635), the synthetic LXR agonist GW3965 (G6295), dimethyl sulfoxide , Dulbecco’s Modified Eagle’s Medium , fetal bovine serum (FBS) (F7524), L-glutamine (G7513), penicillin-streptomycin (P4458), insulin (I9278) and D-(+)-glucose solution (G8769) were purchased from Sigma-Aldrich . Tularik (T0901317) was from Enzo Life Sciences . DMEM, no glucose (11966-025) was purchased from Gibco, Thermo Fisher Scientific . Dual Luciferase® reporter assay system (E1960) was purchased from Promega . All other chemicals were of the highest quality available from commercial vendors.To generate a genome-wide map of ChREBP and LXR binding sites, two published ChIP-seq datasets were reanalyzed—ChREBP ChIP-seq data from mouse liver   and LXR BWA aln (v.0.7.17) with default parameters and alignments with mapping quality less than 30 were discarded using Samtools filter (v.1.9). Peaks were called using MACS (v.2.1.1) with default parameters. Peaks within mm9 blacklisted regions [Reads were mapped to the mm9 reference genome (Assembly number: MGSCv37) using  regions  were disbedtools closest (v.2.26.0) on the peak summits of the ChREBP and LXR peaks and discarding pairs of close peaks with summit-to-summit distance larger than 1000 base pairs. These pairs were linked to their nearest gene by running Rgmatch [--report gene --distance 25 --promoter 5000 as parameters.Pairs of close ChREBP and LXR peaks were selected by running  Rgmatch  with --rhttps://www.ideals.illinois.edu/handle/2142/55240) using the Genomic HyperBrowser [Genomic co-occurrence of the predicted binding sites of the different transcription factors G was mearBrowser .-/-, LXRβ-/- and LXRα-/-β-/-  male mice were housed in a temperature-controlled (22 °C) facility with a strict 12 h light/dark cycle. The mice had free access to water before and during experiments and normal chow before the experiment. The diet contained 18.5% protein, 4% fat and 55.7% carbohydrate . LXRα and β-deficient mice and corresponding controls had mixed genetic backgrounds based on 129/Sv and C57BL/6J strains and were backcrossed in C57BL/6J mice  for six generations. The generation of the LXRα-/-, LXRβ-/- and the DOKO mice have been described previously [LXRαβ wild type, LXRαeviously ,48.Male mice aged 8–12 weeks (weight 25–30 g) were fasted for 24 h (Fasted) or fasted for 24 h and refed for 12 h (Refed). All male littermates in a litter of correct age, weight and genotype were randomly allocated to a given treatment. No particular measures were taken to minimize subjective bias during group allocation. Each genotype-treatment group consisted of 5–8 animals, 53 mice in total, allowing us to detect 2-fold changes, given a coefficient of variation of 25% or less . The mic5 cells/well onto type I collagen coated 12-well plates in attachment medium  with 10% FBS, 1% penicillin-streptomycin and 10 nM insulin. The medium was changed 2 h after plating to overnight media consisting of low glucose DMEM , 5% FBS, 1% penicillin-streptomycin and 1 nM insulin. On the next day, hepatocytes were washed twice with 1 mL medium/well consisting of DMEM with either 1 mM glucose (LG) or 25 mM glucose (HG), 5% FBS, 1% penicillin-streptomycin and 1 nM insulin and then cultured overnight in LG or HG medium supplemented with 0.1% DMSO, 10 µM GW3965 or 10 µM T0901317, respectively. Hepatocytes were harvested after 18 h treatment for RNA isolation.Mouse primary hepatocytes were isolated as previously described with modest changes . Briefly2 in air and routinely tested for mycoplasma contamination. Cells were transfected with indicated plasmids using Lipofectamine 2000 .Huh7 human liver hepatoma cells  and COS-The FLAG-tagged human or mouse LXR expressing plasmids pcDNA3-FLAG-hLXRα, pcDNA3-FLAG-hLXRβ and pcDNA3-FLAG-mLXRα, the untagged human RXRα expressing plasmid pcDNA3-hRXRα and the empty vector pcDNA3-FLAG have been described earlier ,52. To gChrebpβ promoter-driven luciferase reporter pGL3b-ChREBPβ-exon1b-luc (wild-type) and its mutants ChoRE+Ebox-del (both ChoRE and E-box deleted) and ChoRE-del (ChoRE deleted) were kind gifts from Prof. Mark Herman [The k Herman . The pGLk Herman  and the k Herman . The mouk Herman .The multimerized ChoRE and LXRE reporters: pGL3b-2xChoRE-2xLXRE-10 (ChoRE+LXRE-luc), pGL3b-2xChoRE-10 (ChoRE-only-luc) and pGL3b-2xLXRE-10 (LXRE-only-luc) were constructed in two steps—first, the 136 bp inserts were made by gene synthesis . The SacI-BglII-lined inserts were designed to contain two canonical ChoREs (CACGTGatataCACGTG) and two canonical LXREs (AGGTCActctAGGTCA) in the order ChoRE-LXRE-ChoRE-LXRE and with a spacing of 10 bp (gtaatattaa), giving a phasing of ~20 bp center to center between the REs. The inserts were then cut from the production vector pUC57  and subcloned into pGL3 basic vector between SacI and BglII. The schematic representation of synthetic reporters is illustrated in Figure 3A. The Renilla Luciferase reporter pRL-CMV  was used as internal control of transfection efficiency. All plasmids were verified by sequencing.4 cells/well in 24-well plates. After 24 h, cells were transfected with 300 ng of luciferase reporters, 150 ng of LXR/RXR and/or 150 ng of ChREBP/Mlx expressing plasmids or empty controls pcDNA3-FLAG and pCMV4. Renilla luciferase reporter  was included as an internal control for transfection efficiency. All transfections were performed with Lipofectamine 2000. Dual luciferase reporter assay was performed 24 h post transfection as previously described [Huh7 cells were seeded at density 7 × 10escribed . TransfeHuh7 cells were transfected with the ChoRE+LXRE reporter and plasmids expressing ChREBPα, ChREBP-Q or ChREBPβ and Mlxγ. AML12 cells were transfected with plasmids expressing ChREBPα, Mlxγ, with or without LXRα and RXRα. For LXR agonist treatment, 10 µM of GW3965 was applied to cells 6 h post transfection and 0.1% DMSO was used as control. Twenty-four hours post transfection, cells were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by 5 min incubation with 125 mM glycine to quench the reaction. Cells were washed twice in cold PBS and harvested in PBS-T. The cell pellets were lysed in lysis buffer  and 20 mM Tris (pH 8.0). The lysed cells were sonicated to get an average size of 200–500 bp chromatin fragments using a Bioruptor . Chromatin was immunoprecipitated with 4 µg antibody against ChREBP , against LXR (antibody generation described in Reference ), or rab-ΔΔCT method and normalized against the expression of TATA-binding protein (Tbp). All primer pairs showed an efficiency of 90–110% at R2 > 0.98. Primer sequences are listed in RNA was isolated with TRIzol® reagent  according to the manufacturer’s protocol, including a high salt  precipitation step to avoid contaminating polysaccharides to co-precipitate with RNA. Extracted RNA was further purified using RNeasy spin columns . Isolated RNA (500 ng) was reverse transcribed into cDNA using MultiScribe Reverse Transcriptase  and random hexamer primers. RT-qPCR was performed with 1 µL of the cDNA synthesis reaction using Kapa SYBR FAST qPCR Master Mix  on a Bio-Rad CFX96 Touch™ Real-Time PCR Detection System . Assay primers were designed with Primer-BLAST software  . Gene ex3VO4, 1 mM β-glycerophosphate and CompleteTM protease inhibitors , followed by snap freezing on dry ice to disrupt nuclear membranes. The lysates were then thawed on ice, cleared by centrifugation at 17,000× g, 10 min and immunoprecipitated with 2 μg ChREBP , LXRα  or LXRβ  antibodies bound to protein A Dynabeads  for 2 h at 4 °C. Beads were washed three times in wash buffer , 0.1% NP-40) with 5 min rotation in between, before proteins were eluted from the beads with 1× SDS loading buffer at 95 °C for 5 min. Co-immunoprecipitated proteins were analyzed by immunoblotting.COS-1 cells were transfected with plasmids expressing ChREBPα full-length (FL) or N-terminal LID truncation or ChREBPβ, with or without LXRα FL or truncations  or LXRβ. For ChREBPβ transfections we used 6-fold more DNA than of ChREBPα to obtain comparable protein levels in the CoIP. For LXR agonist treatment, 0.1% DMSO or GW3965 (1 µM) was applied to cells 6 h post transfection. Cells were harvested 24 h post transfection and lysed in lysis buffer , 1% NP-40) containing 1 mM NaF, 1 mM NaProteins were separated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)  and blotted onto PVDF membrane . Primary antibodies used were LXRα , LXRα LBD , LXRβ , RXRα , ChREBP , FLAG  and β-actin . Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG or mouse anti-rabbit IgG . p < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prims 8 . All data were presented as means and standard error of the mean (SEM). In addition, all individual data points are plotted. Data distribution and similarity in variance between groups were analyzed by Shapiro-Wilk and Brown-Forsythe tests, respectively. Statistical differences between groups were determined by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests for data with two variables. For data with one variable we used one-way ANOVA with Dunnett correction to compare every mean to the control mean, alternatively Tukey correction for multiple comparisons. For all statistical tests, Rgmatch; corresponding to −5000 to +2500 bp from TSS) were made with the ConsensusPathDB tool from the Max-Planck-Institute for Molecular Genetics (http://cpdb.molgen.mpg.de/) [https://reactome.org). Minimum overlap with input list was set to 3 and the p-value cutoff to p < 0.01.Gene set enrichment analysis of genes with ChREBP-LXR peak pairs with a peak-to-peak-distance <100 bp, residing in promoters, TSS and first exons (based on the mpg.de/) , using thttps://ncbi.nlm.nih.gov/geo) under accession number GSM864670 (LXR), GSM1899651 (FXR) and GSM864671 (PPARα) without any restrictions. The ChREBP ChIP-seq dataset [https://github.com/ivargr/open-the-lid-chip-seq-analysis. The Forbes co-occurrence analysis is available at https://hyperbrowser.uio.no/hb/u/ivar/h/open-the-lid-co-occurence-analysis.The ChIP-seq datasets used in this study is available at NCBI GEO , which affects hepatic gene expression of ChREBP-specific targets like  Chrebpβ . This ca Chrebpβ ,37. LXRα Chrebpβ  and one  Chrebpβ . The seqbona fide LXR or ChREBP target genes (blue dots) located mainly upstream or near the TSS, consistent with the notion that most TF binding sites are found upstream of TSSs [Lpk and Chrebpβ showed significant ChREBP enrichment at the expected sites in the respective promoters and LXR co-occupied the same sites (Rgmatch (https://bitbucket.org/pfurio/rgmatch), we found the peak pairs to be distributed throughout the genome like other NRs [Using the top 20,000 ChREBP peaks and all the LXR peaks we calculated the peak(ChREBP)-to-peak(LXR) distance for all peaks. Interestingly, as many as 11,022 peak pairs showed a peak-to-peak distance of less than 1000 bp and 7928 (71.9%) of these were less than 100 bp apart B. Given  of TSSs ,60. As ame sites E. Lookinther NRs ,61,62. STo assess how the seemingly high genome-wide co-occupancy of ChREBP and LXR compared with the co-occupancy between other hepatic TFs, we reanalyzed ChIP-seq data of mouse peroxisome proliferator-activated receptor-α (PPARα) and farnesoid X receptor (FXR) ,63. We nFinally, we performed gene set enrichment analysis (GSEA) based on the Reactome Pathway Database. We analyzed genes with ChREBP-LXR peak pairs residing either in the promoter, around the TSS or in the first exon and with a peak-to-peak-distance <100 bp (corresponding to peak pairs between −5000 to +2500 bp from the TSS). As expected, the filtered genes were enriched in pathways regulated by both ChREBP and LXR, for example, metabolism of lipids, fatty acids and glucose . NotablyChrebpβ promoter [Lpk promoter [Chrebpβ promoter increased three-fold when the glucose concentration in the media was augmented from 2.5 mM to 25 mM  mice. The mice were fasted for 24 h or fasted for 24 h and refed for 12 h before they were sacrificed. Liver from 5–8 animal per genotype were examined. The feeding-induced hepatic expression of the ChREBP specific target genes Lpk and Chrebpβ was significantly reduced in LXRα-/- and DOKO mice but not in LXRβ-/- mice , LXRα-/- mice B. On the ChREBPα . Taken tAcc, Fasn, and Scd1, which contain both LXREs and ChoREs in their promoter/regulatory regions [Lpk and Chrebpβ promoters, however, only contain ChoREs and no canonical LXREs. Analysis of the Lpk and Chrebpβ reporters using NHR scan (http://www.cisreg.ca/cgi-bin/NHR-scan/nhr_scan.cgi) suggested one weak candidate in the Chrebpβ exon 1B promoter. This was, however, discarded after a deletion scan showing no effect on the promoter activity  revealed that LXRα, LXRβ and FXR show high similarity in multiple patches throughout the LBD (data not shown), suggesting that LXRα might use a similar interface when binding to ChREBPα.The ability of LXRα to co-activate ChREBPα in an LXRE-independent manner naturally brought up the question of what domains of LXRα and ChREBPα that are involved in their interaction. We therefore examined the interaction between full-length LXR and ChREBP and different truncations using CoIP. ChREBPα interacted with LXRα when immunoprecipitating both ways A. On theSrebp1c promoter as well as the Lpk promoter , in which two cysteines (C115 and C118) in the first DNA-binding domain (DBD) zinc finger were mutated to alanine to abrogate DNA binding. The LXRα DBD mutant had lost its ability to transactivate the promoter , the forpromoter . This isAcacb, Fasn and Scd1—which are common targets of LXR and ChREBP—were significantly upregulated by the LXR agonist  or high glucose (25 mM) to induce ChREBP activity and concomitantly stimulated them with the potent, selective LXR agonist GW3965 (10 μM) to induce LXR-activity. The DNL genes  agonist A. The ex pattern B: These reporter . To exclh7 cells . These dLpk and Txnip and the common LXR and ChREBP targets Fasn and Scd1 . In the presence of ChREBPα, all four promoters were robustly immunoprecipitated. Moreover, LXRα did not affect ChREBPα occupancy on any of the promoters  of TFs and coregulators is generally linked to transcriptional repression  and SUMOAn important observation in our study is the different ability of ChREBPα and ChREBPβ to respond to the LXRα trans-coactivation. While ChREBPα and ChREBP-Q are activated by LXRα and repressed by LXR agonists, ChREBPβ is non-responsive to both cues . This isLpk, Chrebpβ, Tixnip, Rgs16), with no obvious LXRE/DR4 close by. In addition, our data demonstrate a physical interaction between the factors and the ability of ChREBPα to recruit LXRα to the promoter ChoRE but not the other way around. This points to a different mechanism underlying the ChREBP-LXR co-occupancy, where ChREBPα recruits LXRα in trans to ChoREs. Our data do not formally rule out the possibility that LXRα on certain sites also contacts chromatin directly, via its DBD, in addition to binding to ChREBPα via its LBD. In fact, such ChREBP-LXR contacts might function as bridging point for smaller or larger chromatin loops [Chrebpα) and exon 1B (Chrebpβ) promoter at the Mlxipl locus, where ChREBPα binding to the Chrebpβ promoter could contact LXRα sitting on the Chrebpα promoter 17 kb downstream and coordinate the expression from both promoters given different nutritional cues. High glucose would enforce the contact, driving Chrebpβ expression [Chrebpα expression. This type of looping is known for other TFs for example, GATA-1 and its co-factor FOG-1. Both factors are required and need to physically interact to produce a DNA loop bringing the β-globin locus control region (LCR) together with the promoter [The high genome-wide co-occupancy of LXR and ChREBP observed in the ChIP-seq data was rather surprising: More than 71% of ChREBP-LXR peak pairs have peaks less than 100 bp apart B, suggesin loops ,87, likein loops , possiblin loops . A tantapression , while hpromoter . Unbiasepromoter ,91 is waChrebpβ transcription and subsequent induction of glycolytic and lipogenic genes is limited, when the local concentration of pyruvate, acetyl-CoA and SFAs, as well as cholesterol/oxysterol reaches a certain level. This hitherto unappreciated role of LXRα may have been overlooked due to LXR’s DNL-promoting function.LXRα and ChREBPα promote glycolytic and lipogenic gene expression in high glucose conditions . High chSeveral LXR-activating drugs have been developed through the years aiming at boosting reverse cholesterol transport ,96. Howe


Exposure of bacteria to sublethal concentrations of antibiotics can lead to bacterial adaptation and survival at higher doses of inhibitors, which in turn can lead to the emergence of antibiotic resistance. The presence of sublethal concentrations of antibiotics targeting translation results in an increase in the amount of ribosomes per cell but nonetheless a decrease in the cells’ growth rate. In this work, we have found that inhibition of ribosome activity can result in a decrease in the amount of free RNA polymerase available for transcription, thus limiting the protein expression rate via a different pathway than what was expected. This result can be explained by our observation that long genes, such as those coding for RNA polymerase subunits, have a higher probability of premature translation termination in the presence of ribosome inhibitors, while expression of short ribosomal genes is affected less, consistent with their increased concentration. Escherichia coli genome, while the genes coding for ribosomal proteins are among the shortest genes. This can explain the observed decrease in transcription capacity that favors the expression of genes whose promoters have a high affinity for RNAP, such as ribosomal promoters.In bacterial cells, inhibition of ribosomes by sublethal concentrations of antibiotics leads to a decrease in the growth rate despite an increase in ribosome content. The limitation of ribosomal activity results in an increase in the level of expression from ribosomal promoters; this can deplete the pool of RNA polymerase (RNAP) that is available for the expression of nonribosomal genes. However, the magnitude of this effect remains to be quantified. Here, we use the change in the activity of constitutive promoters with different affinities for RNAP to quantify the change in the concentration of free RNAP. The data are consistent with a significant decrease in the amount of RNAP available for transcription of both ribosomal and nonribosomal genes. Results obtained with different reporter genes reveal an mRNA length dependence on the amount of full-length translated protein, consistent with the decrease in ribosome processivity affecting more strongly the translation of longer genes. The genes coding for the β and β' subunits of RNAP are among the longest genes in the IMPORTANCE Exposure of bacteria to sublethal concentrations of antibiotics can lead to bacterial adaptation and survival at higher doses of inhibitors, which in turn can lead to the emergence of antibiotic resistance. The presence of sublethal concentrations of antibiotics targeting translation results in an increase in the amount of ribosomes per cell but nonetheless a decrease in the cells’ growth rate. In this work, we have found that inhibition of ribosome activity can result in a decrease in the amount of free RNA polymerase available for transcription, thus limiting the protein expression rate via a different pathway than what was expected. This result can be explained by our observation that long genes, such as those coding for RNA polymerase subunits, have a higher probability of premature translation termination in the presence of ribosome inhibitors, while expression of short ribosomal genes is affected less, consistent with their increased concentration. Bacteria often encounter sublethal levels of antibiotics produced by other microorganisms in their environment. A decrease in growth rate under these conditions can allow a strain to survive long enough until the inhibitor is no longer present or, in some cases, until the bacteria become resistant to the antibiotic via the selection of preexisting mutations or an increase in mutation rates –4. Howev–10–The ppGpp-dependent feedback loop also plays a role in the regulation of ribosome content as a function of growth rate –17. Grow–18–When ribosome activity is inhibited by sublethal concentrations of antibiotics, amino acids are used more slowly and their concentration increases, which can result in a decrease in the intracellular ppGpp pool . The celTo measure the effect that the inhibition of ribosome activity can have on gene expression resulting from a possible repartition of RNAP, we have compared the activity of a ribosomal promoter to that of constitutive promoters with different affinities for RNAP. This approach stems from a well-established protocol developed by Hans Bremer and coworkers of using quantitative measurements of changes in constitutive and ribosomal promoter activity as reporters of changes in the amount of free RNAP and of ppGpp –25.–Escherichia coli genome that, in addition, are found within the same operon.In parallel, we analyzed transcriptomics and proteomics data from the literature on the direct and indirect effects of changing ppGpp concentration and translation limitation on gene expression , 27. TherrnBP1, here called P1, that includes the sequence from −69 to +6 relative to the transcription start site  and then inserted in E.coli chromosome near the gene crl, cynR, or uspE. A kanamycin resistance gene expressed divergently was inserted into the chromosome together with the constructs. (B) The strains carrying GFP were cultivated in a 96-well plate, and their optical density (OD) at 610 nm and green fluorescence were measured with a plate reader (Tecan Infinite 200Pro) every 7 min for at least 30 h. The growth rate μ was derived from the slope of the plot of log OD versus time over the exponential phase, which is defined between a lower and an upper threshold value of OD. GFP concentration ([GFP]) was derived from the slope of the plot of GFP versus OD over the exponential phase. For comparison, bacteria with P5-gfpmut2 and bacteria with P5-lacZ were grown in flasks, and OD and fluorescence were measured with the plate reader every 30 to 50 min. See Li et al.  Chromosomal insertion of the reporter constructs. The reporter gene i et al.  for the Copyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00575-20.10TABLE S1Table S1, XLSX file, 0.01 MB.Sequences of the promoters used in this study. The −35 and −10 regions are underlined. The bold base is the transcription start site. The bases in red in the P1 sequence indicate the discriminator region. Download Copyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rrnBP1-derived promoters /(fc + KLtet), where fc is the free RNAP concentration and a, K5, and KLtet are constants which can be determined by fitting the ratios of GFP production rate to the known values of free RNAP concentrations as a function of growth rate. The free RNAP concentrations as a function of growth rate in the absence of Cm was estimated based on a double exponential function shown by the black line in panel B. (B) The black stars correspond to the estimated free RNAP concentration as a function of growth rate obtained from Klumpp and Hwa (fc = exp [Aexp(–μr/μ)] (black line), where μ denotes the growth rate, fc denotes the concentration of free RNAP, and A and μr are constants. The free RNAP concentration in the presence of Cm is estimated from the data in panel A with the transcription rate ratio mentioned above. (C) The relative ppGpp inhibition effect is estimated as the ratio of GFP production rates from P1 and P5 promoters divided by the ratio of their relative promote activities, i.e., (K5 + fc)(K1 + fc), where fc denotes the free RNAP concentration and K5 and K1 are the dissociation constants for P5 and P1, respectively. The black line indicates a Hill function, i.e., pc is ppGpp concentration and b, pk, and n are constants which are fixed by fitting the estimated ppGpp effect without Cm. ppGpp concentration at the growth rates without Cm was estimated based on an exponential function shown by the black line in panel D. (D) The black stars correspond to the data on ppGpp concentration as a function of growth rate (pc = pc0 exp(–μ/μp) (black line), where μ denotes the growth rate, pc denotes the concentration of ppGpp, and pc0 and μp are constants. ppGpp concentration in the presence of Cm is estimated from the data in panel C with the Hill function mentioned above. See details of the analysis in Materials and Methods. Download FIG S2, EPS file, 0.5 MB.Estimated change in free RNAP concentration from the ratio of GFP production rates from the PLtet and P5 promoters, and estimated change in ppGpp effect on P1 transcription and ppGpp concentration from the ratio of GFP production rates from the P1 and P5 promoters. (A) Measured ratio of GFP production rates from PLtet and P5 as a function of growth rate. The symbols indicate the experimental data. The black line denotes the equation of the ratio of transcription rates from PLtet and P5 without Cm, i.e.,  and Hwa . These dwth rate . These dCopyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gfpmut2 gene rather than at the transcription initiation level. In summary, regulation of transcription initiation by ppGpp cannot solely explain the change in GFP expression from different promoters with increasing sublethal levels of chloramphenicol. However, the shared increase in GFP concentration observed for both the P1 and P5 promoters is consistent with an overall increase in the translation rate, as observed previously in these experimental conditions  is significantly greater than in their presence . Increasnditions , 21.Since the translation rate of GFP is shared by the three promoter constructs, it is possible to obtain an estimate of the magnitude of the promoter-specific effect of ppGpp, and of changes in free RNAP, on the transcription rate by measuring the ratios of GFP production rates. This operation “cancels out” the translation component of gene expression and isolates the transcription-specific effect as the ratio of promoter activities . In the presence of Cm, the fold increase of gene expression from P1 is greater than the one of P5 in the cells that are grown without cAA, consistent with a decrease in ppGpp levels by the addition of the antibiotic . As the in vivo  can be obtained from the respective RNAP binding constants, iK∈{K1, K5, KLtet} using fc/(iK + fc). The differences in binding affinity for each promoter can be estimated based on a statistical-mechanical selection model developed by Berg and von Hippel (E is the difference between the binding energies (see a(K5 + fc)/(KLtet + fc), where a is a scaling factor that accounts for the difference in transcription initiation frequency, and the RNAP concentration at the different growth rates is obtained from a previous study  estimation of the affinity of RNAP-promoter interaction from the promoter sequence, (ii) estimation of the change in ppGpp concentration from the analysis of the effect of Cm on GFP expression from the P5 and P1 promoters, (iii) estimation of the change in transcription and translation rates as a function of Cm, (iv) a quantitative model of the probability of a stalled ribosome as a function of gene length and as a function of chloramphenicol concentration, (v) analysis of previously published data sets showing that a decrease in translation processivity can result in decreased expression of late operon genes, (vi) quantitative model of the probability of a stalled ribosome leading to mRNA degradation that can explain the effect of chloramphenicol on gene expression as a function of gene position in an operon, and (vii) the role of the discriminator region in determining changes of gene expression under translation limiting conditions. Download Copyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00575-20.4FIG S3lacZ gene . The activities of all the proteins were divided by the activity of the wild-type protein. (B) The dependence of growth rate on the Cm concentration can be fit with equation 18 (in Text S1) that we derived based on the work of Dai et al.  Determination of β0 by fitting experimental data of Tsung et al.  in the ai et al. . (C) Thei et al. . (D) Thei et al.  is used.Copyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the x axis. The positions of these black vertical bars relative to the x axis indicate the concentrations of free RNAP.Because of their different affinities for RNAP, the decrease in free RNAP with increasing chloramphenicol has a stronger effect on PLtet first, then on P1, and finally on P5. Note that in order for the ratios obtained from the experimental results to be coherent with the RNAP binding curves, the P5 promoter needs to be nearly saturated by RNAP in the absence of Cm, consistent with the high affinity of the interaction resulting from the consensus −10 and −35 sequences of this promoter. This simple model of RNAP-dependent capacity can explain the data in Therefore, from the values of the RNAP affinities for the two constitutive promoters, PLtet and P5, and the relative changes in promoter activity, it is possible to estimate the change in the amount of free RNAP in the cells as a function of increasing translation limitation. Using the ratio of the promoter activities of P1 and P5, it is possible to estimate the change in ppGpp as a function of growth rate and as a function of Cm , in a faIn summary, the changes in transcription activity measured by the decrease in the PLtet-to-P5 and P1-to-P5 ratios are in line with a decrease in free RNAP concentration. This decrease is independent of a ppGpp-mediated repartition between ribosomal and nonribosomal promoters, since it can be observed to the same extent in the growth media with and without cAA , which hrpoB and rpoC, coding for β and β′, are among the longest genes in E. coli with a length of 4,029 bp and 4,224 bp, respectively .10.1128/mSystems.00575-20.5FIG S4FIG S4, EPS file, 0.1 MB.Increasing concentration of chloramphenicol result in the same change in GFP/RFP independently of the efficiency of the terminator. Fractional change in GFP/RFP as a function of growth rate with increasing Cm concentrations , from the data in Copyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00575-20.6FIG S5P value for overrepresentation of the intersection between the blue and red sets is 1.5 × 10−32, and the P value for underrepresentation of the intersection between the red and green sets is 0.012. (C) Distribution of the similarity of the −8 to −1 promoter sequence compared to the consensus sequence of the discriminator region. In producing the distribution of random sequences, the bias in base usage of E. coli genome (GC content = 43.4%) was considered. The Kolmogorov-Smirnova test for the data set of the R sector inhibited by ppGpp and that of R sector uninhibited by ppGpp plus other sectors gave a two-sided P value of 0.005. Download FIG S5, EPS file, 0.3 MB.A subset of R-sector protein expression is regulated by ppGpp. (A) Schematic diagram of the divergent change of R-sector and P-sector protein concentrations under translation limitation based on reference Copyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00575-20.7FIG S6FIG S6, EPS file, 0.8 MB.Genes whose product belongs to the R sector tend to have promoters with a higher affinity for RNAP, and most of the short R-sector proteins are ribosomal proteins. The list of ribosomal proteins are obtained from Ecocyc . A and  considerCopyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The processivity of translation, the probability of the ribosome reaching the end of the gene before stalling and falling off, has been shown to decrease exponentially with increasing gene length . We reasPhit that a ribosome will be “hit” by chloramphenicol before reaching the end of a mRNA of a given length −1 . RibosoM·min)−1 ], L indiss ratio .gfpmut2 (714 bp), and lacZ , in the presence of increasing chloramphenicol concentrations , while for GFP, it is 6% (238 aa).To test this model, we compared the changes in gene expression from the same constitutive promoter, P5, of two different genes, trations . Using eThe experimental results show that while the concentration of GFP increases as a function of Cm concentration, β-galactosidase concentration decreases . Figure lac operon and those in the ribosomal protein operons S10 and Spc  and GFP, whose genes have been placed within the same operon, where the gfp gene . Increasgfp gene  and to dgfp gene   Gene operon composition as a function of protein sector. R indicates the R sector, NR indicates the other sectors. (C) Difference in the slope of protein concentration as a function of Cm between two genes of the same operon. The slope of the upstream gene is subtracted from the slope of the downstream gene. Note: slope values are not available for all genes in an operon. (D) Frequency of the difference between the arctangent function of the slope of the downstream gene and the upstream gene. (E) Frequency of change in slope sign between genes in the same operon. The sector partition and slopes used in panels A, C, and D are from Hui et al. . DownloaCopyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00575-20.9FIG S8gfpmut2 gene for each promoter construct. The first row is the same data as in K5. The transcription rate was then used to estimate the translation rate that results in the GFP expression rate data in the first row. This translation rate was then used to estimate the transcription rates for PLtet and P1. A similar result is obtained by estimating the transcription rates for PLtet and P1 from their RNAP-promoter dissociation constants. Download FIG S8, EPS file, 0.3 MB.Estimation of the change in transcription and translation rate of the Copyright © 2020 Zhang et al.2020Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Here, we have used the approach developed by Hans Bremer and colleagues –25, usin–Perhaps surprisingly, the translation rate increases in the presence of sublethal levels of antibiotics targeting ribosomes  19). Th. Th19). 40–40–The evidence provided here points to a possible cellular adaptation mechanism leading to a reduction in transcription capacity when ribosome activity is compromised. Decoupling of transcription and translation can have several deleterious effects, including mRNA degradation and R-loop accumulation that can interfere with DNA replication, causing genome instability and increased mutation rates . MoreoveThe increase in translation rate in the presence of sublethal concentrations of chloramphenicol can help explain the similarity in the change in GFP concentration when it is expressed from the two promoters, P1 and P5, that are differentially regulated by ppGpp but with an equivalently high affinity for RNAP. This similarity also indicates that the decrease in ppGpp, while it does have an effect on the transcription from P1 , does noThe repartition of RNA polymerase instead results from the competition among promoters for a smaller pool of available enzyme and therefore depends on the affinity of their interaction, as can be observed on the data obtained comparing GFP expression from the high-affinity P5 promoter with the lower-affinity PLtet promoter. The decrease in the amount of free RNAP estimated by measuring the ratio of GFP production rates from these two constitutive promoters (PLtet/P5) is about 10-fold, independently of the presence of amino acids in the growth medium  and therE. coli, the β and β' subunits , pointing to different strategies of cellular adaptation to changing growth conditions and limitations. Understanding how bacteria modulate their growth rate and resource allocation in response to inhibition of growth has paramount importance in biotechnological and health applications –55. The –gfpmut2 coding for a fast-folding GFP . The promoters include two constitutive promoters   has been described previously (gfpmut2) and β-gal (lacZ) is GAAGGAGAU; for RFP (mCherry), it is AGAGGAGAA.GFP and β-galactosidase  were used as the reporter proteins to measure the rate of gene expression from a specific promoter. The GFP gene used is ding GFP . The β-g plasmid . CompariPLtetO-1 ) and a smut2 for  were insmut2 for  were at eviously . The ribeviously . The RBS610) every 7 min. The autofluorescence measured from the wild-type strain BW25113 was subtracted from the fluorescence of the fluorescent strains at the same OD (dependent on the medium). The experimental procedure of the β-galactosidase assay followed the protocol of Zhang et al. , 0.5% glucose (glu), 0.5% glycerol plus 0.2% Casamino Acids (cAA+gly) and 0.5% glucose + 0.2% Casamino Acids (cAA+glu). The preculture was obtained from the inoculation of one bacterial colony in LB growth medium. After overnight growth, the seed culture was washed once with phosphate-buffered saline (PBS) and diluted 200 times with the corresponding growth medium containing a specific concentration of chloramphenicol (Cm). This culture was diluted again 200 times once it reached exponential phase. The cultures were grown in flasks, shaking at 37°C and 170 rpm, and optical density and fluorescence were measured with a plate reader (Tecan Infinite 200Pro) every 30 to 50 min. Alternatively, the cultures were grown in a 96-well plate, with 150 μl of bacterial culture per well covered by 70 μl mineral oil. The culture plate was kept at 37°C in the plate reader, shaking and measuring fluorescence and optical density at 610 nm /γ and γ is a decay factor from taking into account that the reaction product o-nitrophenol is volatile β-galvolatile . The ratin vivo  of P5 and PLtet asfc denotes the free RNAP concentration, K5 and KLtet are the dissociation constants for P5 and PLtet, respectively, and a is a scaling factor that accounts for the difference in transcription initiation frequency. The transcription initiation frequency, or promoter escape, is higher at PLtet than at P5, resulting in an increased probability of GFP expression for each binding event, and is assumed to be independent of RNAP concentration. This can explain why the GFP production rate (Gpr) PLtet-to-P5 ratio is greater than 1  can be represented as an exponential function of the difference of the corresponding binding energies (jE – iE), i.e., iK) is known, the other one, (jK), can be estimated with the formula The ratio of two dissociation constants  obtained from 4We assume that the translation rate of GFP from the three promoter-If the difference in RNAP binding affinities for P5 and PLtet are known , the change in Gpr ratio with growth rate or with the Cm concentration can be used to estimate the change in free RNAP concentration  can be obtained by fitting the data of Klumpp and Hwa (fc = Aexp(–μr/μ), giving A = 6.82log μm−3 and μr = 0.11 doublings/hour (a and K5 by fitting the Gpr ratio data with a = 10.5 and K5 = 90 μm−3. Finally, we can use It is also possible to estimate the absolute free RNAP concentration in the cell from the available data on the change in the concentration of free RNAP as a function of growth rate  and our  and Hwa , with longs/hour . The changs/hour  is consings/hour . We can


Star transcription, under basal conditions and following a robust endotoxic stress challenge. Rat adrenal glands and blood plasma were collected following i.v. administration of either an ultradian‐sized pulse of ACTH or administration of lipopolysaccharide, as well as under unstressed conditions across the 24‐hour period. Plasma ACTH and corticosterone (CORT) were measured and the adrenal glands were processed for measurement of protein by western immunoblotting, RNA by a quantitative reverse transcriptase‐polymerase chain reaction and association of CRTC2 and CRTC3 with the Star promoter by chromatin immunoprecipitation. An increase in nuclear localisation of CRTC2 and CRTC3 followed increases in both ultradian and endotoxic stress‐induced plasma ACTH, and this was associated with increased CREB phosphorylation and corresponding increases in Star transcription. Both CRTC2 and CRTC3 were shown to associate with the Star promoter, with the dynamics of CRTC3 binding corresponding to that of nuclear changes in protein levels. CRTC isoforms show little variation in ultradian expression or variation across 24 hours, although evidence of long‐term down‐regulation following endotoxic stress was found. We conclude that co‐transcription factors CRTC2 and, more clearly, CRTC3 appear to act alongside phosphorylated CREB in the generation of ultradian pulses of Star transcription, essential for the maintenance of basal StAR expression. Similarly, our findings suggest CRTC2 and CRTC3 mediate Star transcriptional initiation following an endotoxic stressor; however, other transcription factors are likely to be responsible for the long‐term up‐regulation of adrenal Star transcription.The activation and nuclear translocation of cAMP‐response element binding protein (CREB)‐regulated transcription coactivator (CRTC)2 occurs in the rat adrenal gland, in response to adrenocorticotrophic hormone (ACTH) and stressors, and has been implicated in the transcriptional regulation of steroidogenic acute regulatory protein (StAR). We have recently demonstrated the activation of CRTC isoforms, CRTC1 and CRTC3, in adrenocortical cell lines. In the present study, we aimed to determine the activation and expression of the three CRTC isoforms in vivo in relation to  The present study aimed to determine the in vivo dynamics of activation and expression of the three cAMP‐response element binding protein (CREB)‐regulated transcription coactivator (CRTC) isoforms in relation to adrenocorticotrophic hormone (ACTH)‐induced steroidogenic acute regulatory (StAR) transcription. Nuclear localisation and association of CRTC2 and CRTC3 at the Star promoter followed increases in plasma ACTH induced by ultradian and endotoxic stress activity. Corresponding with increased CREB phosphorylation and increases in Star transcription, we conclude that CRTC2 and CRTC3 appear to play a role as co‐transcription factors alongside phosphorylated CREB in the initiation of Star transcription. The activity and expression of these enzymes, particularly that of rate‐limiting steroidogenic acute regulatory (StAR) protein, is tightly regulated by ACTH, predominantly through phosphorylation by the cAMP/PKA pathway.Star transcription is cAMP response element binding protein (CREB), directly regulated by ACTH through phosphorylation.Star transcription in vitroStar promoter in response to ACTH, suggesting a role for these isoforms in mediating the initiation of Star transcription.A key transcription factor involved in the regulation of Under basal (unstressed) conditions, both ACTH and CORT levels fluctuate in both an ultradian and a circadian mannerStar promoter during the initiation and sustained transcription of Star in response to an ACTH ultradian pulse and following acute endotoxic stress. Furthermore, because several key steroidogenic regulators exhibit an ultradian and circadian pattern of expression in basal condition, as well as in response to stress, we investigated whether CRTC 1‐3 also exhibit strong endogenous regulation of transcription and expression.The present study aimed to examine whether the activity and expression of the endogenous CRTC isoforms in vivo in the rat adrenal gland exhibit a similar pattern of activity and expression as observed for other key regulators of CORT synthesis. Specifically, given the differential activation across CRTC isoforms observed in vitro, we aimed to determine the extent to which endogenously expressed CRTC1, CRTC2 and CRTC3 may be activated by translocation and associated with the 22.1am) at 21 ± 1°C with access to food and water available ad lib. Rats were housed four to a cage but were singly housed following cannulation surgery. All animal procedures were approved by Animal Welfare Ethical Review board of University of Bristol and conducted in accordance with Home Office guidelines and the UK Animals (Scientific Procedures) Act, 1986 (Project Licence number: 30/3043).All experiments were conducted in adult male Sprague‐Dawley rats . Animals were given a 1‐week period of acclimatisation prior to surgery or the commencement of experimental procedures. All animals were maintained under a 14:10 hour light/dark photocycle  and diazepam , as described previously.am. Rats were administered with either an ultradian‐sized ACTH pulse  or LPS  via their indwelling catheter. Rats were overdosed with 0.2 mL of Euthatal  via the indwelling cannula at the time points indicated.In the ultradian ACTH stimulation and lipoploysaccharide (LPS) injection experiments, rats were surgically implanted with an indwelling cannula in the jugular vein to allow for i.v. injections, under anaesthetic with a combination of Hypnorm .For investigation of Immediately after death, as indicated above, trunk blood was collected following decapitation. Blood was then processed for measurement by radioimmunoassay of plasma ACTH  and corticosterone, as described previously.2.3A260/A280 range 1.8‐2.1) and 230 nm (A260/A230 range > 1.5). cDNA was reverse transcribed from 1 µg of total RNA using an AMV first‐strand synthesis system kit . qPCR primers were designed for both primary transcripts, spanning an intronic‐exonic region to detect heteronuclear RNA (hnRNA) and mature mRNA, as shown in Table ‐1 for each primer and 2 μL of cDNA, with a final volume of 15 μL, in a StepOne Plus RT‐PCR machine (Applied Biosystems). Briefly, samples underwent denaturation at 50°C for 2 minutes, 95°C for 20 seconds, followed by 40 cycles at 95°C for 3 seconds and 60°C for 30 seconds. Levels were calculated using relative quantification by standard curve, normalised to glyceraldehyde 3‐phosphate dehydrogenase (Gapdh) mRNA, as determined in separate qRT‐PCR reactions. Gapdh mRNA stability was confirmed by statistical analysis, as described below. Absence of detection when omitting the reverse transcription enzyme Superscript III (Invitrogen) indicated a lack of genomic DNA contamination.RNA was extracted using TRIzol reagent and then purified using RNeasy mini kit reagents and column deoxyribonuclease digestion  to remove genomic DNA contamination. RNA was tested using a NanoDrop spectrophotometer  to check quantity via measurement of optical density at 260 nm and quality by via measurement of optical density at 260 nm  in accordance with the manufacturer’s instructions. Nuclear and cytosolic extract from each sample (13 µg) were separated in Tris‐Glycine gels, transferred to polyvinylidene fluoride membranes and blocked for 1 hour in Tris‐buffered saline‐Tween 20 with 5% milk. Immunoblots were then incubated at 4°C, as indicated in Table Whole cell lysate extraction was performed as described previously.2.5‐1; catalogue no. ab109081; RRID: AB_10859591; Abcam, Cambridge, MA, USA), anti‐CRTC3 antibody  or ChIP‐grade non‐specific rabbit IgG . Samples were stored at −80°C prior to DNA quantification by qRT‐PCR, as described above. Primers spanning the three putative CRE sites at −210 bp to −37 bp of the Star promoter  or exon 5 of the Star gene , used as a negative control, were designed using NCBI primer‐blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast) as determined via NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank). Promoter pulldown was quantified using rat genomic DNA, normalised to the total levels in the chromatin input (promoter content in chromatin not subjected to immunoprecipitation).Immediately following collection adrenal glands (n = 3‐4 independent rats per group) were fixed and chromatin was extracted as described previously.2.6P < 0.05 was considered statistically significant, with a trend defined as P < 0.1. prism, version 8  was used for the statistical analysis. For each am‐pm data set, a sine function (with a fixed 24 hours period) was numerically fitted to the experimental data in the least‐squares sense using matlab . The acrophase was then taken as the clock time corresponding to the peak value in the fitted curve.For these experiments, no animals were excluded from the study. Rats were allocated to treatment/timepoint groups using randomisation within blocks, where each block represents a different procedural day. Researchers were blind to treatment/timepoint allocation when performing a radioimmunoassay for plasma ACTH and corticosterone measurement and for RNA quantification by qRT‐PCR. Western immunoblot gels were not blinded, aiming to ensure that, where possible, each time point was represented on each gel; however, quantification of proteins bands was performed using ImageJ; thus, the risk of bias was minimised. All data are expressed as the mean ± SEM of values obtained from a minimum of three independent experiments. Normal Q‐Q plots were used to ensure that data appeared normally distributed, whilst homogeneity of variance was verified using the Bartlett's test. Where data showed equal variance, they were analysed using one‐way analysis of variance (ANOVA) or two‐way ANOVA, as indicated. Where one‐way ANOVA or two‐way ANOVA was significant, this was followed by Tukey’s post‐hoc analysis to compare all means with every other mean. Where Bartlett's test showed significant variance between data groups, Welch’s ANOVA was used, as indicated. Where Welch’s ANOVA was significant, this was followed by Games‐Howell post‐hoc analysis to compare all means with every other mean. The Games‐Howell test has a similar formulation to Tukey's test, although it does not assume equal variances and sample sizes. 33.1F5,14.14 = 2.909; P = 0.052 overall by Welch’s ANOVA)     , we found a significant effect on nuclear levels of both CRTC2  and CRTC3 . Nuclear levels of CRTC2 and CRTC3 were maximal at 5 minutes after the ACTH injection, although this increase was not significant by pairwise analysis ; however, the subsequent decrease in nuclear CRTC2 and CRTC3 levels showed a trend for significance .An ultradian pulse of ACTH was administered in rats (10 ng i.v.) to induce an adrenal response similar to that observed in basal (unstressed) conditions. As recently reported,) Figure  induced ) Figure  that was) Figure  and Star) Figure . Similar) Figure . ACTH ha) Figure . Althoug3.2F7,11.79 = 50.25; P < 0.0001 overall by Welch’s ANOVA)    , although levels of CRTC2  and CRTC3  were significantly decreased by 180‐240 minutes . Changes in nuclear levels of CRTC1 were not detected , whereas there was a significant increase in nuclear localisation of CRTC2  and CRTC3 . Nuclear levels of CRTC2 were maximal by 30 minutes (P = 0.002 0 minute vs 30 minutes by Tukey’s post‐hoc test) and CRTC3 by 60 minutes, although this increase was not significant (P = 0.115 0 minute vs 60 minutes by Tukey’s post‐hoc test), with levels similar to basal by 180 minutes.To activate the adrenal gland response to endotoxic stress, rats were injected i.v. with LPS Figure . As prev) Figure , CORT (F) Figure  and in a) Figure . This wa) Figure . Similar) Figure . Cytosol3.3Star promoter, ChIP assays were performed following injection of 10 ng of ACTH ; however, there was no significant change in levels of CRTC2 binding over time . For CRTC3, binding at the Star promoter was significantly higher than at exon 5  and this binding increased significantly following ACTH administration , differing from that at exon 5 . CRTC3 pulldown of the Star promoter was maximal at 5 minutes, falling by 15 minutes. Comparatively, non‐specific pulldown of Star promoter was not significantly different from that of exon 5 by non‐specific rabbit IgG , with no effect of time  or interaction . Following an endotoxic stressor  and there was no significant overall effect of time  or interaction . Within measurement of binding at the Star promoter only, however, there was a trend for an effect of time on increased CRTC2 binding , with maximal pulldown at 30 minutes (P = 0.067 0 minute vs 30 minutes by Tukey’s post‐hoc test). CRTC3 binding following LPS administration was significantly increased at the Star promoter compared to exon 5 , with an overall significant effect of time  and interaction . Peak binding of CRTC3 to the Star promoter was seen at 30 minutes (P < 0.0001 0 minute vs 30 minutes by Tukey’s post‐hoc test), remaining partially elevated at 120 minutes (P = 0.025 0 minute vs 120 minutes by Tukey’s post‐hoc test). Again, non‐specific pulldown by rabbit IgG saw no significant effect of time  or interaction , although pulldown of Star exon 5 was significantly elevated compared to that of the Star promoter .To determine whether significant increases in adrenal nuclear CRTC2 and CRTC3 in response to ACTH were associated with increased binding of these isoforms at the H Figure  and 25 µH Figure . FollowiH Figure , CRTC2 br Figure , CRTC2 d3.4F5,28 = 1.693; P = 0.169 overall by one‐way ANOVA)    Figure  and CORT) Figure . No sign) Figure .Crtc1  and Crtc3  hnRNA levels but not in Crtc2 . We then investigated whether CRTC transcription will be affected by endotoxic stress , peaking between 10 and 20 minutes and falling below basal by 60‐240 minutes.Interestingly, many transcription factors exhibit an ultradian pattern of transcription in response to a pulse of ACTH.s Figure . hnRNA l4Star transcription, as measured by Star hnRNA levels. Furthermore, using a ChIP assay, we confirm that association of CRTC2 and CRTC3 with the Star promoter occurs in vivo, and demonstrate clear stimulation of CRTC3 binding at the Star promoter in response to ultradian and endotoxic stress stimuli during periods of increased Star transcription. Furthermore, we show that levels of Crtc1 and Crtc3 transcription changes in response to ultradian HPA activity, whereas endotoxic stress leads to a prolonged down‐regulation of Crtc1, Crtc2 and Crtc3 transcription.In the present study, we demonstrate that nuclear translocation of endogenous CRTC2 and CRTC3 in the rat adrenal gland occurs in response to both basal, ultradian HPA axis activity, and following endotoxic stress, thus complimenting the findings from previous in vitro and in vivo studies in which activation of CRTC2 and CRCT3 was shown in response to ACTH and restraint stress.Star transcription are consistent with the results reported previously.Star hnRNA levels closely mimicked that of plasma CORT increasing within 5 minutes in response to a low dose ‘ultradian’ pulse and within 20 minutes following an endotoxic stressor. Similarly, peak nuclear localisation of endogenous CRTC2 and CRTC3 was seen at 5 minutes following an ultradian ACTH pulse, and this is consistent with the activation profile of CRTC2 following administration of a small dose of ACTH in methylprednisolone‐suppressed rats.The observed changes in the levels of Star transcription. This potentially indicates that CRTC1 is less responsive to ACTH signalling than CRTC2 or CRTC3. Elsewhere, CRTC1 has demonstrated a limited increase in nuclear translocation following forskolin treatment in hypothalamic 4B cells, HeLa cells and Hek293 cells.Low dose ACTH and endotoxic stressor induced nuclear translocation of endogenous CRTC1, although with no statistical significance, suggesting that CRTC1 may still play a role in mediating ACTH‐induced Star hnRNA and CREB phosphorylation. This result supports a role for CRTC2 and CRTC3 in the initiation of Star transcription, through co‐activation of CREB. This relationship was further addressed by measuring the dynamics of CRTC2 and CRTC3 binding at the Star promoter. Although binding of CRTC2 at the Star promoter was detected by ChIP, no significant increase was detected following ACTH administration. This may suggest a potential for constitutive binding of CRTC2 under basal conditions. However, previous research investigating CRTC2 binding at the Star promoter in Y1 and ATC7‐L cells in vitro do not support this,Star hnRNA levels was also observed. Thus, our data show that CRCT3 binding to the Star promoter occurs earlier than previously shown in vitro Star promoter by 30 and 15 minutes ACTH treatment, respectively). Similarly, binding of CRTC3 and a trend for binding of CRTC2 at the Star promoter showed peak levels at 30 minutes following an endotoxic stressor. Interestingly, although the amplitude of increases in pCREB levels were far greater following LPS administration compared to 10 ng of ACTH, the size of increase in both nuclear CRTC2 and CRTC3, as well as that of Star hnRNA levels, was of a similar magnitude following both an ultradian stimulus and an endotoxic stressor. This supports the hypothesis that initiation of ACTH‐induced Star transcription, is dependent on CRTC co‐activation of CREB, with phosphorylation of CREB alone being insufficient to potentiate Star transcription.Nuclear localisation of CRTC2 and CRTC3 tightly matches increases in Star transcription, suggesting these isoforms act through a similar mechanism.With a similar pattern of activation seen in the present study for CRTC2 and CRTC3, our findings suggest that these isoforms play a similar role within ACTH‐mediated adrenal transcription, suggesting functional redundancy. We have previously shown that knockdown of either CRTC2 or CRTC3 in vitro resulted in similar levels of reduction of ACTH‐induced Star hnRNA, suggesting that termination of Star transcription is tightly linked to termination of CRTC and CREB activity. However, the delayed depletion of Star hnRNA levels may be the result of the processing time taken for hnRNA levels to be spliced into mature RNA, and this pattern of StAR hnRNA is similar to the 15‐minute delay between the detection of increased Star hnRNA and mRNA levels previously shown in Y1 cells.Star promoter declined after 30 minutes; however, CRTC3 binding was still significantly high at 2 hours and returned to basal by 3 hours, whereas Star hnRNA levels remain significantly high at 3 hours and returned to basal by 4 hours, suggesting that Star transcription continued via mechanisms involving other transcription factors and co‐regulators. Indeed, protein levels of SF‐1 have been demonstrated to increase by 6 hours after LPS injection,Nr4a3 (aka Nor1) and Nr4a1 (aka Nur77).Star promoter by 1 to 4 hours of stimulation with cAMP in MA10 cells, although not at 30 minutes.Star promoter activity in MA10 cells,Star hnRNA splicing to mRNA in MA10 cells have also suggested a delayed secondary onset of transcription factors, such as Nur77 and C/EBPb, after 60 minutes of incubation with cAMP,Star transcription,Star transcription will require further investigation.Deactivation of CRTC2 and CRTC3 also occurred very rapidly following ACTH injection, and prior to decreases in nuclear pCREB and Crtc1‐3 hnRNA levels presented only small initial increases, with hnRNA levels falling significantly by 30 minutes. Conversely, other transcription factors, including SF‐1, Nur77 and Nor1, exhibit circadian variation in expression.Nr5a1, Nr4a1 and Nr4a3 are all increased in response to 10 ng of ACTH, behaving similarly to Star.Crtc1 mRNA has been shown during the day.Crtc1, Crtc2 and Crtc3, as well as levels of both cytosolic and nuclear CRTC2 and CRTC3 protein, decreased significantly below baseline, suggesting negative‐feedback on CRTC isoform expression in the adrenal gland as a result of the long‐term up‐regulation of Star transcription, Further studies are needed to explore this hypothesis.Basal expression of CRTC isoforms appear to show little variation in the adrenal gland, with no significant changes in CRTC1, CRTC2 or CRTC3 protein levels across 24 hours. Similarly, following an ultradian ACTH pulse, Star transcription, in response to both basal ultradian ACTH activity and following stress. Our results support a role for CRTC2 and CRTC3 to function alongside pCREB to mediate rapid adrenal Star transcriptional initiation, aiming to generate ultradian pulses of Star transcription and maintain basal StAR expression levels, as well as initiate Star transcription in response to HPA activation following endotoxic stress. However, these coactivators appear to play less of a role in the long‐term up‐regulation of the stress‐induced transcriptional response of the adrenal gland, indicating the participation of additional transcription factors. Although our findings have focused on measuring the expression of endogenous CRTC1‐3 and their patterns of activation in relation to HPA‐induced Star transcription, future research will require functional studies to allow the direct demonstration of this link.In conclusion, our data show that CRTC2 and, more distinctly, CRTC3 appear to be key co‐transcription factors in the regulation of The authors declare that they have no conflicts of interest.Lorna Smith: Formal analysis; Investigation; Methodology; Writing – original draft; Writing – review & editing. Zidong Zhao: Investigation. Jamie Walker: Formal analysis; Investigation. Stafford Louis Lightman: Conceptualisation; Funding acquisition; Supervision. Francesca Spiga: Conceptualisation; Formal analysis; Funding acquisition; Investigation; Methodology; Supervision; Writing – original draft; Writing – review & editing.Table S1‐S3Click here for additional data file.