TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages – a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs. One group of TFs in this category is the TALE (three amino acid loop extension) family of homeodomain proteins. The TALE family includes Pbx, as well as the closely related Prep and Meis proteins  produces severe embryonic defects (Many transcription factors (TFs) involved in vertebrate embryogenesis are expressed across relatively large time windows that encompass a variety of cellular and morphological changes. While it seems likely that such TFs function by the same mechanism throughout embryogenesis, there is no proteins . Pbx andproteins , locatedproteins . TALE faproteins , Rnx  are also downregulated in our analysis. Similarly, of six genes downregulated in Prep loss-of-function zebrafish  are downregulated in our experiment. This suggests that our RNA-seq analysis captured a comprehensive set of TALE-dependent genes. We conclude that TALE TFs control a gene regulatory network (TALE GRN), which instructs anterior embryonic development and that becomes operative between 6hpf and 12hpf.TALE factors play a key role in early vertebrate embryogenesis, as evidenced by the phenotypes observed in TALE loss-of-function animals. In particular, loss of ac edema , but theh embryo . We usedh embryo . Comparih embryo  when TALh embryo , but exth embryo . Specifih embryo  is alterh embryo , consisth embryo , suggesth embryo , seven  and ~24,200 peaks at 12hpf (Prep12hpf), the majority of which are located within 30 kb of a transcription start site . We noticed that although the Prep12hpf-only peaks do not co-localize with Prep3.5hpf peaks, the two types of sites nevertheless appear to be preferentially located near one another  have become bound by Prep. Since these later sites are also associated with the TALE-GRN, Prep binding is dynamically and continuously associated with the TALE GRN during zebrafish embryogenesis.We conclude that Prep occupies genomic binding sites associated with the TALE GRN as early as late blastula stages. ~60% of these sites are also occupied at segmentation stages, but by this stage a large number of additional binding sites  motif, but not the DECA motif  than at Prep3.5hpf peaks (5%). It is surprising that HEXA sites are not occupied by Prep at blastula stages and we considered the possibility that HEXA sites may not be accessible at this stage. We made use of previously published ATAC-seq data . Since CA motif . HEXA moCA motif  and haveCA motif . AccordiCA motif , at the ly peaks . This Holy peaks , 2000. WXA motif . Consistseq data  to examiseq data , suggestWhile both DECA and HEXA sites have been reported previously, our data show for the first time that there is a temporal order to how TALE factors utilize these motifs during embryogenesis. Specifically, TALE factors occupy primarily DECA sites at blastula stages and these motifs remain occupied at least until segmentation stages, but by segmentation stages additional binding sites become utilized so that TALE factors also occupy HEXA motifs associated with binding sites for tissue-specific TFs such as Hox proteins.12hpf-only peaks are found at highly conserved regions of the genome  that we observe at 12hpf is very likely to represent enhancer elements.Previous analyses of individual DNA elements containing HEXA motifs adjacent to PBX:HOX motifs demonstrated that these act as enhancers in mouse and zebrafish . Converse genome  and are e genome . Finallye genome , 19  identified Prep as bound to DECA motifs ; see als. We findebrafish , indicatebrafish , althougWe noticed that a subset of MPADs shows detectable enrichment for the repressive H3K27me3 histone modification . To examHence, MPADs can be further subdivided such that Class 1 and 2 display active chromatin marks and are found near genes expressed at 6hpf. In contrast, Class 4 MPADs are marked by H3K27me3 and are associated with genes involved in developmental processes, but these are not highly expressed at 6hpf. Class 3 MPADs are only marked by H3K4me1 and genes associated with this class show an intermediate level of expression at 6hpf, but they are not enriched for specific biological functions. We conclude that the chromatin state of MPADs correlates with the biological function of nearby genes and that developmental control genes are primarily associated with repressed (H3K27me3-modified) Class 4 MPADs.We next examined whether chromatin modifications at MPADs change as embryogenesis progresses by comparing their H3K27ac status at the blastula stage (4.5hpf) to that at late gastrula (9hpf) – when the embryonic axes have formed and organogenesis is beginning. We find that Class 1 and 2 MPADs undergo a reduction in the level of H3K27ac modification from 4.5hpf to 9hpf , while RThe fact that developmental control genes are associated with Class 4 MPADs suggests that the TALE GRN genes may fall into this category. Indeed, we find that TALE GRN genes are significantly associated with Class 4 (and Class 3), but not Class 1 or 2, MPADs . A close3.5hpf peaks  die in utero prior to embryonic day 8.5 . EKW zebrafish were kept in groups of 10 individuals under constant water flow at 28°C. To collect embryos, 2 males and three females were crossed for 30 min. Subsequently, the embryos were collected in egg water . After 2 hr, dead and un-fertilized embryos were manually removed and the remainder left to develop until they reached the appropriate developmental stage and then used in the experimental procedures described below.pbx2, pbx4 and prep1.1 as reported previously  or a coeviously . The use embryos , demonst mutants , as expefunction , furtherSample size was not selected based on statistical analysis, but on previous published reports demonstrating that these reagents produce phenotypes in >85% of injected embryos . EmbryosPCS2 + plasmids containing the NF-YDN or PBCAB coding sequence was linearized by NotI digest and purified with a PCR purification kit column (Qiagen). Capped messenger RNAs were synthesized using the SP6 mMessage mMachine kit (ThermoFisher Scientific) from 2 ug of linearized plasmid following manufacturer's instructions. The DNA template was then removed by the addition of 2 µl of TURBO DNase and incubation at 37°C for 15 min. Subsequently, synthesized capped mRNAs were purified on the RNeasy kit columns (Qiagen), quantified on a Nanodrop (ThermoFisher Scientifics) and their quality assessed on a 2% agarose gel.300 pg of mRNA or a mixture of morpholinos  mixed with water and 0.1% phenol red dye were injected into 1 to 2 cell stage zebrafish embryos. Following the injection, embryos were raised to the desired time point and used for experimental procedures.For whole-mount immunostaining, 48hpf embryos were fixed in 4% paraformaldehyde/8% sucrose/1x PBS overnight. Fluorescent staining with the 3A10 primary antibody  and the goat anti-mouse Alexa Fluor 488 secondary antibody  was used to detect Mauthner neurons. For assessment of cartilage formation, 5dpf embryos were fixed in 4% paraformaldehyde/1X PBS overnight, bleached in 30% hydrogen peroxide for 2 hr and stained overnight in 1% HCL/70% ethanol/0.1% alcian blue.Groups of 500 zebrafish embryos  were dissociated in 1XPBS by pipetting and fixed for 10 min in 1% formaldehyde. Fixation was stopped by the addition of glycine to a final concentration of 125 mM and cells were pelleted and frozen in liquid nitrogen. Subsequently, cell pellets were processed following a ChIP protocol described previously . Nuclei Samples were diluted 10 times in dilution buffer  and pre-cleared by the addition of 50 μl protein-A dynabeads (ThermoFisher Scientific) and incubation for 3 hr at 4°C. After removal of the beads, 10 ul of anti-Prep or pre-bleed antiserum was added (Key Resources Table). Immune complexes were precipitated by the addition of 50 μl of protein-A dynabeads (ThermoFisher Scientific) and incubated for 3 hr at 4°C. Beads were washed five times in wash buffer , three times in LiCl buffer  and three times in TE buffer .Chromatin fragments were eluted by the addition of 50 μl of freshly made elution buffer  and incubation at 25°C for 15 min followed by an incubation at 65°C for another 15 min. Then, DNA fragments were reverse cross-linked by adding 2.5 μl of 5M NaCl and incubating at 65°C O/N. Finally, DNA fragments were recovered in 10 μl nuclease free water using a PCR purification mini-elute kit (Qiagen).ChIP DNA fragments and their corresponding input were quantified on a Qubit with the dsDNA HS assay kit (ThermoFisher Scientific). 10 ng of DNA was used for library preparation using the Tru-seq ChIP Sample Preparation Guide (Illumina Inc). For samples containing less than 10 ng of DNA the entire eluted DNA was used. Briefly, sample DNA was blunt-ended and phosphorylated, and a single 'A' nucleotide added to the 3' ends of the fragments in preparation for ligation to an adapter with a single-base 'T' overhang. Omitting the size selection step, the ligation products were then PCR-amplified to enrich for fragments with adapters on both ends. Libraries were sequenced on an Illumina HiSeq2500 Sequencer.The ChIP protocol for ChIP-qPCR is the same as described in the ChIP-seq section above except that a total of 1000 wild-type or injected embryos were collected for NF-YB and Pbx4 ChIPs and 200 embryos for Histone H3 and H3K27ac ChIPs. The following antibodies were used: 10 µl of anti-Prep1.1 and anti-Pbx4 in house sera and their corresponding pre-bleed control sera; 8 µg of anti-NF-YB rabbit polyclonal antibody and control rabbit polyclonal IgG. The relative quantification of select genomic regions was determined by qPCR using specific primers pairs see  and 2 µlTotal RNA from 50 to 100 6hpf or 12hpf zebrafish whole embryos was extracted with the RNeasy kit (Qiagen) following manufacturer's instructions. Total RNA was then used in RNA-seq and RT-qPCR reactions.Total RNA quantification and quality assessment was performed on a Bioanalyzer (Agilent) and only total RNAs with a RNA Integrity Number above nine were further considered. Then, 3 ug of total RNA was used to construct RNA-seq libraries with the Illumina Truseq stranded mRNA library kit after PolyA + RNA enrichment. The quality and size of the fragments was determined on a Bioanalyzer (Agilent) and single-end 100 bp reads were generated on a Hi-Seq sequencer at the molecular biology core of the University of Massachusetts Medical School.500 ng to 1 µg of total RNA was reverse transcribed using the high capacity cDNA kit (ThermoFisher Scientific). The relative quantity of select mRNAs was determined by qPCR: each 25 ul total PCR reaction contained 2 µl of cDNA diluted 10-fold, 0.2 mM of each specific primer see  and qPCR279A, G280A, D281A) located in the conserved NF-YA DNA binding domain, preventing NF-YA DNA binding but not interactions with the other members of the NF-Y complex, were introduced using the Q5 site directed mutagenesis kit (New England Biolabs) and primers bearing the mutations. Plasmids for luciferase reporter assays were generated by amplifying ~500 bp genomic fragments containing the Prep binding sites associated with the tle3a, pax5, prdm14, tcf3a, her6, dachb and fgf8 loci (using the primers listed in Myc-Prep1.1 (NM_131891.3), HA-Pbx4 (NM_131447.1) encoding plasmids were described previously , 2002. FAll the plasmids were validated by Sanger sequencing, amplified in DH5α bacterial cells and extracted using the PureLink HiPure Plasmid Midiprep Kit (ThermoFisher Scientific). All primer sequences can be found in 6 HEK-293T cells were seeded on 10 cm dishes and allowed to grow overnight in antibiotic-free growth medium (DMEM (Gibco) supplemented with 10% FBS (Hyclone)). HEK293T cells were obtained from ATCC (ATCC CRL-3216). These cells were not independently authenticated and were not tested for mycoplasma. The next day, the cells were incubated for 5 hr in Opti-MEM (Gibco) medium containing a mixture of plasmid DNA and Lipofectamine 2000 (Invitrogen) following manufacturer’s instructions. Subsequently, the cells were incubated overnight in fresh antibiotic-free growth medium.3 × 10Transfected cells were lysed in 4 mL of ice cold Co-IP Buffer ) and incubated on ice for 30 min. Cell lysates were centrifuged at 2,000 g for 10 min at 4°C to remove cell debris and pre-cleared by incubation at 4°C after the addition of 50 μL of Protein A/G Agarose Beads blocked in 1% BSA for 1 hr (Roche). To immunoprecipitate the target protein, 8 μg of the appropriate antibody (see Key Resources Table) was added to each sample before incubation at 4°C overnight. The next morning 40 μL of Protein A/G Agarose beads blocked with 1% BSA was added and each sample incubated for 4 hr at 4°C. Non-specific binding was eliminated by five washes in 1 mL of Co-IP Buffer. Finally, the immune-complexes were eluted in 80 μL of 1X Laëmmli Buffer (Biorad) containing 2.5% beta-mercaptoethanol and agitated for five minutes at 95°C.20 μL of each IP sample or 13 μL of each Input sample were loaded onto a 4–20% gradient polyacrylamide gel (Bio-Rad) and the proteins separated at 200V until the dye front reached the end of the gel. The separated proteins were then transferred onto a methanol-activated PVDF membrane at 100V for one hour. After incubation for one hour in blocking buffer ) the membranes were probed with specific antibodies (see Key Resources Table) diluted in TBS-Tween plus 5% BSA and incubated overnight at 4°C. The next day after four washes of 10 min in TBS-Tween the membrane were probed with the appropriate secondary antibody diluted in TBS-Tween plus 5% BSA and incubated at 4°C for two hours. After four washes of ten minutes in TBS-Tween the ECL reaction was performed and chemiluminescence detected with a LAS3000 (Fuji) machine.renilla luciferase. Luciferase was quantified using the DualGlo Luciferase system (Promega E2920) in a Perkin Elmer Envision 2104 Multiplate reader and firefly luciferase levels were normalized to renilla levels. Each assay was performed in triplicate and is presented as mean fold induction ± SD over transfection with empty vector. A vector containing the SV40 enhancer  was used as positive control.For the reporter assays, 100 or 400 ng of each luciferase reporter plasmid was co-transfected (see above for transfection protocol) with 200 ng of each TF  or with 800 ng of control plasmid, as well as together with 50 ng of a plasmid expressing Analysis of expression and ChIP data was done as outlined below using standard bioinformatics packages. Default statistics tools included in each package were used (except as indicated) and the exact parameters for each type of analysis are listed below.p-adj ≤0.05 (Benjamini and Hochberg FDR) and to compensate for the loss of one biological replicate only DEGs with p-adj ≤0.01 were used in all subsequent analyses.Fastq files containing strand specific trimmed and filtered reads were processed using the University of Massachusetts Medical School Dolphin web interface (see Key Resources Table). Reads were quality checked with FastQC aligned to the DanRer10 zebrafish transcriptome and normalized gene expression TPM (Transcripts Per Million) values calculated using RSEM_v1.2.28 with parameters -p4 --bowtie-e 70 --bowtie-chunkmbs 100 . Identifq-value threshold was set to 0.05. Candidate binding regions were then filtered to retain those with a fold enrichment of ≥10. Upon applying these criteria, we noticed that one biological replicate for each ChIP-seq experiment (3.5hpf and 12hpf) underperformed, but more than 95% of the peaks were identified also in the second biological replicate . The mean value and standard error of the mean (SEM) for three independent biological replicates of control and experimental conditions were calculated using Excel. Statistical significance of mean variations between two conditions was calculated using an unpaired t-test in Excel. Two conditions are considered significantly different if p-value≤0.05.Gene expression was determined and normalized to IP Ct value/0.5 Input Ct value). Then the mean value and standard error of the mean (SEM) for three independent biological replicates of control and experimental conditions were calculated using Excel. When necessary the results were expressed as a fold change of specific ChIP signal over control IgG ChIP signal. Statistical significance of mean variations between two conditions was calculated using an unpaired t-test in Excel. Two conditions are considered significantly different if p-value≤0.05.DNA enrichment was determined and normalized to input values using the following formula (0.5GREAT (version 3.0.0 ) allowedDAVID  All TF binding site coordinates used in the following analysis were defined as 200 bp coordinates centered on the ChIP peak summit. Unless otherwise specified, only peaks with an FE ≥ 10 were considered.The distribution of Prep binding sites relative to TSSs was calculated using the windowbed tool from the bedtools suite  in the GA gene was considered associated with a Prep binding site if any of its Ensembl (Zv9) TSS was found within 5 or 30 kb from a Prep peak. Prep-associated genes were defined using the windowbed tool from the Bedtools suite in Galaxy searching for Ensembl TSS  found within 5 or 30 kb of the center of any Prep binding site. Statistical significance of Prep binding association with genes of interest  over a random population of genes was determined with a Pearson correlation test with a statistical significance ≤0.05.The overlap between two populations of ChIP peaks was analyzed using the intersect tool from the Galaxy toolshed. Two Prep peaks  were considered to overlap if their summits were within 50 bp . Prep and NF-YA peaks in mESCs were considered to overlap if their summits were within 500 bp.12hpf-only ChIP-seq peaks were identified by subtracting Prep12hpf peaks overlapping with all Prep13.5hpf peaks identified by MACS2 without applying any enrichment cut-off. This strategy allowed for stringent identification of 11468 Prep12hpf-only binding sites not occurring at 3.5hpf that were used for subsequent analysis.PrepMEME and DREME (MEME-suite version 4.11.1 ) were usAverage conservation score around Prep1 binding sites was computed in the Deeptool suite using Prep1-bound sequences and the UCSC vertebrate PhastCons eight way  wig file as regions of interest and score input files respectively. For Chromatin heatmaps and mean score profiles of Prep binding sites in fish embryos and mESCs were generated with the Deeptools (version 2.0 ) suite o In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior/Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Hugo Parker (Reviewer #1); Licia Selleri (Reviewer #2); Miguel Torres (Reviewer #3).Thank you for submitting your article "TALE factors use two distinct functional modes to control an essential zebrafish gene expression program" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The essential revisions are described below but we also include the full reviews of the three reviewers for further details.Summary:In this manuscript, Ladam et al. investigate how TALE factors contribute to early zebrafish development by characterizing Prep1.1 DNA-binding profiles at blastula and segmentation stages. They correlate these binding profiles with chromatin marks at these loci and with gene expression data from wild-type vs. TALE knock-down embryos. By focusing on two developmental timepoints, they make the interesting finding that the DNA-binding preference of Prep1.1 appears to expand during development. Strikingly, early binding to 'DECA' motifs is a feature at the blastula stage and additional binding to 'HEXA' motifs with adjacent Pbx-Hox motifs is seen by the segmentation stage. Exploring this further, they identify NF-Y as a potential binding partner of Pbx and Prep in blastula embryos. This leads to a model in which TALE factors utilise different modes of DNA-binding at different times in development, depending on the availability of co-factors such as NF-Y and Hox factors.Essential revisions:1) The authors must demonstrate that their model is correct by testing several of the identified elements by reporter assays in zebrafish embryos (or at the very least in cell lines in vitro), coupled with mutation of predicted TALE and NF-Y binding sites to address the importance of these sites for enhancer function.2) The TALE knock-down phenotype needs to be better characterized with appropriate validation of the specificity of the morpholino.3) The authors need to explain the temporal discrepancies of all their assays.4) The authors should provide clarifications/explanations regarding the statement that TALE GRN genes are significantly associated with Class 4 and Class 3, but not Class 1 or 2, MPADs .5) The authors should include a final diagram depicting the model of Prep1.1/TALE DNA-binding dynamics across developmental time and how this relates to the activation of the components of the TALE-GRN, changes in chromatin state and interactions with co-factors.6) Absolute statements should be avoided (e.g. "TALE factors control gene expression by regulating a chromatin transition… at a core set of genes encoding TFs that direct anterior development.")Reviewer #1:This work addresses an important question because Prep and Pbx proteins are crucial factors for vertebrate development and are present throughout embryogenesis but most of the focus up to now has been on their roles during segmentation stages, with their early roles and mode/s of DNA-binding receiving relatively little attention. The finding that these factors exhibit an expansion of their DNA-binding repertoire between blastula and segmentation stages is novel and interesting, representing a significant advance in our understanding of the dynamic roles of these factors during early development. However, I have a few points that I think should be addressed, that would considerably strengthen the conclusions and the manuscript.i) The TALE knock-down phenotype needs to be described/characterised in more detail, to provide a more comprehensive view of the developmental context and to validate the specificity of the morpholino cocktail. For instance, hindbrain segmentation/neuroanatomy and craniofacial morphology should be characterised in the triple morphants to provide more detailed evidence that the knockdown is as expected given the previously characterised MO phenotypes. I also suggest moving the justification for using MO's that is in the Figure 1—figure supplement 1 legend to the Materials and methods section or the main text, where it will be more prominent.ii) An assumption made is that Prep1.1-bound sites, or at least a sub-set of them, represent enhancer elements. The authors must demonstrate that this is true by testing a few such elements by reporter assay in zebrafish embryos, coupled with mutation of predicted TALE and NF-Y sites to address the importance of these sites for enhancer function. This can be done relatively quickly by transient transgenesis, is frequently used for mechanistic dissection of cis-regulatory elements, and will provide crucial evidence for the functionality of these putative enhancers.iii) The authors use data from mouse ESCs to infer evolutionary conservation of the TALE GRN and of TALE-NF-Y co-localised binding, which expands the scope beyond zebrafish. A complementary approach is to address how many Prep1.1-bound peaks overlap with fish-mammal conserved non-coding elements that have been described in the literature. It is also worth checking if any are homologous to elements in the VISTA enhancer browser and have been experimentally validated in transgenic mice. This is straightforward to do and could potentially add weight to the argument that these interactions are evolutionarily conserved.iv) This manuscript would really benefit from a final diagram depicting the model of Prep1.1/TALE DNA-binding dynamics across developmental time and how this relates to the activation of the components of the TALE-GRN, changes in chromatin state and interactions with co-factors.Reviewer #2:The paper by Franck Ladam et al. presents original studies on unexplored mechanisms that underlie very early roles of TALE transcription factors (TFs) in blastula/gastrula versus later roles during segmentation stages of zebrafish development.This interesting study makes a substantial leap forward by identifying binding of TALE factors at genomic Pbx:Prep (DECA) sites during early zebrafish developmental stages, at 3.5 hpf. The authors also report that binding motifs for the maternal NF-Y TF are enriched near DECA sites and that NF-Y can form complexes with TALE proteins. Interestingly, the authors demonstrate that in the later post-gastrula embryo, at segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs with adjacent PBX:HOX sites. Therefore, the authors convincingly demonstrate that TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different developmental stages, which is a novel and as yet unexplored mechanism underlying differential and temporally-restricted functions of TALE TFs in vertebrate embryogenesis.The manuscript comprises an arsenal of high-quality results that are illustrated in complex and impeccable figures. The identification of distinct sequence-based mechanisms that underlie TALE binding to DNA thus directing different developmental functions at successive stages of embryogenesis is per se a fundamental finding. This study will be of high importance and interest for TALE biology, which sorely lacks mechanistic research conducted in vivo in animal models. The original findings reported in this paper can also open newpaths of investigation on roles of TALE factors in the earliest stages of embryogenesis in other organisms, including mammals. In addition, the distinct strategies that are being employed by TALE factors to execute developmental processes at different stages of embryogenesis might be similarly adopted by other TFs with widespread expression patterns, thus opening additional avenues for broader investigations.Concerns that should be addressed:1) Results – General Consideration:The authors conduct multiple genome-wide experiments and/or mine available genome-wide datasets, including RNA-Seq, ChIP-seq for Prep1, ATAC Seq, ChIP-seq for chromatin marks. The time-point analyzed are not fully consistent across these experimental approaches, e.g. RNA-Seq for controls and TALE KD zebrafish embryos is performed at 6hpf (early gastrula) and at 12hpf (segmentation stage); ChIP-seq for Prep1.1 is performed at 3.5hpf (blastula) and at 12hpf (segmentation stage); ATAC-Seq at 4hpf ; and ChIP-seq for chromatin marks at 4.5hpf . While this could be somewhat concerning, the findings and the overall message emerging from the study are strong and do not appear to be weakened by the slight temporal discrepancies. Indeed: a) out of the 13,300 Prep peaks at 3.5hpf, ~60% co-localize with a Prep peak at 12hpf, suggesting that a large fraction of binding sites remains occupied throughout embryogenesis; b) an additional ~16,500 peaks detectable at 12hpf do not co-localize with Prep 3.5hpf peaks, demonstrating that additional binding sites become occupied at later developmental stages; c) Prep binding is dynamically and continuously associated with the TALE GRN during zebrafish embryogenesis; d) TALE factors utilize distinct binding motifs at very early versus late stages of embryogenesis; e) TALE-occupied sites are associated with specific chromatin states at blastula stages; f) developmental control genes are enriched near Modified Prep Associated Domains (MPADs) displaying repressive histone modifications; g) Class 4 MPADs transition to an active chromatin state during later stages of embryogenesis; h) TALE and NF-Y factors have joint roles at very early developmental stages and can form complexes.This reviewer is not particularly concerned about the minor temporal discrepancies present among the various genome-wide assays conducted in this study, given the strong message emerging from all of the reported high-throughput experiments. However, it might be useful to underscore throughout the text and in the Discussion that TALE factors adopt distinct mechanistic strategies in zebrafish blastula and early gastrula versus segmentation stages; in other words to simply cluster together the functions of TALE TFs in blastula and early gastrula within one single group . To this end, it would help to slightly modify the cartoon illustrating the subsequent zebrafish developmental stages in Figure 1A. The authors could group [blastula stages and early gastrula stages] within one single bracket or inside one single box andthe [segmentation stages] inside another bracket or box. Accordingly, this clustering could be clarified in the figure legend.2) Results, subsection “TALE factors control the chromatin state at Class 4 MPADs associated with the anterior GRN”: The authors state that TALE GRN genes are significantly associated with Class 4 and Class 3, but not Class 1 or 2, MPADs . However, RNA-seq  shows that genes associated with Class 3 MPADs  are expressed at similar levels at 12hpf and 6hpf . In contrast, Class 4 MPADs display higher levels of H3K27ac at 9hpf than at 4.5hpf  and their associated genes show the greatest increase in expression between 6hpf and 12hpf. In addition, only class 4 MPADs showed a strong switch to an active chromatin state from 4.5hpf to 9hpf during zebrafish embryogenesis , while class 3 MPADs did not exhibit any significant switch . Collectively, these results are somewhat difficult to reconcile. The authors should qualify these findings and try to explain these differences. Are other factors necessary for activation of Class 3 MPADs? Or do the acetylation changes appear at a later time-point for Class 3 MPADs? Can other scenarios be envisaged? It would be helpful to add these considerations at least to the Discussion section of the paper.3) Results: The authors state: "These findings indicate that TALE factors control gene expression by regulating a chromatin transition – from repressive chromatin in blastula stage embryos to active chromatin in segmentation stage embryos – at a core set of genes encoding TFs that direct anterior development."This statement is very strong and absolute, whereas it does not hold across the entire animal kingdom. In fact, in the mouse TALE factors substantially affect "posterior" development, as shown by the presence of severe posterior developmental defects in various mouse models with LOF for different TALE TFs. For example, in Pbx LOF mouse embryos it has been reported that the posterior axial skeleton, the hindlimb, and the urogenital system are severely compromised, among other posterior structures. In addition, also in the zebrafish embryo, Prep binds many loci in addition to ones associated with the anterior GRN and some of these additional sites are near genes that regulate other developmental processes known to involve TALE function. For instance, Prep peaks are found near genes involved in myogenesis  and expression of myogenic genes is disrupted in TALE KD embryos, as the authors report in the Discussion section of the paper. Given all of these considerations, the statement above should be at least qualified and also toned down:"These findings indicate that TALE factors control gene expression by regulating a chromatin transition – from repressive chromatin in blastula stage embryos to active chromatin in segmentation stage embryos – at a core set of genes encoding TFs that direct primarily anterior development in the zebrafish embryo."4) Results, subsection “NF-Y proteins regulate TALE GRN expression and form complexes with TALE factors”, first paragraph: The authors describe a NF-Y motif that is specifically enriched at Prep3.5hpf peaks . NF-Y is also maternally deposited in zebrafish , consistent with a joint role for TALE and NF-Y factors at very early developmental stages. This is a critical finding, as it identifies a potential new cofactor for TALE proteins. The authors could better emphasize this exciting finding, for example drawing a parallel between the roles of NF-Y in zebrafish and in mouse embryonic development. In fact, also in the mouse NF-Y has been shown to have critical functions for very early embryonic development . This parallel would broaden the impact and breadth of the reported findings and would relate them also to other species, e.g. mammals.5) Discussion: Given the wealth of high-quality results and novel findings that are reported in this interesting study, it would greatly help to add one additional figure – or figure panel – with a cartoon that summarizes and illustrates in a succinct manner the most salient findings. This would leave the reader with a strong and easy-to-remember 'take-home' message.Reviewer #3:The manuscript by Ladam et al. reports a ChIPseq analysis of Prep proteins at two developmental stages of the zebrafish embryo -blastula and segmentation- and the correlation of these data with transcriptome modifications associated to combined downregulation of Prep and Pbx factors using a morpholino approach. This work identifies a shift in binding preference by Prep factors between the two stages analyzed. While in the blastula the preferred binding site is a Prep-Pbx combined site, in the segmenting embryo the preference shifts to binding sites in which Prep binds to the Prep-alone binding site or the Pbx-Hox binding site (to which is can bind forming trimers). Classification of the blastula binding sites by their Histone Modification profile identifies a subset of targets that show repressive marks at the blastula stage and become active at the segmentation stage in a regionally-restricted manner. This class is enriched in genes whose expression and Histone modification profile is sensitive to the loss of Prep-Pbx function, indicating a pioneer function for Prep-Pbx in this tissue-specific genes.In addition to this, authors analyze the occurrence binding sites for the pioneer transcription complex, which appear strongly associated to the Prep-Pbx binding motifs. They characterize association of Prep-Pbx to subunits of the NF-Y complex and they demonstrate that NF-Y activity is required for the transcriptional and epigenetic activation of Prep-Pbx responsive genes.This is an important work mainly for two conclusions; one is that TALE transcription factors show stage-specific preference for binding sites defined by binding sequence. The data are compelling on this conclusion and suggest that it is the activation of tissue-specific transcriptional cofactors at later stages what directs this specificity.The second finding is the identification of a cooperative complex between two pioneering complexes Prep-Pbx and NF-Y. This is an important finding, although some aspects remain only superficially explained or are not fully conclusive. My main concern on this point is that the authors claim to have discovered the function of the Prep-Pbx-NF-Y binding sites, however they do not perform any functional assay on this sequence. In my opinion, the data shown are only correlative with respect to this point. While elimination of Prep-Pbx function or elimination of NF-Y function affects a common set of targets, this does not demonstrate that the effect is through the action of these proteins as a complex on the Prep-Pbx-NF-Y binding sequence. Also, is the Prep/Pbx or NF-Y function different for those sites/genes in which only the Prep-Pbx sites are present versus those in which the Prep-Pbx-NF-Y site is found?. Also, given that the set of sensitive genes are tissue-specific and their activation associates with the colonization of nearby Prep-only and Pbx-Hox sites, could the function of these new sites be the one that is relevant for chromatin opening and transcriptional activation and not the interaction at the Prep-Pbx site?. Therefore, if possible, experiments in which the Prep-Pbx-NF-Y binding sequence is functionally analyzed for factor binding and enhancer activity should be included to make conclusions stronger.In connection to this, a more comprehensive description of the prevalence of the Prep-Pbx and Prep-Pbx-NF-Y sites among the classes studied would help understanding whether there is specific association to subclasses. This would include the classification according to stage  and MPAD classes. Also a detailed description of the occupancy of specific sites associated to the TALE GRN containing the Prep-Pbx-, Prep-Pbx-NFy, Prep-only or pbx-Hox binding sites and how these evolve between blastula and segmentation would be a very valuable addition to the manuscript. Essential revisions:1) The authors must demonstrate that their model is correct by testing several of the identified elements by reporter assays in zebrafish embryos (or at the very least in cell lines in vitro), coupled with mutation of predicted TALE and NF-Y binding sites to address the importance of these sites for enhancer function.There are two types of elements identified by our analyses – one where Prep occupies monomeric HEXA binding sites associated with PBX:HOX motifs (this type of element is much more frequently occupied at 12hpf than at 3.5hpf) and one where Prep occupies DECA sites associated with NF-Y motifs (this is the predominantly occupied motif at 3.5hpf).hoxb1a locus (that contains a Prep+Pbx:Hox motif) drives gene expression in vivo and in vitro and that the TALE and Hox sites are essential . Furthermore, motif discovery in CNEs combined with functional testing in zebrafish recently identified HEXA+PBX:HOX motifs as being essential for enhancer activity . Hence, previous work has demonstrated that HEXA+PBX:HOX containing elements act as enhancers. This is now discussed in the Results subsection “Some TALE-occupied sites are associated with chromatin marks at blastula stages”, first paragraph. The revised manuscript now also includes additional data showing that the HEXA+PBX:HOX elements identified in our study are evolutionarily conserved  and associated with known enhancer marks . Finally, we now show that, of 74 enhancers known to be active in the zebrafish hindbrain , 19  coincide with Prep12hpf-only peaks. Since enhancer activity was assayed at 2-3 days of development, but our ChIP data is derived from 12hpf (and we applied a stringent FE>10 cutoff for the Prep peaks), this represents a substantial overlap. These results are now discussed in the aforementioned Results subsection. Hence, our analyses of HEXA+PBX:HOX elements are consistent with previous reports defining these elements as enhancers.Elements containing HEXA+PBX:HOX motifs have been studied previously. Analyses of individual such elements in the mouse demonstrated that they act as enhancers and that the Prep, Pbx and Hox binding sites are essential for enhancer function . Accordingly, we have previously shown that an element from the zebrafish sufficient to convey enhancer activity. In fact, we have previously shown both in vivo and in vitro that TALE factors are not sufficient to drive gene expression , indicating that TALE factors alone do not mediate enhancer activity. Furthermore, Prep, Pbx and NF-Y are all ubiquitously expressed, but their binding is associated with genes that are expressed in a tissue-restricted manner, indicating that there must be additional regulatory input at enhancers for these genes. Accordingly, in the original manuscript, we did not propose that the DECA+NFY elements act as enhancers. Instead, our observation that TALE and NF-Y occupy many genomic sites prior to the appearance of enhancer marks  and are required for the deposition of H3K27ac modifications at TALE-dependent genes , led us to suggest that these factors are instead required at a step prior to establishment/action of enhancers (possibly in a ‘pioneer’ role). This is now clarified in the second paragraph of the Discussion and in a new model diagram in Figure 7H. Nevertheless, as requested by the reviewers, we have now cloned and tested genomic regions containing DECA+NFY sites in HEK293 cells. We have previously used transfection in HEK293 cells to demonstrate that HEXA+PBX:HOX containing elements from the zebrafish hoxb1a locus acts as an enhancer , so this is a reasonable system to assay zebrafish enhancers. We find that only one of the seven elements drives expression of a luciferase reporter , indicating that DECA+NFY elements may not function as classical enhancers. This is consistent with our proposed model that Prep, Pbx and NF-Y instead act at these elements to play an earlier role (possibly as pioneer factors) to permit the subsequent action of tissue-specific TFs (such as Hox TFs) at nearby enhancers. This model is clarified in the new Figure 7H.Since the DECA+NFY containing elements are novel, they have not been previously assayed for enhancer activity. Our data show that many such elements are associated with chromatin marks indicative of enhancers. In particular, our analysis in Figure 4A, B uses H3K4me1 (a modification that is well established as a mark of enhancers) to define MPADs at 4.5hpf and Figure 4F shows that many MPADs are marked by H3K27ac (a mark found at active enhancers) at 4.5hpf. Notably, many MPADs that lack H3K27ac at 4.5hpf, gain it over the next several hours . In fact, even some Prep-bound sites that lack H3K4me1 at 4.5hpf (non-MPADs) gain enhancer marks during embryogenesis . Hence, many DECA+NFY elements appear to be (or become) associated with enhancer marks. We also find that these elements are enriched at regions of the genome that are conserved with mammals , consistent with DECA+NFY elements playing an evolutionarily critical role. Importantly, even though DECA+NFY motifs are associated with enhancers, these motifs are not necessarily expected to be 2) The TALE knock-down phenotype needs to be better characterized with appropriate validation of the specificity of the morpholino.Several published studies indicate that complete removal of Pbx or Prep function in zebrafish converges on the same phenotype . Accordingly, we expect that simultaneous disruption of Pbx and Prep function will also produce this phenotype. The published phenotype has been characterized morphologically, as well as by alcian blue staining for head cartilage, by immunostaining for hindbrain neurons and by in situ hybridization/microarray analysis for changes in gene expression. In the original submission of this manuscript, we show that our TALE KD phenotype is morphologically indistinguishable from the published phenotype . We have now also assayed head cartilage formation and differentiation of hindbrain Mauthner neurons – we find that both are affected as expected based on the published reports ). This analysis is now covered in the Results subsection “TALE factors control a gene network regulating formation of anterior embryonic structures in zebrafish”. Lastly, we compared our RNA-seq data from TALE KD embryos to gene expression changes in two published reports . It is difficult to do a direct quantitative comparison since our RNA-seq was done at 12hpf after KD of both Prep and Pbx, while French et al. knocked down only Pbx and assayed gene expression (by microarray and in situ hybridization) at 18hpf and Deflorian et al. knocked down only Prep and assayed (only by in situ hybridization) at 20-24hpf. In spite of these caveats, of the 13 genes downregulated in French et al., seven were downregulated in our analysis and of the six genes downregulated in Deflorian et al., four were downregulated in our experiment (this is covered in the aforementioned subsection). We note that gene expression changes in TALE KD embryos become more pronounced over time , likely explaining why not all previously reported TALE-dependent genes are detected by our RNA-seq. In sum, we conclude that our TALE KD produces a phenotype very similar to the published phenotype resulting from Pbx or Prep knockdown.As requested, the MO justification has been moved to the Materials and methods subsection “Interference with protein function in embryos”.3) The authors need to explain the temporal discrepancies of all their assays.Our analyses center on two timepoints – late blastula (3.5-4.5hpf) and early segmentation (12hpf). These timepoints were chosen because they represent a stage prior to zygotic gene expression (late blastula) and a stage coincident with the onset of tissue morphogenesis (early segmentation). These stages are now indicated in Figure 1A and the rationale for their selection is explained in the legend to Figure 1.For the late blastula stage, we use chromatin ChIP-seq (4.5hpf) and ATAC-seq (4hpf) datasets for comparisons to our 3.5hpf Prep ChIP-seq data. The use of datasets from slightly different timepoints allowed us to use published datasets instead of replicating previous work. We agree with reviewer 2 that the difference between 3.5hpf, 4hpf and 4.5hpf is unlikely to affect our conclusions for two key reasons. 1) at these stages all zebrafish blastomere cells appear to be pluripotent and capable of differentiating into any tissue derivative (Ho and Kimmel 1993). 2) genome-wide analyses at these stages require large numbers of embryos (hundreds to thousands) that cannot be individually staged. Since zebrafish development is not perfectly synchronized, embryos will develop at slightly different speeds – even in a clutch from a single female. Therefore, when large numbers of embryos are collected, they are likely to contain a range of slightly different stages, meaning that ChIP-seq/ATAC-seq analyses done at 3.5hpf, 4hpf and 4.5hpf will actually have considerable overlap in the actual stages of the embryos analyzed. This fact is now mentioned in the Materials and methods subsection “Analysis of chromatin features”. We did not carry out RNA-seq at 3.5hpf because zygotic gene expression is not yet active at this timepoint. Instead, we selected the 6hpf timepoint (early gastrula), when zygotic gene expression is more robust. However, we do not detect any differentially expressed genes at 6hpf, so this RNA-seq dataset is not used to identify TALE-dependent genes.4) The authors should provide clarifications/explanations regarding the statement that TALE GRN genes are significantly associated with Class 4 and Class 3, but not Class 1 or 2, MPADs .Our data show that TALE-dependent genes are associated with Class 3 and 4, but not Class 1 or 2 MPADs . The reviewer comments that Class 3 MPAD-associated genes appear to behave more like Class 1 and 2 MPAD-associated genes in some regards and asks us to address this issue. However, our data indicate that, while the Class 3 MPAD-associated genes undergo smaller changes than Class 4-associated genes, they nevertheless show a greater increase in expression and higher H3K27ac levels than genes associated with Class 1 and 2 MPADs , suggesting that they differ from Class 1 and 2 genes in these regards. These differences are now emphasized on lines 296-297. We do not know why Class 3 MPAD-associated genes show less pronounced signs of activation than Class 4 MPAD-associated genes, but we note that Class 3 MPAD-associated genes are more highly enriched for functions related to muscle differentiation . This process is just beginning at the timepoint we used to assay gene expression  and H3K27ac , suggesting that activation of Class 3 MPAD-associated genes may still be ongoing at this stage. This possibility is now discussed in the fifth paragraph of the Discussion.5) The authors should include a final diagram depicting the model of Prep1.1/TALE DNA-binding dynamics across developmental time and how this relates to the activation of the components of the TALE-GRN, changes in chromatin state and interactions with co-factors.A diagram is now included as Figure 7H.6) Absolute statements should be avoided (e.g. "TALE factors control gene expression by regulating a chromatin transition… at a core set of genes encoding TFs that direct anterior development.")The statement referenced by reviewer 2 has been revised as requested (subsection “TALE factors control the chromatin state at Class 4 MPADs associated with the anterior GRN”). We have also reviewed the text to make sure that our statements do not imply general conclusions, unless warranted.Reviewer #1:[…] i) The TALE knock-down phenotype needs to be described/characterised in more detail, to provide a more comprehensive view of the developmental context and to validate the specificity of the morpholino cocktail. For instance, hindbrain segmentation/neuroanatomy and craniofacial morphology should be characterised in the triple morphants to provide more detailed evidence that the knockdown is as expected given the previously characterised MO phenotypes. I also suggest moving the justification for using MO's that is in the Figure 1—figure supplement 1 legend to the Materials and methods section or the main text, where it will be more prominent.This issue is addressed under Essential revisions – point 2 above.ii) An assumption made is that Prep1.1-bound sites, or at least a sub-set of them, represent enhancer elements. The authors must demonstrate that this is true by testing a few such elements by reporter assay in zebrafish embryos, coupled with mutation of predicted TALE and NF-Y sites to address the importance of these sites for enhancer function. This can be done relatively quickly by transient transgenesis, is frequently used for mechanistic dissection of cis-regulatory elements, and will provide crucial evidence for the functionality of these putative enhancers.This issue is addressed under Essential revisions – point 1 above.iii) The authors use data from mouse ESCs to infer evolutionary conservation of the TALE GRN and of TALE-NF-Y co-localised binding, which expands the scope beyond zebrafish. A complementary approach is to address how many Prep1.1-bound peaks overlap with fish-mammal conserved non-coding elements that have been described in the literature. It is also worth checking if any are homologous to elements in the VISTA enhancer browser and have been experimentally validated in transgenic mice. This is straightforward to do and could potentially add weight to the argument that these interactions are evolutionarily conserved.This issue was addressed as part of Essential revisions – point 1 above. Specifically, the revised Figure 4E now shows that MPAD sites  coincide with genomic regions conserved among vertebrates. In addition, we selected a subset of elements for more in depth analyses of conservation and this data is now presented in Figure 4—figure supplement 1D. Lastly, we find that many enhancers shown to be active in the hindbrain , coincide with Preppeaks .iv) This manuscript would really benefit from a final diagram depicting the model of Prep1.1/TALE DNA-binding dynamics across developmental time and how this relates to the activation of the components of the TALE-GRN, changes in chromatin state and interactions with co-factors.A diagram is now included as Figure 7H.Reviewer #2:[…] Concerns that should be addressed:1) Results – General Consideration:[…] However, it might be useful to underscore throughout the text and in the Discussion that TALE factors adopt distinct mechanistic strategies in zebrafish blastula and early gastrula versus segmentation stages; in other words to simply cluster together the functions of TALE TFs in blastula and early gastrula within one single group . To this end, it would help to slightly modify the cartoon illustrating the subsequent zebrafish developmental stages in Figure 1A. The authors could group [blastula stages and early gastrula stages] within one single bracket or inside one single box andthe [segmentation stages] inside another bracket or box. Accordingly, this clustering could be clarified in the figure legend.This issue is addressed under Essential revisions – point 3 above.2) Results, subsection “TALE factors control the chromatin state at Class 4 MPADs associated with the anterior GRN”: The authors state that TALE GRN genes are significantly associated with Class 4 and Class 3, but not Class 1 or 2, MPADs . However, RNA-seq  shows that genes associated with Class 3 MPADs  are expressed at similar levels at 12hpf and 6hpf . In contrast, Class 4 MPADs display higher levels of H3K27ac at 9hpf than at 4.5hpf  and their associated genes show the greatest increase in expression between 6hpf and 12hpf. In addition, only class 4 MPADs showed a strong switch to an active chromatin state from 4.5hpf to 9hpf during zebrafish embryogenesis , while class 3 MPADs did not exhibit any significant switch . Collectively, these results are somewhat difficult to reconcile. The authors should qualify these findings and try to explain these differences. Are other factors necessary for activation of Class 3 MPADs? Or do the acetylation changes appear at a later time-point for Class 3 MPADs? Can other scenarios be envisaged? It would be helpful to add these considerations at least to the Discussion section of the paper.This issue is addressed under Essential revisions – point 4 above.3) Results: The authors state: "These findings indicate that TALE factors control gene expression by regulating a chromatin transition – from repressive chromatin in blastula stage embryos to active chromatin in segmentation stage embryos – at a core set of genes encoding TFs that direct anterior development."This statement is very strong and absolute, whereas it does not hold across the entire animal kingdom. In fact, in the mouse TALE factors substantially affect "posterior" development, as shown by the presence of severe posterior developmental defects in various mouse models with LOF for different TALE TFs. For example, in Pbx LOF mouse embryos it has been reported that the posterior axial skeleton, the hindlimb, and the urogenital system are severely compromised, among other posterior structures. In addition, also in the zebrafish embryo, Prep binds many loci in addition to ones associated with the anterior GRN and some of these additional sites are near genes that regulate other developmental processes known to involve TALE function. For instance, Prep peaks are found near genes involved in myogenesis  and expression of myogenic genes is disrupted in TALE KD embryos, as the authors report in the Discussion section of the paper. Given all of these considerations, the statement above should be at least qualified and also toned down:"These findings indicate that TALE factors control gene expression by regulating a chromatin transition – from repressive chromatin in blastula stage embryos to active chromatin in segmentation stage embryos – at a core set of genes encoding TFs that direct primarily anterior development in the zebrafish embryo."This issue is addressed under Essential revisions – point 6 above.4) Results, subsection “NF-Y proteins regulate TALE GRN expression and form complexes with TALE factors”, first paragraph: The authors describe a NF-Y motif that is specifically enriched at Prep3.5hpf peaks . NF-Y is also maternally deposited in zebrafish , consistent with a joint role for TALE and NF-Y factors at very early developmental stages. This is a critical finding, as it identifies a potential new cofactor for TALE proteins. The authors could better emphasize this exciting finding, for example drawing a parallel between the roles of NF-Y in zebrafish and in mouse embryonic development. In fact, also in the mouse NF-Y has been shown to have critical functions for very early embryonic development . This parallel would broaden the impact and breadth of the reported findings and would relate them also to other species, e.g. mammals.A more extensive discussion of previous work on NF-Y in embryogenesis (including the Bhattacharya reference) is now included in the Results subsection “NF-Y proteins regulate TALE GRN expression and form complexes with TALE factors”, second paragraph.5) Discussion: Given the wealth of high-quality results and novel findings that are reported in this interesting study, it would greatly help to add one additional figure – or figure panel – with a cartoon that summarizes and illustrates in a succinct manner the most salient findings. This would leave the reader with a strong and easy-to-remember 'take-home' message.A diagram is now included as Figure 7H.Reviewer #3:This is an important work mainly for two conclusions; one is that TALE transcription factors show stage-specific preference for binding sites defined by binding sequence. The data are compelling on this conclusion and suggest that it is the activation of tissue-specific transcriptional cofactors at later stages what directs this specificity.[…] The second finding is the identification of a cooperative complex between two pioneering complexes Prep-Pbx and NF-Y. This is an important finding, although some aspects remain only superficially explained or are not fully conclusive. My main concern on this point is that the authors claim to have discovered the function of the Prep-Pbx-NF-Y binding sites, however they do not perform any functional assay on this sequence. In my opinion, the data shown are only correlative with respect to this point. While elimination of Prep-Pbx function or elimination of NF-Y function affects a common set of targets, this does not demonstrate that the effect is through the action of these proteins as a complex on the Prep-Pbx-NF-Y binding sequence.We have now carried out a functional analysis of these binding sites .In the original manuscript, we were careful not to claim that a Prep:Pbx:NFY complex represents the functional unit controlling the TALE GRN – since this is difficult to prove experimentally. Our data show 1) that TALE and NF-Y TFs occupy binding sites (DECA and CCAAT) that are close to each other (average distance = 20bp), 2) that these TFs regulate a shared set of genes and 3) that they can form a complex. Hence, “We conclude that NF-Y binds adjacent to TALE factors at DECA sites and that both factors are required for regulation of the TALE GRN, possibly by functioning in a complex.”Also, is the Prep/Pbx or NF-Y function different for those sites/genes in which only the Prep-Pbx sites are present versus those in which the Prep-Pbx-NF-Y site is found?We partially addressed this point in the original manuscript , showing that TALE-occupied sites with adjacent NF-Y motifs have higher levels of H3K27ac, reduced nucleosome occupancy and reduced levels of H3K27me3 compared to ones lacking NF-Y motifs. However, these effects are relatively subtle and we did not examine them further. The main limitation with this avenue of inquiry is that we do not have NF-Y ChIP-seq data in zebrafish. Hence, we do not know which NF-Y sites are occupied , which makes it difficult to draw conclusions about NF-Y activity at these sites.Also, given that the set of sensitive genes are tissue-specific and their activation associates with the colonization of nearby Prep-only and Pbx-Hox sites, could the function of these new sites be the one that is relevant for chromatin opening and transcriptional activation and not the interaction at the Prep-Pbx site?.The experiments in Figure 6F and 7F, which demonstrate that TALE and NF-Y are required for increased H3K27 acetylation, specifically assay the chromatin state at MPADs (that contain DECA+NFY motifs) and were done at a stage (9hpf) prior to expression of most tissue-specific genes. This suggests that TALE and NF-Y acting at DECA+NFY motifs play an important role.Therefore, if possible, experiments in which the Prep-Pbx-NF-Y binding sequence is functionally analyzed for factor binding and enhancer activity should be included to make conclusions stronger.This issue is addressed under Essential revisions – point 1 above.In connection to this, a more comprehensive description of the prevalence of the Prep-Pbx and Prep-Pbx-NF-Y sites among the classes studied would help understanding whether there is specific association to subclasses. This would include the classification according to stage  and MPAD classes. Also a detailed description of the occupancy of specific sites associated to the TALE GRN containing the Prep-Pbx-, Prep-Pbx-NFy, Prep-only or pbx-Hox binding sites and how these evolve between blastula and segmentation would be a very valuable addition to the manuscript.We do not find any significant differences in the distribution of DECA versus DECA+NFY sites among the various regulatory elements (MPADs and non-MPADs), nor do we find a difference in their association with TALE GRN genes. This is now shown in Figure 7—figure supplement 1D and discussed in the second paragraph of the subsection “NF-Y proteins regulate TALE GRN expression and form complexes with TALE factors”.


Escherichia coli, the best-studied model prokaryote, are coupled and regulated in coordinate fashion. Accordingly, the growth rate-dependent control of the synthesis of RNA polymerase (RNAP) core enzyme (the core component of transcription apparatus) and ribosomes (the core component of translation machinery) is tightly coordinated to keep the relative level of transcription apparatus and translation machinery constant for effective and efficient utilization of resources and energy. Upon entry into the stationary phase, transcription apparatus is modulated by replacing RNAP core-associated sigma (promoter recognition subunit) from growth-related RpoD to stationary-phase-specific RpoS. The anti-sigma factor Rsd participates for the efficient replacement of sigma, and the unused RpoD is stored silent as Rsd–RpoD complex. On the other hand, functional 70S ribosome is transformed into inactive 100S dimer by two regulators, ribosome modulation factor (RMF) and hibernation promoting factor (HPF). In this review article, we overview how we found these factors and what we know about the molecular mechanisms for silencing transcription apparatus and translation machinery by these factors. In addition, we provide our recent findings of promoter-specific transcription factor (PS-TF) screening of the transcription factors involved in regulation of the rsd and rmf genes. Results altogether indicate the coordinated regulation of Rsd and RMF for simultaneous hibernation of transcription apparatus and translation machinery.Transcription and translation in growing phase of  Escherichia coli in rich media at an optimum temperature  under sufficient supply of oxygen exhibit a progression of constant steady-state growth as measured by either counting of the viable cells or measuring the cell turbidity. Traditionally, the cell growth has been classified into three phases: non-replicative lag phase; replicative exponential phase; and stationary phase of replication cessation. The growing-phase E. coli has long been used as a model organism relying on the belief that its laboratory culture is homogenous in cell populations. Most of our knowledge of modern molecular genetics such as the mechanisms and regulation of gene expression was established using such apparently homogenous planktonic cell cultures.Batch cultures under optimal laboratory conditions of the well-characterized model bacterium E. coli. A variety of protection systems against such hazardous environments are induced for survival by changing the cell organization at both the molecular and cellular levels . Likewise, the factors for ribosome silencing differ between E. coli and other bacteria. For instance, non-gamma proteobacteria form 100S ribosome but lack RMF and contain long HPF homologues (Hibernation of the Translation Machinery). As to the silencing of transcriptional apparatus and translational machinery, we focus on the well-characterized E. coli K-12 systems in this review.Up to the present time, a set of anti-sigma factors have been identified, each sequestering each of all seven  factors . Similarmologues  . This minor heterogeneity might correspond to the difference in the cycle of cell division   , while Un (UspG) . The stocoli see Figure 3E. coli K-12 AD202 strain lacking the ompT gene encoding outer membrane protease 7, which exhibits strong protein hydrolysis activity during cell lysate preparation once liberated from the outer membrane. In the experiments shown in As noted above, the pattern of genome expression in the stationary-phase changes for adaptation and survival as measured by genome-wide expression patterns of mRNA and protein products using the modern omics systems. In this section, we focus on the expression and degradation of the whole set of stationary proteins during the prolonged culture after the stationary phase up to 8 days. For protein separation and identification, we employed the radical-free highly reducing (RFHR) system of two-dimensional (2D) gel electrophoresis , and HPF (renamed YhbH), which all are involved in ribosome hibernation; for details, see Hibernation of the Translation Machinery.The RFHR system is in particular useful for analysis of small proteins, allowing the identification of these small-sized ribosome-associated proteins. The CR  fraction contained the newly identified 50S proteins, L35 (RpmI) and L36 (RpmJ) , and 30Scis-trans isomerase activity) and StpA (H-NS-like nucleoid protein with RNA chaperone function), and two of six E. coli UspGs, UspD and UspG. All these proteins are involved in repair and refolding of RNAs and proteins , Mdh , GuaB (inosine 5′-monophosphate dehydrogenase), and ZapB (cell division factor). HchA is involved in repair of glyoxal- and methylglyoxal-glycated proteins  and nuclagenesis . ZapB plagenesis . Under ssurvival . These 8Both the sequential increase in cell buoyant density and the sequential synthesis of stationary-phase proteins are apparently under a single pathway, but it should be noted that the pathway for entry into the stationary phase is multiple. During the prolonged culture, the heterogeneity in the cell population should also be amplified due to generation of various types of cells on different pathways, such as persister cells, mutant cells, and dead cells .Escherichia coli K-12, seven different species of the sigma subunit exist, each recognizing a specific set of promoters , thereby interfering with anti-sigma activity . RecentlHere, we propose the hibernation of growth-phase RNAP holoenzyme through conversion of RpoD sigma by Rsd anti-sigma factor. The RNAP core enzyme can then be used for assembly of RpoS holoenzyme for transcription of stationary-phase genes. It should be noted that excess free core enzyme, if present, should form transcriptionally inactive dimers or oligomers  for storE. coli K-21, large (50S) and small (30S) subparticles associate with each other to form the functional 70S ribosomes. The 50S subparticle is composed of two species of rRNA (23S and 5S) and a total of 33 species of the ribosomal protein, referred to r-protein (L1 to L36), whereas the 30S subparticle is composed of 16S rRNA and a total of 21 species of r-proteins (S1 to S21)  . Under oDuring this exponential phase, the ribosome profile detected by sucrose density gradient centrifugation (SDGC) includes 70S ribosomes as the major component and in addition, small amounts of 30S and 50S subparticles, and polysomes . Upon enE. coli is formed by the binding of two factors, the RMF  is also markedly modulated. For this alteration, the transcription apparatus is altered by binding of anti-sigma factor Rsd to the RpoD sigma for sigma replacement with stationary-phase-specific RpoS (see above) while the translation machinery is modulated by binding of RMF and HPF to 70S ribosome to form the inactive 100S ribosome dimer (see above). Until recently, however, little was known how the expression of factors involved in hibernation of transcription apparatus and translation machinery is regulated. We have then performed a systematic search for TFs involved in regulation of the promoters of two key regulators, Rsd for hibernation of RNAP and RMF for hibernation of ribosomes, by using the newly developed promoter-specific transcription factor (PS-TF) screening system (The formation of transcriptional apparatus and translational machinery are tightly coupled and coordinated, showing the growth rate-dependent synthesis of RNAP core enzyme  and ribog system .rsd and rmf promoter probes and a total of about 200 purified TFs from E. coli K-12 W3110, we performed PS-TF screening (rsd and rmf probes, although the binding affinity was different between these TFs (rmf and rsd genes are under the control of multi-factor promoters (in vitro and in vivo of both the rsd and rmf genes (Using creening . A totalhese TFs , suggestromoters . After rmf genes  (Figuremf genes . Resultsmf genes , which amf genes .rsd and rmf probes, a small number of TFs bound only to either the rsd or rmf probe (rsd- or rmf-specific TFs might be involved in independent regulation of either transcriptional apparatus or translational machinery under as yet unidentified specific environmental conditions. This review proves the initial stage of molecular basis of the hibernation of E. coli, focusing on the transcription apparatus and the translation machinery. The whole set of TFs involved in the regulation of rsd and rmf genes will be described elsewhere.Besides the large set of TFs with binding activity to both mf probe Figure 7HY: data collection, writing, and figure preparation in Hibernation of the Translation Machinery and Coordinated Hibernation of Transcription Apparatus and Translation Machinery sections. TS: data collection and figure preparation in Discontinuous Change of the Cell Buoyant Density and Coordinated Hibernation of Transcription Apparatus and Translation Machinery. AW and YM: data collection, writing, and figure preparation in Growth-Dependent Change of the Protein Expression Pattern section. AI: design of this review article, data collection, writing, and figure preparation in Introduction, Discontinuous Change of the Cell Buoyant Density, Hibernation of the Transcription Apparatus, and Coordinated Hibernation of Transcription Apparatus and Translation Machinery. All authors read and approved the manuscript.This study was supported by MEXT Cooperative Research Program of Network Joint Research Center for Materials and Devices to AI, and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities to AI.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 human population is getting ageing. Both ageing and age-related diseases are correlated with an increased number of senescent cells in the organism. Senescent cells do not divide but are metabolically active and influence their environment by secreting many proteins due to a phenomenon known as senescence associated secretory phenotype (SASP). Senescent cells differ from young cells by several features. They possess more damaged DNA, more impaired mitochondria and an increased level of free radicals that cause the oxidation of macromolecules. However, not only biochemical and structural changes are related to senescence. Senescent cells have an altered chromatin structure, and in consequence, altered gene expression. With age, the level of heterochromatin decreases, and less condensed chromatin is more prone to DNA damage. On the one hand, some gene promoters are easily available for the transcriptional machinery; on the other hand, some genes are more protected . The structure of chromatin is precisely regulated by the epigenetic modification of DNA and posttranslational modification of histones. The methylation of DNA inhibits transcription, histone methylation mostly leads to a more condensed chromatin structure (with some exceptions) and acetylation plays an opposing role. The modification of both DNA and histones is regulated by factors present in the diet. This means that compounds contained in daily food can alter gene expression and protect cells from senescence, and therefore protect the organism from ageing. An opinion prevailed for some time that compounds from the diet do not act through direct regulation of the processes in the organism but through modification of the physiology of the microbiome. In this review we try to explain the role of some food compounds, which by acting on the epigenetic level might protect the organism from age-related diseases and slow down ageing. We also try to shed some light on the role of microbiome in this process. Ageing is defined as a continuous accumulation of detrimental changes in the whole organism due to the impaired functioning of many tissues and organs. A constant loss of function of the muscle and skeletal systems, memory disabilities and a higher vulnerability to, for example, infections, are observed with age . Age is The observations of some beneficial effects exerted by a diet are supported by a great deal of evidence from scientific studies. Certain mechanisms of such an impact have been already defined and certain molecular factors have been recognized. It has been proven that the ageing process is malleable, and that by appropriate proceedings, we can influence the pace and the course of ageing and ARD . HoweverIt has been documented that one of the most recognized causes of ageing is cellular senescence. Firstly, it has been observed that senescent cells accumulate with ageing in many tissues ,9,10 andOne of the most spectacular and significant changes observed in senescent cells is modification of the chromatin structure . The rolAgeing is associated with some features, which are characteristic for all elderly; however, age-related diseases do not affect all of them equally. To common age-related health problems belong: impaired visions and hearing, sarcopenia, osteoporosis, increased vulnerability to infections, impaired wound healing and decreased skin elasticity. With age increases the risk of appearance of ARD, which include: neurodegenerative diseases , cardiovascular diseases , type II diabetes, lung diseases , osteoarthritis, cataract, glaucoma and certain types of tumors  . It has Cellular senescence is not always detrimental, as it may appear based on its engagement in ageing and ARD. The function of this basic cellular process is complex and the relevance of senescence depends on the age of the organism . SenesceCellular senescence can occur as a result of telomere shortening, and is then called replicative senescence, or as a response to stress and is then termed stress-induced premature senescence (SIPS) ,36. SeneCellular senescence is listed as one of the nine cellular hallmarks which contribute to ageing: genomic instability, telomere attrition, epigenetic alterations, stem cells exhaustion, altered intercellular communication, cellular senescence, mitochondrial dysfunction, loss of proteostasis, deregulated nutrient sensing . HoweverSenescent cells are characterized by multiple alterations in cell morphology, physiology and biochemistry , which reduces the calorie intake without causing malnutrition, and some activities mimicking it, such as certain micro-nutrients or mild physical activity ,46. DietThe main signalling pathways involved in longevity, and thus proposed to be a target for ageing intervention, include mTOR–S6K signalling and the GH/IGF-1 axis, which require AMPK and specific sirtuins . They arThe genetic material of all Eukaryotes is confined to a highly organized nucleus measuring only a few microns. To fit in such a restrained volume, 146 kb long segments of double stranded DNA are wound on histone octamers forming the basic structure of a nucleosome. The nucleosomes are then further condensed by linker histones and scaffold proteins into higher order chromatin. The chromatin is enclosed by a double nuclear membrane spiked with complexes of transmembrane proteins—nucleoporins. The interior membrane is lined with a mesh of intermediate filaments, namely lamins, that constantly interact with chromatin domains ,61.Epigenetics is defined as the study of heritable changes in gene function that do not entail any alteration in DNA sequence . The epiOne of the prominent examples of epigenetic modification is DNA methylation, which is characterized by covalent transfer of methyl group from S-adenosylmethionine (SAM) to the fifth position of cytosine ring (5mC) within cytosine-phosphate-guanine (CpG) dinucleotide. DNA methylation in vertebrates is mainly restricted to CpG sites (CpGs) and 60–80% of CpGs in the human genome are methylated. Approximately 7% of CpGs are located in CpG islands (CGIs), which are regions of high CG density . DependiDNMT1, DNMT2, DNMT3A, and DNMT3B have been identified [DNMT1, DNMT3A, DNMT3B) and the function of DNMT2 is a little controversial. Moreover, DNMT-like regulatory enzyme DNMT3L (DNA methyltransferase 3 like) is worth to be mentioned. DNMT1 was the first methyltransferase identified and is responsible for replicating the methylation pattern on newly synthesized DNA strands based on the hemi-methylated template strand, hence it plays a maintenance role. On the other hand, DNMT3A and DNMT3B are believed to methylate DNA de novo. Nevertheless, recent studies have suggested that these three enzymes can play complementary roles in mammals [DNMT2 to DNA methylation. Khalil et al. show that the activity of DNMT2 in aged mouse macrophages is considerably increased, which leads to hypermethylation in promoter regions of autophagy genes Atg5 and LC3B. This results in reduced autophagy, which is one of the causes of chronic inflammation, a characteristic feature of aged organisms [DNMT2 is shown to be upregulated in replicatively senescent human fibroblasts, which suggests its role in longevity regulation. Interestingly, silencing of DNMT2 results in changes in proliferation-related and tumor suppressor miRNAs level and leads to proliferation inhibition and induction of cellular senescence mediated by oxidative stress [DNMT2 silencing in mouse fibroblasts leads to, inter alia, telomere shortening, elevation of cell cycle inhibitors and DNA damage, resulting cell senescence [In humans, four DNA methyltransferases (DNMT) genes, namely entified . Three o mammals . As far  mammals . Last bu mammals . It is m mammals . Howeverrganisms . Moreovee stress . DNMT2 snescence . It is bnescence . The metnescence . These pnescence . How thenescence ,88, and nescence . This innescence  althoughnescence . The samnescence .Moreover, chromatin accessibility is regulated via a crosstalk between DNA methylation and histone modifications. Methylated DNA recruits histone deacetylases and histone methyltransferases e.g., SuV39H1 which, by methylating H3K9 (histone H3 lysine 9), tightens the chromatin structure . MoreoveThe next level of nuclear organization and gene expression control concerns chromatin structure predominantly controlled by posttranslational modifications (PTMs) of histones. Histones are highly conserved DNA-binding proteins, that form a nucleosome core. Each nucleosome consists of two copies of canonical histones H2A, H2B, H3 and H4. The double stranded DNA is wound on the established histone octamer and the whole complex is stabilized by linker histone H1 . Each coHistones can be post-translationally modified on the N-terminal tails protruding from the globular histone core. Depending on the type of modification the impact on chromatin accessibility and stability differs considerably. Among the plethora of PTMs the most prevalent are methylation, acetylation, phosphorylation and ubiquitination . These mWith the help of histone modifiers chromatin can adopt two dynamic conformations: that of transcriptionally inactive heterochromatin, condensed during the interphase and replicated in the late S phase, and that of euchromatin accessible to transcription, decondensed during the interphase and replicated in the early S phase . HeterocSirtuins, through histone deacetylation, take part in the formation of constitutive and facultative heterochromatin. The most studied sirtuins are SIRT1 and 6. SIRT1 is involved in deacetylation of lysine K9 in histone 3 (H3K9), which leads to its trimethylation (acetylation of K9 protects it from trimethylation) and facilitates the interaction with heterochromatin protein 1 α (HP1α). Such interaction keeps the chromatin in a closed state resulting in gene silencing ,104. SIRMicroRNAs are small (21–25 nucleotides) RNAs, which bind to complementary nascent mRNAs and trigger their degradation leading to inhibition of expression of specific genes. It has been estimated that 1%–4% genes in the animal genome encode miRNAs  and compHigher order 3D chromatin architecture involves containment of certain chromatin regions inside particular domains, called topologically associated domains (TADs). These provide favorable environment for gene expression regulation via enhancer-promoter interactions. TADs are enclosed by CTCF and cohesin that insulate them from the neighboring domains . ChromatMechanisms responsible for establishing and maintaining the chromatin structure are summarized in In ageing, the DNA methylation pattern is altered. The most clear alteration is related to a global decrease in DNA methylation observed in different species such as mouse, rat, cow, hamster and humans ,118,119.Senescent cells are characterized by global loss of canonical histones both nucleosome forming (a “loss” of nucleosomes) and linker histones , which mGenerally, senescent cells exhibit global loss of HDACs and sirtuins ,132 althOn the other hand, during ageing, a global loss or redistribution of heterochromatin is observed, leading to increased genome instability and altered gene expression . The losIt is believed that an impact on chromatin compaction  could be sufficient to induce cell senescence due to its influence on the activation of the DDR pathway (ataxia telangiectasia mutated (ATM)- and ATR-dependent), which is not associated with DNA damage . On the C. elegans miRNA lin-4 dramatically reduced the lifespan of the nematode and overexpression of this miRNA extended it [lin-14, elongated lifespan, which is disturbed by the overexpression of this type of miRNA. Both these miRNAs, lin-4 and lin-14, regulate the insulin/IGF-1 pathway. Other miRNAs, such as miR-375, which regulates insulin secretion in murine pancreatic islet cell [miR-122 in murine liver, are related to this signaling pathway [MicroRNAs and their shuttles (extracellular vesicles in particular) regulate a large number of cellular functions and physiological processes and are currently recognized as regulators of healthy and unhealthy ageing . In partended it . The loslet cell  or miR-1 pathway . Some mi pathway  and, theChromatin structure is also influenced by changes in the nuclear envelope. The inner surface of the nuclear envelope is lined with two types of lamins—A and B. Senescence is associated with the depletion of lamin B1 and mutations in the lamin A gene resulting in accumulation of defective prelamin A . This leAll age/senescence-related changes in chromatin structure and nucleus architecture are summarized in An increasing amount of evidence derived from both clinical and experimental studies indicates that epigenetic deregulation, especially of DNA methylation, is frequently associated with ageing and may underlie the etiology of chronic diseases e.g., diabetic complication, CVD (atherosclerosis), cancer, metabolic disorder and neurodegeneration ,169,170.However, the role of epigenetics in ageing is much more complex and is not limited only to one generation . It has IGF-2 gene. Moreover, peri-conceptual folic acid supplementation influenced methylation of IGF-2 promoter [The consequences of a diet for health can be observed on a long-term basis, counted even in decades. Diet has an impact on the onset of ADR and longevity, but some effects can be effectively counteracted/eliminated by certain approaches . Epigenepromoter . Dietarypromoter . DR is gpromoter . This ispromoter ,183,184.promoter . It is bpromoter .Nutrients directly regulate both the transcription and translational processes and interfere with metabolism by affecting DNA methylation, histone modifications and post transcriptional gene regulation by non-coding RNAs. High fat, low protein or energy restricted diet can alter the epigenetics marks ,186,187.Nutrition may affect both the establishment and maintenance of the DNA methylation pattern, which can exert long-lasting health effects. Gene expression can be modulated by both nutrient and non-nutrient dietary compounds. Currently, the major interest is focused on the effects of specific micronutrients and non-nutritional dietary components. To this group belong natural compounds, e.g., polyphenols, the most abundant phytochemicals in fruits, vegetables and plant-derived beverages . There i12, choline and betaine) [Nutritional and dietary factors have been postulated to affect DNA methylation by changing the availability of the methyl donors and altering the activity of the DNMT enzymes. To the first group belong micronutrients which are co-factors for enzymes involved in one-carbon metabolism, including folate, vitamin B6, vitamin B12, choline and methionine and those which can affect one-carbon metabolism indirectly . The diebetaine)  or genisbetaine) . DNA metbetaine) ,202.Gene regulation can be mediated by secondary DNA structures, e.g., G-quadruplexes (G4) . It is bIn some situations, the application of phytochemicals, which alter DNA methylation, can lead to adverse epigenetic changes. Such observation has been made in the case of premenopausal women receiving isoflavones (to which genistein belongs) in every day diet . This caThere are a lot of data showing that histones can be modified by nutrients and dietary compounds. It is related to the inhibitory potential of some dietary constituents interacting with enzymes involved in histones decoration. Histones marks can be altered by changed abundance and/or efficacy of the enzymes involved in their decoration or by impaired availability of the enzyme substrate. As it was mentioned before the chromatin status is controlled by HATs, HDACs, HMTs and HDMs. However, histones are not only methylated and acetylated. Histones tails can be modified by phosphorylation, ribosylation, ubiquitination, sumoylation and biotinylation , which vNumerous inhibitors of HDACs are recruited from polyphenolic compounds. A lot of data are related to sirtuins, especially 1 and 6 . HoweverThe fact that sirtuins exert multiple beneficial effects in the organism, such as reduction of the symptoms of CVD, diabetes and neurodegeneration as well as are linked to longevity, , it is rHDAC inhibitors can be one of the most promising prevention strategies concerning epigenetic changes including reduction/reversal of the already existing detrimental alternations. The restoration of the transcription of genes involved in proper metabolism, which decreases with age, can delay the functional decline of the organism . Indeed,Some limitations are imposed by the lack of specificity of HDAC inhibitors. This poses a real problem since inhibition of most HDACs is considered as beneficial, while sirtuins have to be activated to exert their anti-ageing/anti-ARD role. To this aim, careful studies to find out some unique compounds to be involved in the selective regulation of HDAC activity are necessary.Polyphenols, e.g., EGCG, curcumin, resveratrol and quercetin, are also regulators of miRNAs, as has been shown in cancer cells ,239,240.HDAC4 and NCOR2 genes, which had an impact on the adipose tissue metabolism [GLUT4 transcription in adipocytes, and correlates with insulin resistance [GLUT4 expression [The remodeling of the epigenome can be carried out by environmental factors other than nutrients, for example, by exercise. A large body of data indicates that physical activity (PA) can modulate gene expression through epigenetic alterations. This could be a beneficial strategy in chronic diseases, such as metabolic syndrome, diabetes, cancer , cardiovascular and neurodegenerative diseases, reviewed in . Physicatabolism . HDAC4 asistance . HDAC4 lpression . In turnpression . It has pression . PA actspression . In the pression ,251,252.pression . MicroRNpression . During pression . MicroRNpression . In thispression .Ulk1/ATG1, Ulk3/ATG1, Atg9A/ATG9, Lc3/ATG8, GabarapL2/ATG8, and vacuolar membrane protein Vmp1). Histone H3 hypoacetylation , via spermidine inhibition of histone acetyltransferase activity, increased expression of genes involved in autophagy such as ATG7, ATG11, ATG15. Moreover, methyltransferase EHMT2/G9a, by targeting H3K9, repressed transcription of autophagy genes [Atg9a) [Autophagy is considered as an anti-ageing catabolic process and is linked with/ cell survival and longevity . It is ogy genes . Conversgy genes . Autophagy genes . Reducedgy genes . The catgy genes ,262. Spegy genes . The secgy genes . Upregul [Atg9a) . MIR34 a [Atg9a)  and mTOR [Atg9a) .The human body makes an excellent environment for a highly dynamic and complex ecosystem of bacterial archaeal, fungal and viral species, which is referred to as human microbiota. These microorganisms constitute lifelong symbiotic companions that are present from birth until our final days. The exact starting point of microbial colonization, however, is currently a subject of heated debate . It was The largest and most prominent microbial reservoir is residing in the lower gastrointestinal tract (GIT). Gut microbiota play significant role in human health and diseases ,291. TheThere are about 1000–1500 recognized bacterial species living in the gut and each person harbors about 150 different species, which together contain more genes than the human genome and constitute a sort of a second genome . GIT is 11 to 1012, making their genome nearly 150 times larger than a human [Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Along with mucosal secretions, the bacteria form a rather thick and resistant biofilm, that line the epithelium, working as a mediator between the lumen and host’s body [Bifidobacteria and Enterobacter decrease in elderly, while Bacteroidetes increase in comparison to young adults [Lactobacillus plantarum, Escherichia coli, Nissle, and Bifidobacterium infantis, which increase expression of proteins forming tight junctions, and as a result, strengthen the intestinal barrier [GIT is composed of small and large intestines. Small intestine is responsible for nutrient absorption, facilitated by metabolic action of microbial cells that are rather low in abundancy but stable in composition . During  a human . Bowels t’s body . These ct’s body . In ageit’s body . Bifidobg adults . Moreoveg adults  and leadg adults . Intestig adults  that indg adults , the devg adults ,316,317. barrier . Althoug barrier , however barrier . This alPropionibacterium) on cheeks and forehead of healthy elderly decreases as compared to young adults [Malassezia spp. and bacteria Streptomyces spp., that produce complex volatile organic compounds, which in turn may serve as a noninvasive diagnostic tool of early Parkinson’s disease [Porphyromonas gingivalis, is a serious risk factor for developing Alzheimer’s disease [Ageing has a tremendous influence on the microbial composition within all human body. For instance, human skin harbors fairly different colonies between dry volar forearm, quite moist axillary vault and sebaceous face , where tg adults ,324, whig adults . The ski disease . Similar disease . Patient disease  with com disease ,330 as c disease . Elderly disease , notably disease ,334. The disease .As mentioned earlier, the role of the microbiome is strongly associated with the pace of ageing and the quality of life of the elderly. Some data show that gut microbiota is implicated in the pathogenesis of metabolic disorders, such as diabetes and obesity, and promotes atherosclerosis, and vascular endothelial dysfunction ,338,339.The interaction between nutrients (micro and macro) and microbiota is very tight. Such a relation concerns, e.g., polyphenols ,343,344.12 [Recent data have shown that SIRT1 can mediate the interaction between the host and the microbiome . Mice wi12 , which a12 . An asso12 .Maintaining a proper diet is important for individuals of all age. Moreover, the nutritional needs of elderly do not substantially differ from those of younger adults. However, it is particularly important to promote some nutritional strategies among those of older age because of the risk of malnutrition due to impaired absorption, loss of appetite and chewing difficulties, which may affect the proper nutritional status . These sWell-recognized anti-ageing strategies have been for a long time linked to a proper diet. Recently, it has been well documented that diet can modify chromatin structure. Moreover, it was proven that certain epigenetic modifications are long-lasting and can be inherited while others can be quite easily reversed. Therefore, nutrients can determine our health and disease even if they were consumed a long time ago, e.g., in the prenatal period. They can play a pivotal role in the ageing process and longevity. To the puzzle composed of two elements, i.e., nutrients and epigenetics, the microbiome should also be added. The interaction of these three seemingly independent elements creates a coherent picture that may be useful for developing potential anti-ageing strategies. The awareness of this relationship allows the adoption of a holistic approach to ageing with only one aim in mind: to synchronize all strategies in order to optimize the conditions necessary to healthspan elongation. The age-related epigenetic changes can be reversed by some macromolecules from the diet, the bioavailability of which depends on the microbiome, which per se affects the age-related dysfunctions. The urgent issue to address is how the current knowledge can be transferred into practice. More broadly, it is a question of how to target the individual needs of people, based on their epigenetic history and the composition of the microbiome, taking simultaneously into account age and gender, and not neglecting the impact of environment, which modulates the epigenetic landscape. The current view suggests that the epigenetic age, defined as the presence of characteristic markers, could be a more powerful predictor of future health decline and ARD than chronological age. The most important scope for future studies is to develop the strategies and promote such dietary habits, which will be effective in modulating the epigenome (directly or by the impact on microbiome) to eliminate or delay the expression of genes associated with the initiation and progression of ARD and ageing disabilities . Such nutritional habits/strategies must be adapted to age. It is essential to identify and characterize the most potent dietary factors with a strong and defined impact on the epigenome and microbiome, and to recognize the mechanism of their action.