Plant and animal embryogenesis have conserved and distinct features. Cell fate transitions occur during embryogenesis in both plants and animals. The epigenomic processes regulating plant embryogenesis remain largely elusive.cis-elements for fate patterning. Embryonic maturation is characterized by increasing H3K27me3 and decreasing chromatin accessibility, which likely participates in restricting totipotency while preventing extensive organogenesis. Finally, epigenomic signatures are correlated with biased expression among homeolog triads and divergent expression after polyploidization, revealing an epigenomic contributor to subgenome diversification in an allohexaploid genome.Here, we elucidate chromatin and transcriptomic dynamics during embryogenesis of the most cultivated crop, hexaploid wheat. Time-series analysis reveals stage-specific and proximal–distal distinct chromatin accessibility and dynamics concordant with transcriptome changes. Following fertilization, the remodeling kinetics of H3K4me3, H3K27ac, and H3K27me3 differ from that in mammals, highlighting considerable species-specific epigenomic dynamics during zygotic genome activation. Polycomb repressive complex 2 (PRC2)-mediated H3K27me3 deposition is important for embryo establishment. Later H3K27ac, H3K27me3, and chromatin accessibility undergo dramatic remodeling to establish a permissive chromatin environment facilitating the access of transcription factors to Collectively, we present an invaluable resource for comparative and mechanistic analysis of the epigenomic regulation of crop embryogenesis.The online version contains supplementary material available at 10.1186/s13059-022-02844-2. Embryogenesis, during which the fusion of parental gametes following fertilization generates an entirely new organism, represents the beginning of development and ensures the life cycle for plants and animals –4. AdvanEpigenomic dynamics accompany a series of developmental events during mammalian early embryogenesis, including maternal-to-zygotic transition (MZT), zygotic genome activation (ZGA), lineage specification and fate determination, and tissue fate differentiation , 7, 8. DIn comparison, our knowledge of the epigenomic dynamics, including histone modifications, histone variant, chromatin accessibility, and their potential implications in developmental regulation, such as MZT, lineage specification, fate transition, and embryonic maturation, remains largely unknown in plants, especially for crops with complex genomic structure . CompariAs an important crop that provides 20% of the calories and protein daily consumed by humans, wheat embryogenesis is of particular interest for elucidating the genomic and epigenomic regulation of the ontogenetic of polyploidy. Hexaploid wheat (AABBDD) results from two times hybridization and polyploidization events of three putative diploid wild grass progenitor species , 25. A rR > 0.94) and high correlation with published chromatin immunoprecipitation (ChIP)-seq results (R > 0.8) but also high repeatability . Second, we validated that the methods were sensitive to lower nuclei input. Robust CUT&Tag results were obtained using 1000 nuclei at a sequencing depth of ~ 1/3–1/20 of ChIP-seq experiments  and ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing) to map the histone modification and chromatin accessibility during plant embryogenesis , 27. A pHaving the techniques established, we generated a ‘reference epigenome’ of wheat embryogenesis across eight characteristic stages . Embryo sacs were used for early embryogenesis (DPA0-4), while dissected embryos were sampled for later stages (DPA6-22). The epigenome includes seven histone modifications , occupancy of histone variant H2A.Z and RNA polymerase II, and chromatin accessibility, as well as transcriptomes  are transcribed from TEs loci  using ChromHMM , 32. TotIn mice and humans, H3K4me3 and H3K27me3 are extensively reprogrammed immediately after fertilization, which is pivotal for early embryogenesis . Thus, wTogether, we revealed species-specific dynamic patterns of histone modification reprogramming during early embryogenesis in wheat Fig. b, S4c. DAP2, signaling-related gene MAPKKK17, and stigma expressed gene IAA1 , with decreased H3K27me3 and increased H3K27ac, and ATAC-seq signal, while a limited change of H3K4me3 from DPA2  is responsible for deposition of H3K27me3; consisting of four subunits including the single-copy gene encoded FERTILIZATION-INDEPENDENT ENDOSPRM (FIE) [FIE orthologues  in embryo at DPA6, while gradually reduced at DPA8 and DPA12  were genotyped by sequencing , all the triads harbored severe mutation with frameshift, while for homozygous lines (fie_aabbdd) B69 and C87, each had a weak mutation for either A or B triads, respectively  . In wheaCollectively, reprogramming of histone modification, particularly H3K27ac and H3K27me3, and establishing chromatin accessibility at early embryo development correlates with the proper transition from maternal to zygotic process and re-gain of PRC2 deposition of H3K27me3 is vital for the embryo development.Arabidopsis, were enriched in early built ACRs, such as the binding motifs of LEC1, MYB118, WUS, etc. , did not participate in embryonic development  have been reported to reset during embryonic development in bidopsis . Here, w, H3.3 habidopsis . While icis-elements binding occur at mid-stages  mainly functioned during mid-embryogenesis, which matched the “stage-specific transcriptional divergence” model.Embryo development is one of the most fundamental and remarkable processes in biology. The genetic and epigenetic control of embryogenesis has long been elusive in plant. Here, we generated a reference epigenome for embryo development in wheat, including chromatin state, accessible chromatin regions, and temporal transcription factors-genes regulatory networks. Our data highlighted conservative and species-distinct epigenomic remodeling kinetics during early embryogenesis in wheat, especially for reprogramming of H3K27ac and H3K27me3. By integration of transcriptome and epigenomic data, we uncovered an orchestrated transcriptional regulation by three-phase regulatory models: chromatin accessibility, histone modification, and The common wheat is an allohexaploid plant with three sub-genomes and high proportion TEs occupancy. The epigenomic modification and gene expression were extensively divergent among subgenomes. In addition, the distinct chromatin environment was associated with the biased transcription of homeolog triads. The different TEs occupancy at promoter regions has also potentially influenced the genes’ fate following polyploidization. These insights extend our understanding of the genetics and epigenetic regulation of embryonic development of wheat and provided a wealthy data resource and candidate regulators for functional genomic study.Triticum aestivum) Chinese Spring was used in this study. All plant was grown in growth room under a 16-h light/8 h dark cycle with light intensity 1000 μmol m−2 s−1 and temperature 15–26 °C depending on growth stage. The stamens are removed before the pollen maturation. Then, we conduct artificial pollination and record the number of days to ensure the accurate time of seed development. Embryo and embryo sac at the specific developmental stage were sampled for later use of total RNA extraction and nuclei isolation. The dissection method of embryo and embryo sac was largely based on the previous description with some modifications [A spring wheat . RNA-seq libraries construction is specified according to different aims. For mRNA sequencing, oligo (dT) was used for enriching the mRNA from total RNA. For non-coding RNA sequencing, rRNA is subtracted by using Ribo-Zero Plus rRNA depletion kit (Illumina), and then fragmentation and random primer was used for reverse transcript process. Sequencing is performed via the Illumina NovaSeq platform by Annoroad Gene Technology .2O. The library was amplified 17 cycles by Q5 high fidelity polymerase  and purified by AMPure XP beads . Finally, the library was sequenced using an Illumina Novaseq platform by Annoroad Gene Technology. All antibody information was list in Additional file The CUT&Tag experiment was performed as reported with minor modifications , 55. In The method of nuclei extraction was performed as before , 55. Afthttps://github.com/s-andrews/FastQC) to ensure the high quality of reads.All fastq data, including DNA and RNA sequencing, were generated based on Illumina Hiseq-PE150. Raw data were filtered by fastp (v0.20.0) with “–detect_adapter_for_pe” parameter for reads filter, low-quality bases trimming, and adapters removing . FurtherTriticum aestivum (Chinese Spring) genome assembly (IWGSC RefSeq v1.0) [https://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Assemblies/v1.0/. The IWGSC Annotation v1.1 was used as the gene annotation reference. For DNA sequencing data, BWA-MEM (v0.7.17) algorithm was used for alignment with “-M” parameter to avoid shorter split hits [Both DNA sequencing including CUT&Tag, and ATAC-seq and RNA sequencing data were aligned based on eq v1.0) , which wlit hits . For RNAlit hits .2 Fold Change ≥ 1 and FDR ≤ 0.05 [Sam files generated from hisat2 were converted to bam files without deduplication. FeatureCount v1.6.4 was used for reads quantity per gene . An R paR ≤ 0.05 . The rawR ≤ 0.05 . TPM valR ≤ 0.05  was usedR ≤ 0.05 . For nonR ≤ 0.05 . GffCompR ≤ 0.05 . The de Cut&Tag data analysis was largely based on the previously provided pipeline . In brieThe bam file process and bigwig conversion steps in ATAC-seq are the same as that in Cut&Tag. For ATAC-seq peak calling, only MACS2 was used with parameters “–cutoff-analysis –nomodel –shift -100 –extsize 200”. ChIPseqSpikeInFree was not applied in ATAC-seq data process. For both Cut&Tag and ATAC-seq peaks, if a peak overlapped with the proximal of a gene, including 3 kb upstream and 2.5 kb downstream, we assigned the peak to the gene. If multiple genes meet the condition, a position priority strategy (promoter > exon > intron > 5′UTR > 3′UTR > downstream) and nearest gene principle was used for target genes assign. An R package ChIPseeker was used for this peaks annotation process .Reads count under special peaks of Cut&Tag and ATAC-seq were calculated by FeatureCount . For CutFor chromatin state analysis, chromHMM was used , 32. “Bihttps://jaspar.genereg.net/) was used as motifs set [HINT (Hmm-based IdeNtification of Transcription factor footprints) was used for ATAC-seq footprints identity . JASPAR tifs set . CustomeZ-score. We further calculated the PC1 and PC2 for every gene using the expression values of eight samples. Because the standard expression values of all genes can form a circle, the atan2 function in R was used to return the angle in radians for the tangent PC2/PC1, which were further used for gene expression order ranking. As a result, the psuedotime expression of genes was ranked and visualized by complexHeatmap in R [Only six-module genes generated from WGCNA were used for the analysis in Fig. map in R . GO and map in R .p-value < 0.05 were used. We further calculated the Pearson correlation between TFs and corresponding targets, and only significant pairs  were retained. To simplify the GRN, we focused on the TFs-TFs network. As a result, several TFs contained multiple genes, we combine those as TF modules, including TCPs, ARFs, MYBs, and WOXs.For gene regulatory network building, the TFs for significantly enriched motifs with TaBBM, TaZHD5, TaLEC2, TaMYB118, and TaAMT1;1) promoter was amplified and fused in-frame with the CP461-LUC vector to generate the reporter construct target-pro: LUC. The candidate B3 domain TF binding motifs of TaLEC2 were disrupted by site-directed mutagenesis at the promoter of TaBBM, and the TaBBMmut-pro:LUC reporter construct was obtained to further confirm the regulatory effect of TaLEC2 on TaBBM  using a syringe with the needle removed. The Nicotiana benthamiana were cultivated for 2–3 days at a temperature of 22 °C and a light cycle of 16 h light/8 h dark. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured using a dual luciferase assay reagent (Promega).The pACRs genomic sequence of downstream targets , TaFIE-7B (TraesCS7B02G377900LC), and TaFIE-7D (TraesCS7D02G305100), gene-specific primers were designed around the gRNA target site. Primers F1 and R were used to amplify TaFIE-7A, F2 and R were used to amplify TaFIE-7B, and F3 and R were used to amplify TaFIE-7D. PCR products were genotyped by Sanger sequencing.For wheat genome editing, plasmid constructs pJIT163-Ubi-Cas9  and pU6-https://www.kulzer-technik.de), according to the manufacturer’s instructions. Sections (2 mm thick) were cut using a UC7&2265 microtome  and stained with 0.02% toluidine blue.Seeds of different DPA time were fixed under a vacuum in FAA solution . The samples were then dehydrated through a graded ethanol series and embedded in Technovit 7100 resin  was used to compute statistics and generate plots. For two groups’ comparison of data that fit a normal distribution, Student’s t-test was used, such as Fig. U test was used, such as Fig. If not specified, R . Figure S1. Quality control for CUT & Tag and ATAC-seq. Figure S2. Features of various modifications. Figure S3. Dynamic ACRs during wheat embryogenesis and difference among sub-genomes. Figure S4. Histone modifications dynamic and subgenome comparison. Figure S5. Histone modifications reprogramming at pro-embryo. Figure S6. Potential function of PRC2 in embryogenesis. Figure S7. Inhibition of deep organogenesis in late embryo at chromatin level. Figure S8. Chromatin landscape affected sub-genome bias expression. Figure S9. Epigenetic modification on different types of homoeologs bias expressed genes. Figure S10. Comparisons of embryogenesis among different ploidy wheat.Additional file 2: Dataset S1. The stage-specific pACRs.Additional file 3: Dataset S2. DEGs between DPA2 and DPA0.Additional file 4: Dataset S3. H3K27ac and H3K4me3 decreasing at DPA2 compared with DPA0.Additional file 5: Dataset S4. The correlation between up-regulated genes expression and up-regulated pACRs in DPA2 compared with DPA0.Additional file 6: Dataset S5. The resetting and re-building of H3K27me3.Additional file 7: Dataset S6. Overlap between promoter H3K27me3 resetting and H3K27ac/ATAC-seq up-regulation.Additional file 8: Dataset S7. The synchronization of ATAC-seq and RNA-seq dynamic.Additional file 9: Dataset S8. Selected key modules genes.Additional file 10: Dataset S9. The expression of organ identity genes.Additional file 11: Dataset S10. The expression patterns of candidate organ identity TFs.Additional file 12: Dataset S11. Pearson correlation of gene expressions between hexaploid wheat and ancestor.Additional file 13: Dataset S12. The genes expression rank of homoeologs.Additional file 14: Dataset S13. The deviation scores of key motifs during embryonic development.Additional file 15: Table S1. Antibody and Primer information used.Additional file 16: Dataset S14. Mutation of BBM promoter sequence.Additional file 17. Review history.


Pax (paired), Nkx, Otx (orthodenticle), Gsx/Gsh (genetic screened), and Dlx  homeobox gene families. This comprehensive review outlines the integral role of key homeobox transcription factors and their target genes on forebrain development, focused primarily on the telencephalon. Furthermore, links of these transcription factors to human diseases, such as neurodevelopmental disorders and brain tumors are provided.Forebrain development in vertebrates is regulated by transcription factors encoded by homeobox, bHLH and forkhead gene families throughout the progressive and overlapping stages of neural induction and patterning, regional specification and generation of neurons and glia from central nervous system (CNS) progenitor cells. Moreover, cell fate decisions, differentiation and migration of these committed CNS progenitors are controlled by the gene regulatory networks that are regulated by various homeodomain-containing transcription factors, including but not limited to those of the  Early brain development is marked by the formation of different compartments through the segmentation of the neural tube that is guided and defined by specific regional expression of transcription factors. The developing brain is sectioned into three contiguous parts, the prosencephalon in the most anterior area, which then matures into the forebrain; the mesencephalon following posteriorly, which give rises to the midbrain; and further posteriorly the rhombencephalon, the early form of the hindbrain. These areas further partition, where the prosencephalon separates into primary prosencephalon  and secondary prosencephalon  , 2018, aThe regions adjacent to the ventricular surface in the brain are the ventricular zone (VZ), followed by the subventricular zone (SVZ), and the mantle zone (MZ) . The VZ Nkx2.1 expressing progenitor cells in the MGE and PoA (pva+), somatostatin (sst+), and 5ht3a+ interneurons subtypes  are produced in both dorsal and ventral telencephalon; NPCs from the dorsal telencephalon give rise to projection neurons (glutamatergic) and NPCs from the ventral telencephalon differentiate into inhibitory interneurons (γ-amino butyric (GABA)-ergic) , 2002b.  and PoA , and mig and PoA . These vsubtypes . Many ssn the MZ . The main the MZ . Some kend Meis2 . The dif neurons . EBF1 th neurons .Pax6+ radial glia , mediated by ErbB4 attracts and regulates the final destination of GABAergic interneurons in the cortex (Nrp1) and neuropilin-2 (Nrp2), which are expressed in migrating interneurons  containing a helix-loop-helix-turn-helix structure . The 60 n the HD . Apart fn the HD . Proteinn the HD . Homeobon the HD , and cann the HD .E-box motif, and is specific for different transcription factors (Basic helix-loop-helix (bHLH) proteins are another superfamily of transcription factors present in most eukaryotes, with critical functions during embryonic development, such as neurogenesis and myogenesis. bHLH domains contains two alpha helices, helix 1 and helix 2. These helices are connected by a short loop, and at the amino-terminal end of helix 1 is a basic region . This ba factors . Upon bi factors . The uni factors .Ascl1, Olig1, Olig2, and Olig3) and forkhead (Foxg1) genes required for neurodevelopment. For this reason, we have included Ascl1, Olig1/2/3, and Foxg1 in In the following major section of this comprehensive review, detailed summaries of 21 homeobox genes  that encode homeodomain containing transcription factors are provided. These genes were selected due to their essential role in forebrain development. However, we acknowledge that this selection of genes excludes several other important homeobox genes as well as key bHLH  paired-like HD transcription factor is the vertebrate homolog of the Drosophila aristaless  gene, which is essential for appendage formation  signaling. The expression of SHH downstream targets, Gli1 and Ptch3, are increased in Arx cKO mice dorsal telencephalon. Thus ARX represses these SHH downstream signals, and in turn represses Olig2 expression ], previously called CCAAT-displacement protein (CDP) or Cut-like homeobox 1/2 (Cut1/2), that are the mammalian homologs of the Drosophila gene cut locus (ct)  . Ct is rosophila  and playosophila . CUX1 is cancers , while C1-q24.12 . CUX trard helix , and onerd helix . Howeverrd helix . CR1/CR2rd helix  and the rd helix , 1995. C domains . Proteol domains . While t domains . CUX prol domain . CUX tral domain , and phol domain , CKII  of the cortex  mice display abnormal dendrites and synapses indicating a critical role for Cux genes in dendritogenesis , caused by an increase in the number of neuronal progenitor cells   in a number of human cancers (CUX1 activates the phosphoinositide-3-kinase (PI3K) signaling pathway as a result of transcriptional downregulation of the PI3K inhibitor, PIK3Ip1 (CUX1 results in increased tumor growth and increased susceptibility to PI3K-Akt inhibition (Mutations in nt (GDI)  while CUdisorder . CUX1 ha cancers . Loss of PIK3Ip1 . This muhibition . CUX1 hahibition .Distalless (dll) was discovered in Drosophila for its essential role in limb development ; however, only Dlx1, Dlx2, Dlx5, and Dlx6 are expressed in the forebrain  mice die prematurely at postnatal day 0 (P0) with minor abnormalities in GABAergic neuron formation, demonstrating DLX1 and DLX2 are somewhat functionally redundant  genes are the mammalian homologues of the Drosophila gene empty spiracle (ems), which is responsible for head structure development  and increased ventral marker gene expression . Emx2/Pax6 double homozygous mutants demonstrate a stronger phenotype with a lack of dorsal identity, showing these two homeobox factors function cooperatively to specify dorsal telencephalic identity  genes encode a family of transcription factors important for patterning of the ventral telencephalon. Gsx genes are the mammalian orthologues of the Drosophila intermediate neuroblasts defective (ind) genes; mutation in Drosophila induces a loss of intermediate neuroblasts  of Gsx2 upregulates the oligodendrocyte precursor cell (OPC) marker Pdgfrα, and promotes premature oligodendrocyte differentiation  olfactory bulb interneurons are specified in the dLGE  is a TALE HD containing transcription factor , and expression shifts rostrally to the diencephalon from E10.5 , a region in the diencephalon that releases SHH signaling molecules for the patterning of prethalamus and thalamus , a bHLH transcription factor in the hypothalamus represses Irx3 expression, as Sim1 KO mice exhibit ectopic expression of Irx3 in the anterior hypothalamus  genes, due to the role of thalamus . Single-thalamus . Sim1 hothalamus . The neuthalamus ,b. In Sihenotype .Lhx transcription factors belong to the LIM-HD family of homeobox genes that have both a LIM zinc finger domains and a HD  are important for differentiation and migration of interneuron in the developing telencephalon , where Lmo1 competes with Lhx5 to bind with the Lhx binding partner LDB, thereby inhibiting Lhx function  and PSB (antihem) development up to E9.5 and E10.5, respectively , sst, and pva expression. Reduction in GAD1 does not downregulate LHX6 and vice versa; hence, upstream factors likely contribute to the regulation of these genes  gene family belongs to the TALE class of homeobox proteins, a homolog of the Drosophila homothorax gene, which is essential for directing the localization of Pbx Drosophila homologue extradenticle , and all contain a conserved homothorax domain  to modulate MEIS2 transcriptional activities  GABAergic interneurons, which populate different regions of the brain. Type I Htr3a+ are enriched in transcription factors expressed in the LGE, including MEIS2, and these interneurons populate the deep cortical layers , and is also known as the thyroid transcription factor 1 and thyroid specific enhancer binding protein, since it also plays a role in thyroid, lung and pituitary development  gene  is complementary to that of Pax6   gene . Nkx2.1 x domain . In embrse areas . Nkx2.2  species . The dor . Within with SHH .Nkx2.1 expressing progenitor cells give rise to GABAergic and cholinergic neurons, which populate the neocortex and striatum, respectively  expressing interneuron subtypes, resembling interneuron populations derived from the caudal GE  is an ortholog of the Drosophila orthodenticle transcription factor, with OTX1 located in the human chromosome region 2p13 and OTX2 located in the human chromosome region 14q21-22 -8 and Gbx2, another homeobox gene that is required for caudal brain patterning and formation  gene family based on its homology to the Drosophila gene eyeless. There are nine PAX transcription factors identified in mammals; all contain a paired domain and can be further categorized according to the presence or absence of additional domains, usually a HD. Pax6 contains two DNA binding domains, a paired domain and a HD, as well as a proline-serine/threonine rich domain in the carboxyl-terminal  and Gsx2-expressing  progenitor cells at the PSB , a bHLH transcription factor , in late corticogenesis (E15.5) there is an addition of differentiated neurons acquiring a GABAergic interneuron phenotype  gene family encodes a transcription factor family (Pou1f-Pou6f) of which Pou3f2 (Brn2) encodes a neural transcription factor that is necessary for mammalian CNS development and also for the production of corticotropin-releasing hormone  of the hypothalamus  DKO mice display an abnormal brain phenotype with decreased neocortical thickness and significant reduction of upper layer cells  expression in some melanomas by binding to its promoter region, which drives the cells to adopt a more stem-like and aggressive phenotype (isorders . POU3f2 isorders . When POisorders . POU3F2 s (TRIM8  as well M8 (VRK2 , other gM8 (VRK2 . While PM8 (VRK2 . Upregulhenotype . This uphenotype .non-cell autonomous roles as well as by combinatorial modes of action. As an example, PAX6 exhibits non-cell autonomous activity in the development of other organs such as the eye and spinal cord (Pax6 mutants (Gadd45b, a gene that may play a role in epigenetic gene activation, as a downstream target for modulating visual cortex plasticity (Other than the regulatory functions discussed above, homeodomain-containing transcription factors can regulate forebrain development through nal cord . This acnal cord . This sunal cord . PAX6 exnal cord . OPCs, a mutants . OPCs we mutants . Another mutants . Extraceasticity . These ecombinatorial code has been well documented and studied in the spinal cord (Pax6, Emx2, and Nr2f1 (In addition, although transcription factors display highly specific expression patterns, many are co-expressed at early stages of development, and work in a combinatorial manner. This concept of a nal cord . The orgnal cord . A similnal cord . In partnd Nr2f1 . Also, tnd Nr2f1 . Future In this review of vertebrate forebrain development , selecteRL, AG, ER, MF, JW, and DE conceived and wrote the manuscript. RL, MF, and AG designed and generated the figures. All authors contributed to editing and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The fact that overexpression of the yeast Ser/Thr protein phosphatase Ppz1 induces a dramatic halt in cell proliferation was known long ago, but only work in the last few years has provided insight into the molecular basis for this toxicity. Overexpression of Ppz1 causes abundant changes in gene expression and modifies the phosphorylation state of more than 150 proteins, including key signaling protein kinases such as Hog1 or Snf1. Diverse cellular processes are altered: halt in translation, failure to properly adapt to low glucose supply, acidification of the cytosol, or depletion of intracellular potassium content are a few examples. Therefore, the toxicity derived from an excess of Ppz1 appears to be multifactorial, the characteristic cell growth blockage thus arising from the combination of various altered processes. Notably, overexpression of the Ppz1 regulatory subunit Hal3 fully counteracts the toxic effects of the phosphatase, and this process involves intracellular relocation of the phosphatase to internal membranes. Overexpression of a given protein can cause harm to the cell for multiple reasons. For instance, intrinsically disordered regions in overexpressed proteins display a tendency to produce protein aggregation by making promiscuous molecular interactions . The stoSaccharomyces cerevisiae Ppz1 and Ppz2 enzymes are type 1-related Ser/Thr protein phosphatases. They are composed of a C-terminal catalytic domain ~60% identical to the yeast PP1 catalytic subunit (PP1c) Glc7 and contain a long N-terminal extension of about 350 residues [Candida albicans [Aspergillus fumigatus [The residues ,10. Wherresidues , and thealbicans  and Aspeumigatus  has raisS. cerevisiae, the role of Ppz1 is more prominent than that of Ppz2. Ppz1 regulates monovalent cation homeostasis in two ways: by inhibiting K+ uptake in both Trk-dependent and independent manner [+/ K+-ATPase encoded by the ENA1 gene [In t manner ,15 and bNA1 gene ,17. A roNA1 gene . SimilarNA1 gene  and in tNA1 gene .Ppz1 function is regulated in vivo by two proteins that act as inhibitory subunits, Hal3 and Vhs3. Both proteins bind to the catalytic domain of the phosphatase and inhibit its enzymatic activity ,22,23, aPPZ1 was expressed from an episomal plasmid, from either its own native promoter or the strong inducible GAL1-10 promoter, growth was severely or fully blocked [PPZ1 is one of the genes for which the cell has the lowest tolerance limit, indicating that the cell cannot endure the accumulation of the phosphatase. Accompanying PPZ1, this work also uncovered genes coding for protein kinases such as TPK1, TPK2, TPK3, or PRK1, as well as for the protein phosphatase CDC14. These results emphasized that deregulation of the protein phosphorylation status, likely leading to alteration of diverse signaling pathways, can have dramatic effects on cell growth and survival.Early evidence suggested that Ppz1 function must be subjected to tight regulation and that excessive Ppz1 activity was detrimental to the cell. It was reported that when  blocked ,25. Late blocked , based oIn this review, we will describe how work developed in the last few years and based on diverse and complementary experimental approaches has provided a clearer picture of the specific molecular basis underlying the toxicity stemming from an excess of Ppz1 activity.S. cerevisiae genome encodes two PPZ paralogs, PPZ1 and PPZ2, which are very closely related in sequence in their C-terminal catalytic domain . However, their N-terminal extensions are far more divergent. Early evidence showed that strong overexpression of the C-terminal catalytic moiety of Ppz1 (348 residues) was also highly toxic [PPZ2 would also be detrimental to cell growth. Yet, expression of Ppz2 under the same conditions used for Ppz1, driven from a tetO-regulatable promoter in a multicopy plasmid, showed very little effect, if any, on cell growth [PPZ2 than PPZ1. It must be noted, though, that the levels of Ppz2 protein in the tetO-based experiments were significantly lower than that of Ppz1, raising the possibility that the lack of toxicity could be due to insufficient accumulation of the phosphatase. However, a hybrid version carrying the N-terminal half of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1 even if it was expressed at levels similar to that of native Ppz2 [ppz1 mutant strain. Current efforts in our laboratory aim to identify the precise structural features in the Ppz1 N-terminal region that are crucial for toxicity.As mentioned above, the ly toxic  and, morly toxic . In addily toxic . Therefol growth . In factl growth  revealedive Ppz2 . This inive Ppz2 , who repPPZ1 altered the ribosomal function, causing an important reduction in the polysome content, which was normalized when the Ppz1 inhibitor Hal3 was overexpressed. We demonstrated that a relatively delayed effect of Ppz1 overexpression led to an increase in the phosphorylation of the eIF2α translation initiation factor at Ser51, likely mediated by the Gcn2 protein kinase [I required for translation initiation. Another inhibitory mechanism is the sequestering of eIF4E by the eIF4E-binding proteins Eap1 and Caf20, which interferes with the formation of the closed-loop complex and represses translation. Our observation that deletion of GCN2 improves, to some extent, the growth of cells overexpressing Ppz1, but lack of Caf20 or/and Eap1 does not, suggests that the excess of Ppz1 affects the formation of the ternary complex rather than that of the closed-loop complex. Nevertheless, in a recent phosphoproteomic study using the yeast strain ZCZ01, in which the genomic PPZ1 copy is controlled by the strong GAL1-10 promoter, we found Eap1 T284 dephosphorylated after 1 h of Ppz1 induction, and it remained in this state up to 4 h [https://www.yeastgenome.org, accessed on 27 January 2022), this is the first report describing the phosphorylation of T284 and, therefore, the consequences of this phosphorylation are unknown.It has been recently demonstrated that the excess of Ppz1 activity impairs protein synthesis, probably at the initiation step. This might be due to a possible role of Ppz1 in regulating ribosome biogenesis and function since the endogenous (non-overexpressed) Ppz1 protein has been co-purified with ribosomal proteins and localized in a wide range of ribosomal fractions covering the 40S and 60S ribosomal subunits, the 80S ribosome, and polysomes . In addin kinase . Phosphop to 4 h . AlthougFurther evidence about the effect of an excess of Ppz1 activity on protein synthesis came from the identification of changes in the phosphorylation state of diverse proteins involved in this process . As showDOT6 came out in a screen looking for multicopy suppressors of growth arrest caused by the excess of Ppz1 [DOT6, the above-mentioned multicopy suppressor screen yielded NOC2, important for ribosome biogenesis, as well as several genes coding for ribosomal proteins  [Dot6 is one of the transcriptional repressors that, when phosphorylated by the TORC1 substrate Sch9 kinase in response to nutrient limitation, releases its repressor effect on genes coding for ribosomal proteins (RP) and proteins required for ribosomal biogenesis (RiBi) . Interes of Ppz1 . Seventy of Ppz1 . We have of Ppz1 . We cann RPL37B) .PPZ1 ORF under the control of the doxycycline-repressed promoters 2tetO and 7tetO, present in strains MLM03 and MLM04 [The effects of mild expression of Ppz1 in carbon sources other than galactose were investigated by placing the nd MLM04 . Remarkand MLM04 . These rPPZ1 from the 7tetO-driven multicopy pCM190 plasmid. Once the expression of PPZ1 was induced for 3.5 h, the transcriptional response after 1 h of the shift from 2% to 0.25% glucose was determined in both strains. Assuming only those genes whose mRNA increased at least two-fold in the presence of 0.25% glucose, our mRNA-Seq data revealed that 85 genes were induced by low glucose in cells containing the empty pCM190 vector (unpublished results). As expected, the most over-represented Gene Ontology (GO) category for these genes, according to the YeastMine tool [p-value 7.4 × 10-9). Interestingly, the expression level for this set of 85 genes was clearly lower in many cases in cells overexpressing Ppz1 than in cells with the empty plasmid (average of 3.00-fold vs. 1.66-fold induction) . No significant GO categories were found among the set of 25 genes unaffected by an excess of Ppz1. These results suggested that overexpression of Ppz1 under the tetO promoter from the pCM190 plasmid does impair the normal transcription adaptation to conditions in which glucose becomes limiting by interfering with the proper activation of a set of genes mainly involved in carbon metabolism.To further investigate this possibility, we have used mRNA-Seq to determine the transcriptional response triggered by the shift from high to low glucose in normal cells and in cells overexpressing ine tool , was carduction) . To idenependent . GO analTo further analyze our transcriptomic data, the Yeastract+ portal  was usedPPZ1 expression was induced, as well as concomitant dephosphorylation of Snf1 T210. These changes were accompanied by substantial retention of Mig1 in the nucleus [Nuclear localization of the Mig transcriptional repressors is controlled by phosphorylation and by the interaction with Hxk2. In the presence of high glucose, unphosphorylated forms of Mig1 and the major glucose kinase Hxk2 localize to the nucleus and interact with each other, allowing repression of transcription of genes required for use of other carbon sources . Under g nucleus .Snf1 is also dephosphorylated and inactivated by the Glc7-Reg1 complex, and, as a result, Snf1 can no longer phosphorylate the Mig transcriptional repressors ,41. AbouGAL promoter is triggered by β-estradiol, that cell growth is completely blocked even in the presence of 2% glucose [Based on these results, it could be argued that disruption of the normal mechanism for adaptation to glucose scarcity could be a main reason for the growth defect caused by higher-than-normal Ppz1 levels. However, while this could be a limiting factor for mild overexpression of the phosphatase, we have observed, using a ZCZ01 derivative (strain MAC01), in which expression from the  glucose . Since sCLN1, CLN2, CLB1, and CLB5, which are repressed, consistently with the previously reported halt in the G1 phase caused by excess of Ppz1 [Genome-wide transcriptomic and phosphoproteomic analyses of the short-term response to Ppz1 overexpression using the ZCZ01 strain  revealed of Ppz1 ,46. SimiCells overexpressing Ppz1 suffer oxidative stress. This conclusion was deduced from the transcriptomic data (strong induction of known oxidative stress-responsive genes) and further confirmed by the determination of reactive oxygen species (ROS). Because it is well known that oxidative stress is responsible for DNA damage , this cop-value: 1.23 × 10-9) are contained within the cell cycle process category (GO:0022402). These include three protein kinases  that are involved in septin organization and bud emergence, a process that is blocked in cells overexpressing Ppz1 [The proteomic study of changes induced by Ppz1 overexpression revealed that very few proteins became more abundant, in agreement with the reported negative effect on protein synthesis . In conty GO:002202. Theseing Ppz1 . SeveralTwo other protein kinases were investigated further. The first one was Snf1, whose role in response to carbon source limitation is described in the previous section. However, Snf1 has also been related to the regulation of the cell cycle. Inhibition of the Snf1 activity leads to a decrease in the expression of G1-specific genes and the accumulation of cells in the G1 phase ,51. A siA second kinase whose phosphorylation state changed upon overexpression of Ppz1, in this case by increasing phosphorylation at its key T174 and Y176 residues, was the Hog1 kinase. Phosphorylation at these residues is known to activate Hog1, and such activation induces a delay in the G1-S transition, which occurs by two mechanisms: i) phosphorylation of Sic1, the cyclin-dependent kinase (CDK) inhibitor, at T173, and ii) inhibition of transcription of G1 cyclins by targeting Whi5, which acts as a repressor of G1-specific transcription, and the Msa1/2 transcription factors ,55. In appz1 and ppz1 ppz2 deletion strains were highly tolerant to sodium and lithium cations [ENA1 Na+,K+-ATPase gene, involved in response to salt stress [The relationship between Ppz1 and monovalent cation homeostasis was explored long ago, prompted by the finding that the  cations . This eft stress ,17, but t stress . Becauset stress , it was trk1 trk2 strain. This result clearly argued against Trk inhibition being the basis of Ppz1 toxicity. Interestingly, deletion of the gene encoding the Na+,K+/H+ plasma membrane antiporter Nha1 resulted in improvement of growth in Ppz1-overexpressing cells, placing again the focus on monovalent cation homeostasis. Nha1 is a housekeeping protein that exports Na+ and K+ in exchange for protons. In conjunction with the Ena1 ATPase, it enables cell growth in the presence of high concentrations of toxic monovalent cations [NHA1 deletion vanishes when cells are grown at pH near neutrality. All this evidence suggests that high levels of Ppz1 lead to the hyperactivation of Nha1 and a subsequent exacerbated influx of H+ in exchange for K+ ions. Several pieces of evidence support this scenario: (i) Nha1-mediated potassium efflux activity in cells overexpressing Ppz1 is far higher than in control cells, and (ii) as expected, expression of native Nha1 eliminates the growth improvement derived from the NHA1 deletion, whereas expression of mutated versions of Nha1 (such as the D77N variant), unable to mediate H+/cation exchange, did not.However, we recently showed  that cel cations ,59. This cations ,60 and i cations . Indeed, cations . In addiIt has been reported that Nha1 S481 is responsible when phosphorylated by an unknown kinase for binding to yeast 14-3-3 proteins and that this decreases Nha1 activity ,63. ThisNHA1 did not fully normalize growth rate and K+ and H+ intracellular contents in cells overexpressing Ppz1 indicates that additional targets affected by Ppz1 overexpression and relevant for monovalent cation homeostasis must exist. Although their nature is still obscure, several pieces of evidence suggest that the essential H+-ATPase Pma1 could be one of them. We observed that cells overexpressing Ppz1 failed to acidify the medium properly even in cells where NHA1 was deleted and that this effect was not due to lower-than-normal Pma1 levels [The fact that deletion of 1 levels . This su1 levels ,65. Rema1 levels . On the 1 levels ,67,68 or1 levels  results 1 levels , and thiIn conclusion, as exemplified in As mentioned above, the role of Hal3 in salt tolerance and cell cycle regulation ,72 was eGAL promoter [In agreement with the inhibitory role of Hal3 on Ppz1 function, expression of Hal3 from a multicopy plasmid fully restored normal growth in cells overexpressing Ppz1 either from its own promoter or from the strong promoter ,27. Ppz1promoter ,28,79,80promoter  that epiVPS27 gene aggravated the cell growth defect even when Ppz1 was overexpressed at moderate levels and greatly impaired the ability of Hal3 to normalize the growth of these cells [VPS27 encodes a component of the ESCRT-0 complex required for various cellular functions, including the trafficking of membrane proteins to the vacuole for degradation [SPO20, coding for a meiosis-specific subunit of the t-SNARE complex that mediates vesicle fusion required for the prospore membrane formation [PPZ1 [Translocation of Ppz1 to internal membranes is a crucial event for the ability of Hal3 to counteract Ppz1 toxicity. Indeed, mutation of the se cells . VPS27 eradation . Interesormation , partialWhereas the mechanism underlying Ppz1 relocalization is still obscure, current evidence suggests that this is more an exceptional instrument to avoid Ppz1 toxicity, linked to excessive phosphatase activity, than a normal regulatory process. Relocalization of the phosphatase does not occur with a non-catalytic version of Ppz1 bearing the R451L mutation, which normally binds to Hal3, suggesting that critical toxic events must occur. The nature of these events is not known. However, as described above, overexpression of Ppz1 results in a fast and strong drop in intracellular pH , and thiTo sum up, higher-than-normal levels of Ppz1 cause a profound alteration in many cellular mechanisms. These include potassium and intracellular pH homeostasis, redox homeostasis and oxidative stress, halt in protein synthesis, or adaptation to carbon source scarcity. Such alterations derived from or are reflected in widespread changes in the phosphoproteome that affect key components of intracellular signaling such as the Hog1 or Snf1 kinases and likely result in the amplification of the effects. Therefore, the Ppz1 phosphatase is an excellent example of how overexpression of a given protein can drastically affect cellular homeostasis. We also described here a very specific contribution of the non-catalytic domain of Ppz1 in cellular toxicity and an unpredicted mechanism by which the Ppz1 regulatory subunit Hal3 contributes to alleviating the deleterious effects of the excess of the phosphatase.


Being a Ty suppressor, spt23 can also interact with transposons (Tys) that are contributors to yeast’s adaptive evolution. Nevertheless, few studies have investigated whether and how much spt23 can exert its regulatory functions through transposons. In this study, expression quantitative trait loci (eQTL) analysis was conducted with thermal-tolerant Saccharomyces cerevisiae strains, and spt23 was identified as one of the most important genes in mutants. spt23-overexpression (OE), deletion (Del), and integrative-expressed (IE) strains were constructed. Their heat tolerance, ethanol production, the expression level of key genes, and lipid acid contents in the cell membranes were measured. Furthermore, LTR -amplicon sequencing was used to profile yeast transposon activities in the treatments. The results showed the Del type had a higher survival rate, biomass, and ethanol production, revealing negative correlations between spt23 expression levels and thermal tolerance. Total unsaturated lipid acid (TULA) contents in cell membranes were lower in the Del type, indicating its negative association with spt23 expression levels. The Del type resulted in the lower richness and higher evenness in LTR distributions, as well as higher transposon activities. The intersection of 3 gene sets and regression analysis revealed the relative weight of spt23’s direct and TY-induced influence is about 4:3. These results suggested a heat tolerance model in which spt23 increases cell thermal tolerance through transcriptional regulation in addition to spt23-transposon triggered unknown responses.spt23 is a key gene for heat tolerance, important for LA contents but not vital.• Deletion of spt23 decreases in yeast’s LTR richness but not in evenness.• The relative weight of spt23’s direct and TY-induced influence is about 4:3.• The online version contains supplementary material available at 10.1007/s00253-022-11920-3. Saccharomyces cerevisiae  oleic oil content within cell membranes  amplicon sequencing), cell membrane fatty acid content evaluation, and qRT-PCR were used to identify the two pathways that spt23 is involved in during heat shock stress.An obstacle to assessing these mechanisms is the accurate determination and quantification of yeast transposon activity because Tys typically comprise repeat sequences that are interspersed in genomes and render it too complex for accurate genomic profiling  platform. LTR diversity dynamics are then evaluated to characterize transposon activities and inform population distribution analyses. Using these methods, we observed changed LTR alpha diversity in yeasts cultivated at 30 and 37 °C, suggesting the practicability of this method. After quantifying transposon activities, the level of S. cerevisiae 1015 (S. cerevisiae CGMCC 2.4748) is an ethanol-yielding strain, and strain 101530 is a thermal-tolerant mutant, obtained through UV irradiation with 20 W of light power and a vertical distance dimension of 20 cm and then screening by grown in YPD medium at 45 ℃ for 72 h. The yeast expression vector pNC891 was kindly provided by Hackett et al.  following the manufacturer’s instructions, and genome sequencing was performed on an Illumina MiSeq platform using 300 bp paired-end sequencing. FastQC (v.0.11.7) was used for reads quality control, and Trimmomatic (v.0.38) was used to filter out low-quality reads and trim reads   on the Illumina NovaSeq sequencing platform. Read mapping and counting were conducted with HTSeq (1.99.2) , wild type (WT) spt23 was cloned into the pPH4 vector that is used for yeast overexpression  cassette was synthesized with fusion PCR. The pADH1-GFP fragment was then amplified from the pNC891 vector. A fusion fragment with homologous recombinant arms to spt23 was co-transformed with pCrispr-con-gRNA into spt23Δ strains. Transformants were then selected on a solid YPD medium with 300 mg/mL G418. Colonies were evaluated for GFP expression with fluorescence microscopy at an excitation wavelength of 438 nm. The recombinant strains were denoted as spt23::GFP-spt23 or integrative-expressed (IE).Two target sites in the 5′ direction of Extraction of pure yeast cell membranes followed Panaretou and Piper  but withmga2 is a homolog of spt23. dog2 and stf2 are indicators of yeast hyper-osmosis and ROS (reactive oxygen species) tolerance response initiation. ole1, erg1, and acc1 are important factors involved in yeast lipid acid synthesis, and rfu1 participates in ubiquitin homeostasis that is critical for proteasome functioning /(LTRref/TYref) for five transposon families (n = 1–5). Ta1 is copy number-sensitive and incapable of LTR sequence diversity profiling. Ta2 is typically calculated and also considers the diversity of LTR sequences, with calculation by (SI/TY)/(SIref/TYref), where SI is the Shannon index value for LTRs in a given sample.Specific primers were designed to amplify TY1-TY5 and their LTRs Fig.  and annospt23-affected genes during thermal tolerance adaptation. Another set of genes comprising the first out-going neighbors of spt23-affected transposons was denoted as the T-CORE gene set. Thus, the intersecting genes of the H-CORE and M-CORE sets reflect the weight of spt23’s role in thermal tolerance. In addition, the intersecting genes of the H-CORE and T-CORE sets reflect the weight of transposon effects in thermal tolerance. From the perspective of influence weights, TULAs  and transposon activity were considered variables representing direct regulation by spt23 and indirect regulation by transposons during yeast thermal tolerance. A regression model was then built where the partial correlation coefficients reflected the importance of variables.A set of genes that were differentially expressed independently of strain and growth phase were identified and referred to as the H-CORE gene set. Genes from the PheNetic inferred core interaction networks were denoted as M-CORE and represented spt23Δ recombinant strain was constructed and confirmed with PCR using degenerate sequence analysis and Sanger sequencing ) was achieved at 24 h, representing 18 and 28.8% higher levels than that of strain 1015 at 30 or 37 °C. The growth of strains 101530 and 1015 at 30 °C was faster than at 37 °C. Better growth performance for strain 101530 was observed at 37 °C. Recombinant 101530 strains  exhibited higher biomass accumulation than 1015 (+ 0.02 × 108 − 0.1 × 108) at 30 °C. Biomass peaks for strain 101530 types Del and OE were observed at 0.01 × 108 CFU/mL when grown at 37 °C, representing 0.27 × 108 CFU/mL higher values than biomass peaks for strain 1015. In contrast, the IE type exhibited lower peak biomass than strain 1015 (− 0.51 × 108). Accumulated biomass generally followed the sequence of Del > WT > OE > IE for both strains 101530 and 1015  and 12.77 + 0.2% (V/V), respectively, and for strain 1015 were 14.13 + 0.12% (V/V) and 11.87 + 0.19% (V/V), respectively  at 30 °C and 14.0 ± 0.14% (V/V) when grown at 37 °C  and 1015 , in addition to 55 °C for 101530 (p = 0.0022). The Del types of strains 101530 and 1015 exhibited lower survival than the WT at 45 °C, 50 °C, and 55 °C, with the exception of strain 101530 at 50 °C. The OE and IE types exhibited lesser heat tolerance than the WT at all times . Significant differences were observed in survival among recombinant types at 45 and 50 °C for both strains 101530  did not support the presence of a valid difference between the WT and other recombinants for strain 101530. The survival rate of the over-expression type decreased by 35.44% because it was incapable of growing under 0.33% hydrogen peroxide stress. During 50% caramel stress, only the over-expression 101530 type exhibited a significant increase in caramel tolerance . A total of 1900 genes were identified with sense mutations from SNPs and indels, making them important for drive mutation analysis. The total number of differentially expressed genes is shown in Table Intersection analysis of the four-gene set groups in Table spt23 was considered the 1st (at 16 h) and 2nd (at 40 h) most important influencer of the transcriptomes. Genes in the network were extracted and denoted as M-CORE  when the IE type was treated with heat. The dog2, ole1, spt3, mga2, and stf2 expression levels exhibited similar increases in the IE type. Deletion of spt23 caused a sharp decrease in acc1, erg1, rfu1, stf2, and swi5 expression levels when yeast was exposed to heat treatment  , while rfu1 levels were negatively correlated with spt23 levels (R2 > 0.7)  contents, indicating a non-dominating influence of spt23 (R2 = 0.3)  contents increased (by + 1.8 and 18.3% for strains 1015 and 101530) when WT strains were cultured at 37 °C relative to levels when grown at 30 °C. The Del type exhibited the same SLA trend increase as the WT (+ 2.8%) due to increased palm oleic acid and decreased oleic oil contents. Although all the WT and Del types exhibited lower SLA contents when grown at 37 °C, varying compositional changes were observed. WT strain 101530 exhibited an increase in SLA by decreasing longer chain lipid acid contents , while WT strain 1015 primarily exhibited decreased short-chain lipid acids . In contrast, the IE type exhibited decreased SLA contents (− 7.2%) , while significant decreases were observed for OTUs (p = 0.029) that represented clustered groups of LTRs. Significant differences were not detected for the Shannon diversity index values or Chao richness estimations  with spt23 expression levels were not significant  between 30 and 37 °C grown cultures were significant  and spt23-transposon-regulated core genes .To evaluate how p = 0.015, Table R (0.903) indicated a strong regression relationship. The adjusted R2 = 0.741 represents the total explanatory power of the two variables for the total variance and represents the combined direct and indirect influences of spt23. The regression coefficients of TULA and Ta2 exhibited p = 0.005 and p = 0.043, and thus neither factor should be excluded in the model. The partial correlation coefficients for TULA and Ta2 were − 0.90 and 0.77, respectively. Weights were evaluated with squared partial correlation coefficients, revealing that TULA and Ta2 explain 0.810 and 0.591 of the total residuals, respectively . Thus, TULA was the most important factor conferring thermal tolerance, while transposons exhibited lower influences. The regression model was established as Survival45 =  − 2.39⋅TULA + 69.98⋅Ta2 + 152.43.A regression model of survival rate to TULA contents and transposon activity (Ta2) was statistically significant  in lipid acid metabolism along with proteosomes  mechanism . This result implies that transposon activities are highly associated with temperature shifts  aside from spt23 expression levels. If transposon activity was independent of spt23 regulation, it should be an intrinsic characteristic of yeast strains. It is also possible that transposons are regulated by other genes or co-regulated with spt23 under various stresses. Nevertheless, further studies are needed to verify this hypothesis since the sample count is inadequate for rigorously evaluating it.A contradictory result is the high level of correlation between Schizosaccharomyces pombe Below is the link to the electronic supplementary material.


Mesenchymal stem/stromal cells (MSCs) hold great promise and clinical efficacy in bone/cartilage regeneration. With a deeper understanding of stem cell biology over the past decade, epigenetics stands out as one of the most promising ways to control MSCs differentiation. Trithorax group (TrxG) proteins, including the COMPASS family, ASH1L, CBP/p300 as histone modifying factors, and the SWI/SNF complexes as chromatin remodelers, play an important role in gene expression regulation during the process of stem cell differentiation. This review summarises the components and functions of TrxG complexes. We provide an overview of the regulation mechanisms of TrxG in MSCs osteogenic and chondrogenic differentiation, and discuss the prospects of epigenetic regulation mediated by TrxG in bone and cartilage regeneration. RUNX2, OSX, DLX3, and SOX9, these TrxG proteins play a prominent role in the epigenetic regulation of osteogenic and chondrogenic differentiation.TrxG family is one of the most important mechanisms for gene expression activation in epigenetics. The COMPASS family, ASH1L, and CBP/p300 are associated with histone methylation and acetylation activities. The SWI/SNF complexes have ATP‐dependent chromatin remodelling activities. By targeting differentiation‐related key genes such as  Bone regeneration is required under various physiological and pathological situations that cause bone loss including trauma, infection, skeletal abnormality or tumour.HOX) genes and other target genes.There is mounting evidence suggesting that epigenetic regulation of gene expression plays an essential role in stem cell fate determination during development.2TrxG proteins mediate epigenetic activation in the form of both complexes (COMPASS family and SWI/SNF family) and individual proteins (ASH1L and CBP/p300).Another group of TrxG complexes has ATP‐dependent chromatin‐remodelling activities. The SWI/SNF (switching/sucrose non‐fermentable) complex genes were identified to have counteractive roles against Polycomb‐mediated repression of HOX genes in Drosophila.Other members of TrxG family have not been demonstrated to exist in stable complexes, though physical and functional interactions occur among proteins. The SET‐domain containing KMT, absent small or homeotic disc1 (ASH1), is reported to be associated with the histone acetyltransferase (HAT) CREB binding protein (CBP) in Drosophila.33.1As a core subunit of COMPASS KMTs, WRAD is required for complex assembling and functioning. Structurally, DPY30 directly binds to ASH2L via a DPY‐30 binding‐motif (DBM) on the C‐terminus of ASH2L, while RBBP5 interacts with the SPRY domain of ASH2L and bridges it to WDR5.Myc) and Runx2.Osx−/− mice, the occupancy of WDR5 and H3K4me3 level at the promoter of OSX target gene bone sialoprotein 2 (Ibsp) was significantly reduced, correlating with Ibsp expression decrease and repression of osteoblastic differentiation.HOTTIP interacted with WDR5, and knockdown of HOTTIP impacted WDR5 nuclear translocation. WDR5 directly bound the promoter regions of catenin beta 1 (CTNNB1) mediating transcriptional activation. The HOTTIP‐WDR5 interaction activated Wnt/β‐catenin signalling during osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).The regulatory role of WRAD subunits, especially WDR5, on stem cells differentiation has been well investigated. WDR5 belongs to a large protein family containing the conservative WD‐repeats domains and is involved in multiprotein complex assembling, localization and regulation.Col1a1) promoter, caused a phenotype of larger skeleton, acceleration of endochondral and intramembranous bone formation, and increased hypertrophic chondrocyte layer size. In vitro experiments indicated that WDR5 enhanced expression of a perichondrium‐specific gene, twist basic helix–loop–helix transcription factor 1 (Twist1), leading to fibroblast growth factor 18 (Fgf18) downregulation, and resulting in promotion of chondrocyte proliferation and differentiation.Furthermore, it has been reported that overexpression of WDR5 in mice perichondrium, under the control of collagen type I alpha 1 chain  and MLL2 (KMT2B), are mutually exclusive in human MLL1/2 COMPASS‐like complex which additionally contains WRAD core structure, MEN1 and HCFC1.HOX genes, while MLL2 is the major methyltransferase responsible for H3K4me3 on bivalent genes in mouse embryonic stem cells.HOXD4 and HOXC8 promoters, associated with reduced HOX gene expression.HOX genes, distal‐less homeobox 3 (DLX3), promotes osteogenic differentiation of stem cells by targeting RUNX2. In vitro experiments in human dental follicle stem cells (DFCs) showed that activation of DLX3 expression was regulated by MLL1/2 complexes and SUMO specific peptidase 3 (SENP3). Decreased deposition of MLL1/2 complexes components and reduction of H3K4me3 marks were found on the DLX3 gene when SENP3 was knockdown, leading to inhibited osteogenic differentiation of DFCs.The two mammalian homologues of Drosophila Trithorax gene, MEN1 gene knockout mice were found to be embryonic lethal and 20% of embryos showed cranial and facial developmental defects.Men1 inactivation by antisense oligonucleotides (AS‐oligo) compromised the activity and expression of alkaline phosphatase (Alpl), Col1a1, Runx2 and osteocalcin (Bglap) induced by BMP2 in C3H10T1/2 murine mesenchymal progenitor cells and ST2 stromal cells. Mechanistically, it was found that menin physically and functionally interacted with the BMP‐signalling downstream factors SMAD1, SMAD5, and RUNX2 in uncommitted MSCs and activated transcription of differentiation‐related genes.Col1a1 and Bglap.miR‐26a), which had a negative post‐transcriptional control on SMAD1. Menin activated miR‐26a expression by occupying its promoter. MEN1 inhibition by siRNA resulted in downregulation of miR‐26a and upregulation of SMAD1 protein in osteoblastic‐differentiated human adipose tissue‐derived stem cells (hADSCs), which might explain the suppression role of menin during late osteogenic differentiation.Menin 1(MEN1) is considered as a tumour suppressor associated with multiple endocrine neoplasia type 1 sydrome.Men1 in Men1f/f;Bglap‐Cre mice showed a reduction of osteoblasts numbers and significant decreases of bone mass and volume in both trabecular and cortical bones. Men1‐deficient calvarial osteoblasts exhibit impaired mineral apposition and reduced transcriptional responsiveness to BMP2, leading to downregulation of osteogenesis‐related genes expression.Men1 knock‐out mice also showed remarkable deficiency in osteoclastogenesis. On the contrary, overexpression of menin specifically in osteoblasts driven by the 2.3‐kb Col1a1 promoter increased bone mass accumulation but did not affect osteoclast differentiation in vivo.Men1 in the osteoblast lineage using Men1f/f;Runx2‐Cre and Men1f/f;Osx‐Cre mice displayed strong decreases of trabecular bone mass resembling osteoporosis. However, they found no significant changes in osteoblast number and osteoblast function in young‐ and middle‐aged mice lacking menin in the osteoblast lineage. Menin deficiency specifically in osteocytes upregulated the expression of C‐X‐C motif chemokine 10 (Cxcl10) and led to enhanced osteoclastogenesis, suggesting menin modulated osteocyte–osteoclast crosstalk in osteoporosis.Men1f/f;Runx2‐Cre mice displayed lesion of ossifying fibroma (OF) in mandibular bone, with elevated levels of early osteoblast differentiation markers such as ALPL, COL1A1, RUNX2, and OSX. The protein levels of the late osteoblast differentiation marker OCN, however, remained unchanged. The jaw bone‐derived primary mesenchymal stromal cells isolated from the OF tumour (OFMSCs) showed a significant reduction of Men1 mRNA level and reduced ALP activity and mineralization. These results suggested Men1‐dificient OFMSCs were arrested at preosteoblastic differentiation stage, leading to the disordered bone formation in OF lesion.Kanazawa et al. found that osteoblast‐specific deletion of Runx2/p57. In addition, MEN1, MLL2, and MLL3 (but not MLL1 or MLL4) bind to Runx2 P1 promoter, regulating the H3K4me3 state that promotes and sustains the expression of this bone‐master gene in osteoblasts.Mll4 showed precocious chondrocytes differentiation. Mechanistically, Mll4 deletion decreased H3K4me3 modifications on the short stature homeobox 2 (Shox2) gene, leading to increased expression of Sox9 and inhibition of chondrocytes transdifferentiation to osteoblasts.MLL4 in hTERT‐immortalised human adipose‐derived MSCs showed significantly impaired chondrogenic differentiation and partially impaired osteogenesis, which could be rescued by inhibition of the nuclear mechanosensor, ATR serine/threonine kinase.Mll4 knockout mice, which was generated by Wnt1‐Cre, the hypertrophic chondrocyte differentiation and bone deposition in structures of cranial base were significantly decreased, representing a deficiency in endochondral ossification.MLL3 (KMT2C) and MLL4 (KMT2D) are homologues of a Drosophila H3K4 monomethyltransferase, Trithorax‐related (Trr).KDM6A overexpression in human MSCs promoted osteogenesis by activating expression of osteogenic genes and inhibited adipogenesis, whereas KDM6A knockdown by siRNA had the reverse effect.Kdm6a in osteoblast cells, resulting from the increased level of H3K27me3 on the promoter regions of Runx2 and Osx.Dkk1) caused by glucocorticoid treatment and was indispensable in averting glucocorticoid‐impaired osteogenesis. Mice treated with glucocorticoid and KDM6A inhibitor GSK‐J4 exhibited a reduction of bone mineral density and trabecular bone loss.KDM6A expression was upregulated in osteogenic human periodontal ligament stem cells (hPDLSCs), along with increased ALPL, RUNX2, and OPN expression. As a target of miR‐153‐3p, KDM6A overexpression reversed the microRNA's inhibitory effect on the osteogenic differentiation of hPDLSCs.KDM6A belongs to the H3K27me3 demethylase KDM6 family which also contains lysine demethylase 6B (KDM6B), antagonising the PcG repressive H3K27 methyltransferase EZH2.KDM6B instead of KDM6A was increased in chondrogenic differentiation. Knockdown of KDM6A and KDM6B by siRNA inhibited expression of chondrogenic‐related markers such as aggrecan (ACAN), collagen type II alpha 1 chain (COL2A1), collagen type X alpha 1 chain (COL10A1) and SRY‐box transcription factor 9 (SOX9), though KDM6A knockdown had a lesser effect. Demethylase inhibitor GSK‐J4 treatment also showed reduced total collagen and glycosaminoglycan (GAG) during MSCs chondrogenesis.KDM6A via shRNA repressed proteoglycans and collagen formation in both monolayer and micromass culture of hPDLSCs. The mRNA levels of SOX9, COL2A1 and ACAN were decreased in KDM6A‐deleted hPDLSCs, and H3K27me3 was increased at SOX9 promoter. The EZH2 inhibitor EPZ‐6438 decreased H3K27me3 level and rescued the impaired chondrogenic potential, suggesting the dynamic balance of H3K27 methylation is an important facet in the regulation of stem cell chondrogenesis.Kdm6a and Sox9 expression. Treatment with cocultured BMSCs and ACCs via knee joint cavity injection on OA rats lessened the cartilage lesions, which might reveal a promising direction for OA treatment.It is known that H3K27me3 levels are regulated during MSCs chondrogenesis. Yapp et al. investigated the role of KDM6A and KDM6B in chondrogenic human bone marrow‐derived MSCs. The expression of 3.2In recent years, SWI/SNF complexes have gained much attention for their essential roles in gene expression regulation, chromatin modification maintaining, and DNA repair.Brm‐null mice developed normally to adulthood in contrary to the early embryonic lethality of Brg1‐null mice.Brm‐null mice showed significant increases in Alpl and Fgfr2 expression, but the level of RUNX2 and OSX remained unchanged compared to wildtype, suggesting that Brm deletion merely mediated the early commitment of mesenchymal stem cells to the osteoblast lineage rather than induced differentiation into mature osteoblast. Moreover, adipogenesis was impaired in Brm‐deleted C3H10T1/2 cells as well as Brm‐null BMSCs. Brm‐null mice showed a phenotype of adiposity reduction in the bone marrow and resistance to age‐related osteoporosis.The two independent ATPases have distinct roles in mammalian development: Brg1 expression was confirmed in the developing skeleton and primary osteoblasts. Transgenic NIH‐3 T3 cells expressing a mutant BRG1 protein showed inhibited BMP2‐induced expression of alkaline phosphatase (APase), indicating that SWI/SNF chromatin remodelling activity is essential for osteogenic lineage induction.Bglap promotor. The ATPase activity of SWI/SNF is required for Bglap transcription.Osx promoter in osteogenic‐differentiated C3H10T1/2 cells and the catalytic activity of SWI/SNF is required for Osx expression.Runx2/p57 was independent of SWI/SNF complexes activity as observed in C2C12 cells.Studies by Young et al. assessed the link between SWI/SNF complex subunits and BMP2‐induced osteoblast differentiation. Bglap, Fasl, and Per3. BRM deficient MC3T3 cells showed enhanced osteogenic differentiation resisting the repressive effect of dexamethasone. These results indicate that glucocorticoid‐mediated inhibition of osteogenic genes is highly dependent on BRM‐SWI/SNF complexes.Brm and Brg1 gene depletions in MC3T3‐E1 cells have shown that Brg1 deficiency impeded osteogenic differentiation, while disruption of BRM accelerated mineralization accompanied higher expression levels of osteogenic markers. BRG1‐specific SWI/SNF complexes were required for activation of tissue‐specific genes. On the contrary, BRM‐containing complexes were found to associate only with the repressed promoter along with the corepressor HDAC1.Pbrm1, Arid2, and Brd7, respectively, impaired BMP‐Smad1/5/8‐signalling‐induced expression of osteogenic early responsive genes including Alpl, Osx, Runx2, and attenuated long‐term osteogenesis in OP9 murine MSC cell line and human BMSCs. Mechanistically, co‐localization of PBRM1/ARID2/SMARCC1/EP300 and H3K27ac was found on the transcription start site (TSS) of Alpl, Bmpr1b, and Tgfbr2 genes, indicating a direct role of PBRM1/PBAF in transcriptional activation.BRM was required for glucocorticoids to get access to the promoters of osteogenesis favoured or adipogenesis inhibiting genes such as Fgfr3) was upregulated by transcription factor SP1. The regulatory mechanism of this process was revealed as BRG1 exposed SP1 binding sites at Fgfr3 promoter.The SWI/SNF‐mediated chromatin remodelling mechanism has been proved to exist in cartilage tissue. 11 genes of SWI/SNF complexes including BRG1 and BRM were identified by comprehensive mapping of human cartilage‐expressed transcripts.3.3Ash1l expression with bone mass. Ash1l knockdown suppressed osteogenic and chondrogenic differentiation of C3H10T1/2 cells via decreasing H3K4me3 deposition on the promoter region of Hoxa10, Osx, Runx2, and Sox9 genes. These results hint the role of ASH1L in osteogenesis and chondrogenesis of MSCs is dependent on its HMTase activity.HOX genes and was responsible for H3K4 trimethylation.Absent, small, or homeotic disc1 like (ASH1L) is another member of the Trithorax group containing a SET domain and is known as an epigenetic transcriptional activator counteracting Polycomb repression.3.4CREBBP/EP300 genes, exhibit HAT activity that is responsible for H3K27 acetylation (H3K27ac).The Trx complex named TAC1 in Drosophila contains CBP which interacts with Ash1 and confronts Polycomb silencing.Crebbp heterozygous mice show various tissue defects involving the skeletal, haematopoietic and nervous system. It has been found that heterozygous Crebbp‐deficient mice showed a significant decrease in trabecular bone volume mainly related to osteoclastogenesis of bone marrow stromal cells since the mineral apposition or bone formation rates are unperturbed.CREBBP or EP300 cause the Rubinstein‐Taybi syndrome, which is autosomal dominant inheritance and exhibits congenital abnormalities such as distinctive facial features, skeletal dysplasia, and intellectual disability.Homozygous null mutations of CBP or p300 in mice cause early embryonic lethality, and RUNX2, OCN, OSX, and matrix metallopeptidase 13 (MMP13).SOX9), which is considered a master regulator in chondrocyte differentiation. SOX9, SMAD3, and p300 formed a transcription complex on the SOX9‐binding‐site of COL2A1 gene enhancer region, and facilitated COL2A1 expression in human MSCs.CNMD), a cartilage specific protein, in human MSCs. The PcG member YY1 transcription factor repressed chondromodulin expression in undifferentiated MSCs by recruiting histone deacetylase HDAC2. As a co‐activator, p300 is associated with SP3 transcription factor and bind the core‐promoter region of CNMD gene. The combination of siRNA inhibition of YY1 and forced expression of p300 and SP3 markedly induced chondromodulin expression.The mechanism of CBP/p300 in gene expression activation has been well established. CBP/p300 associates with gene enhancer and TSS regions, bind to various transcription factors bridging them to RNA polymerase II, and relax the chromatin structure at target gene promoters via their HAT activity.4As important components of epigenetic regulation, TrxG proteins mediate transcriptional activation via histone methylation, acetylation, and modifications on chromatin structure. This paper summarises the regulatory roles of TrxG proteins in MSCs osteogenic and chondrogenic differentiation, as shown in Table The authors declare no conflict of interest.Q.M. C.S. and B.Y. collected the related paper and drafted the manuscript. C.S. and Y.S. revised the manuscript. L.Y. designed the review and revised the manuscript. All authors read and approved the final manuscript.


The histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences.Recently, we developed isogenic CRISPR-edited DIPG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway, in particular, ASCL1 and NEUROD1.Altogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild-type.The online version contains supplementary material available at 10.1186/s13072-022-00447-6. One of M-mutant . The K27M-mutant –9. CurreM-mutant , 9–17. AM-mutant .The exact scope of oncogenic mechanisms are still to be defined, but it is established that H3.3K27M disruption to epigenetic marks are involved in the maintenance of tumor properties , 19. SevImportantly, the H3.3K27M mutation, and subsequent chromatin mark changes, in DIPG results in modifications to the transcriptome that are mostly unique from those associated with H3.1K27M DIPG and H3-WT glioblastoma including increased expression of genes related to neural development, neurogenesis, NOTCH signaling, and differentiation , 26, 27.Here we build upon our previous work, where we used CRISPR–Cas9 to gene-edit the H3.3K27M point mutation in established DIPG lines to generate isogenic H3.3 wild-type cell lines . We asseTo study how the oncohistone H3.3K27M affects chromatin dynamics and how chromatin landscape changes impact gene expression we used Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) –33 on ouH3F3A mutation status, that is H3.3K27M lines clustered together, while wild-type H3.3 clustered together , and intergenic regions of the genome for each cell line. Interestingly, the percentage of accessible peaks mapped to the promoter regions decreased upon reversion of H3.3K27M to wild-type  analysis of biological processes, cellular components, molecular function, and KEGG pathways. Both parental lines (XIII and XVII) showed enrichment for nervous system development, homophilic cell adhesion, and actin binding terms in the genes in their respective open chromatin regions highlighting processes that are likely important to DIPG biology more broadly  transcription factors with known functions in neuronal cell fate and differentiation of glioblastoma stem cells (GSCs) as well as normal neuronal cells [A similar pattern was also observed following overlaying the regulatory enhancer and super-enhancer regions with the open chromatin regions that we identified. XIII and XVII cells were enriched for enhancers and super-enhancers related to NOTCH signaling and nervous system development genes including, but not limited to, al cells –57. Our al cells , 56, 58.al cells , 60.COBL, which is upregulated and has accessible enhancer and super-enhancer regions in H3.3K27M cells. COBL is a calcium and calmodulin-dependent actin nucleator in developing neurons, particularly in the cerebellum, that increases neurite formation and branching [Fitting with our GO results, motif analysis revealed that open chromatin regions in H3.3K27M cells are enriched for ASCL1 and NEUROD1 binding motifs. Those motifs are located in genes related to neuronal development including ranching , 35, 61.H3F3A status resulting in some degree of sample clustering by principal component analysis based on patient origin rather than completely based on H3F3A status. It has been extensively documented that while histone H3 and IDH1 mutation status are the main identifiers for pediatric high grade-glioma (HGG) and DIPG subgroups there are other secondary mutations within these subgroups that drive disease progression [While there were consistencies between the two parental K27M DIPG cell lines and separately between the two isogenic H3.3 wild-type lines, some distinct differences remained even between cells of like gression , 62–65. OLIG2 enhancer region for both H3.3K27M DIPG lines compared to matched isogenic H3.3 wild-type lines, consistent with a previous study [OLIG2 in line XIII and its promoter in line XVII. OLIG2, like ASCL1 and NEUROD1, is a basic helix–loop–helix transcription factor and a marker of the oligodendrocyte lineage, one of the proposed cells of origin for DIPG [OLIG2 has been shown to be necessary for DIPG cell tumorigenesis and proliferation within orthotopic mouse xenograft models containing OLIG2 knockdown demonstrating reduced tumorigenesis and increased mouse survival [OLIG2 expression mark the potential cell population of origin but also presents a potential drug target for DIPGs [OLIG2 is an upstream activator of ZEB2 and increased OLIG2 levels have been shown to activate ZEB2 during development leading to oligodendrocyte precursor cell (OPC) maturation and myelination [ZEB2 is an established regulator of EMT and has been linked to increased invasiveness and migration, poor prognosis for glioma patients, and is upregulated in glioblastoma and in DIPG as demonstrated in this study [ZEB2 is also linked to Notch signaling during Schwann cell development and acts as a repressor of Notch-Hey2 signaling [ZEB2 may be interacting with NOTCH or one of its downstream targets in a way that drives disease progression in DIPG. Further investigation of ZEB2 in DIPG and its role in NOTCH signaling could help inform new therapies, especially those that target EMT. Our model suggests that H3.3K27M results in decreased H3K27me3 at the OLIG2 enhancer ultimately resulting in increased expression of OLIG2 and driving disease progression  and triturated until cells were lysed. Samples were incubated on ice for 3 min, washed with 1 mL of the buffer , and centrifuged at 500 RCF for 10 min at 4 °C. The pellet was resuspended in 50 uL of transposition mixture containing 25 uL of 2× TD Buffer , 5 uL Transposase (Illumina Nextera Kit), 0.5 uL 0.1% digitonin, 0.5 uL 10% Tween-20, and PBS, and incubated at 37 °C for 60 min, and DNA recovered using Zymo DNA Clean and Concentrator-5 Kit. Libraries were generated by PCR in 50 uL reactions  . The PCusegalaxy.com to define the gene coordinates of the peaks [ATAC-seq samples were sequenced in duplicate using the HiSeq with paired-end 150 bp sequencing. Adaptors were removed from raw paired-end sequencing files using bbduk (BBMap version 38.70 BBDuk) . The reshe peaks . Gene onhe peaks , 78, 79.he peaks , 81 and he peaks . Modelinhe peaks .To identify a K27M-specific transcriptional network, accessible chromatin regions in line XIII Parental cells, were intersected with genes significantly upregulated in line XIII parental (K27M) cells compared to WT cells. FIMO was used to scan these regions for the motifs identified as enriched in K27M ATAC-seq peaks (version 5.4.1) , 81. A nIn performing ChIP, cells were crosslinked with 1% formaldehyde, lysed, and sonicated using a Bioruptor Pico (Diagenode) to generate chromatin fragments < 500 bp. For each ChIP, 20–30 μg of sonicated chromatin was used and immunoprecipitated using an H3K4me3 antibody (Millipore 04-745), on magnetic Dynabeads (Invitrogen). For spike-in normalized ChIP-qPCR, 5 μg of sonicated Drosophila chromatin was added to each ChIP sample prior to immunoprecipitation. qPCR enrichment was normalized to Drosophila values across all samples. Primers are listed (5ʹ-to-3ʹ) as follows:COBL and OLIG2, normalized to GAPDH) on a Stratagene Mx3005P. Primers are listed (5ʹ-to-3ʹ) as follows:RNA was extracted from cells using the NucleoSpin RNA Kit (Macherey–Nagel) from which cDNA was made using the iScript cDNA Synthesis Kit (Bio-Rad). RT-qPCR was performed using the PowerUp SYBR Green Master Mix , Student’s Additional file 1: Figure S1. Genome-wide profile of accessible chromatin regions in H3.3K27M and H3.3-WT DIPG tumor samples. Table S1. Summary of HOMER and DiffBind results for ATAC-seq samples. Figure S2. H3.3K27M DIPG cells are enriched for open chromatin peaks genes related to the nervous system and GTPase activity compared to their isogenic gene-edited wild-type counterparts. Table S2. Overlap of gene expression changes and ATAC-seq peak changes between WT and K27M cells. Table S3. Overlap of enhancer and super-enhancer regions with ATAC-seq peaks in XIII and XVII cell lines. Figure S3. H3.3K27M DIPGs specific transcription factor binding sites are enriched in nervous system and neuronal development genes. Figure S4. Determination of enriched transcription factors shared between H3.3K27M and H3.3-WT peaks. Figure S5. H3.3K27M DIPG cells differentially express transcription factor ZEB2.Additional file 2: Table S4. A. Super-enhancers with open-chromatin and increased gene expression in parental DIPG lines . B. Supn et al.  and supen et al. ).