Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark enhances the interaction of architectural protein with chromatin regulatory sites and identify determinants that facilitate the localization of HMGN proteins at regulatory sites to modulate cell-type specific gene expression. The nucleosome-binding proteins HMGN1 and HMGN2 prefer acetylated nucleosomes at H3K27ac and the loss of this interaction alters binding of transcription factors to chromatin. The interaction of TFs with chromatin is dynamic; TFs continuously move throughout the nucleus and reside temporarily at their specific binding sites5. The binding of TFs to chromatin is facilitated by nuclear factors that help TFs access their binding sites and perturbed by factors that impede the binding of TFs to specific chromatin sites, especially at enhancers and promoters6, chromatin regions enriched in H3K27ac nucleosomes7. Possible regulators of TF chromatin binding include architectural proteins, such as the linker histone H1 and high mobility group (HMG) proteins; these ubiquitous structural proteins are known to affect chromatin organization in many cell types. Histone H1 facilitates the formation of higher-order chromatin organization and stabilizes compact chromatin structures that inhibit transcription10, while HMG proteins are mostly associated with reduced chromatin compaction and increased gene expression from chromatin templates12. Given the global effects of H1 and HMGs on chromatin structure and gene expression, it is likely that these ubiquitous structural proteins do modulate the binding of TFs to chromatin, a possibility that has not been studied in detail. Here we focus on the high mobility group N (HMGN) proteins and show that the major members of this family, HMGN1 and HMGN2, bind preferentially to nucleosomes containing the H3K27ac epigenetic mark, and affect the binding of TFs to chromatin.Correct binding of transcription factors (TFs) to their specific DNA motifs in chromatin plays a key role in establishing an epigenetic landscape that facilitates cell-type-specific gene expression necessary for the maintenance of cell identity14. The interaction of HMGN proteins with chromatin is highly dynamic; HMGNs bind to nucleosomes with short residence times and can be readily dislocated from their chromatin binding sites15. The binding of HMGNs to nucleosomes reduces chromatin compaction, most likely because it alters the interactions of linker histone H1 with chromatin18 and binds to the nucleosome acidic patch, a region thought to stabilize interactions between neighboring nucleosomes20. Although HMGNs bind to chromatin without DNA sequence specificity, they preferentially localize to enhancers and promoters, chromatin regulatory sites that are easily digested with DNaseI and enriched in epigenetic marks of active chromatin, including H3K27ac modified histones22. Changes in HMGN levels are frequently associated with a wide range of cell-type-specific changes in gene expression and phenotypes23. Genetically altered mice lacking both HMGN1 and HMGN2 proteins (DKO mice) are born and survive but show multiple phenotypes21, (https://www.mouseclinic.de/). MEFs isolated from DKO mice can be reprogramed into pluripotent cells by exogenous TFs more efficiently than MEFs isolated from WT mice suggesting that loss of HMGNs destabilized the maintenance of cell identity24. In Down syndrome, one of the most prevalent human genetic diseases, the presence of an extra copy of HMGN1 has been directly linked to increased levels of H3K27ac and to gene expression changes and to increased incidence of acute lymphoblastic leukemia26. Taken together, the available data suggest that HMGNs modulate and fine-tune cell-type-specific gene expression programs.HMGN is a family of abundant and evolutionarily conserved proteins that bind to nucleosomes without specificity for the underlying DNA sequence27, it is not clear how these structural proteins, that interact with chromatin without DNA-sequence specificity, can nevertheless affect gene expression and cellular phenotypes in a cell-type-specific manner. Likely, HMGNs affect cell-type-specific gene expression by preferentially localizing to chromatin regulatory regions, as indicated by genome-wide analyses that show high HMGN occupancy at chromatin regions containing high levels of H3K27ac22, an epigenetic modification that marks promoters and enhancers29. Conceivably, the presence of HMGN at enhancers and promoters may affect the interaction of TFs with these sites, thereby impacting cell-type-specific gene expression. The factors that target HMGN to enhancers and promoters and the possible effect of these proteins on TFs chromatin binding have not yet been investigated.Because the amount of HMGN in a cell is enough to bind only about 1% of the nucleosomesHere we identify the major determinants that facilitate the preferential binding of HMGN proteins to chromatin regulatory sites and show that HMGNs affect the binding of transcription factors to chromatin. Using bioinformatic analyses we demonstrate that HMGN1 and HMGN2 preferentially localize to nucleosomes containing the H3K27ac residue. We show that the presence of H3K27ac, an epigenetic mark of active chromatin, strengthens the binding of both HMGN1 and HMGN2 to the modified nucleosomes, and that loss of HMGNs alters H3K27 modifications and H1 occupancy at enhancers and promoters. We analyze the genome-wide binding of several TFs in cells derived from WT and DKO mice and find that loss of HMGNs alters the binding of TFs to chromatin, especially at enhancers. Our studies provide insights into factors that affect the recruitment of ubiquitous architectural chromatin binding proteins to regulatory chromatin to modulate transcription factor accessibility and fine-tune cell-type-specific gene expression programs.29, but not with H3K27me3, an epigenetic mark of silent chromatin. Furthermore, the intensity of both the HMGN1 and HMGN2 signal correlates positively with the intensity of the H3K27ac signal but shows no correlation with the H3K27me3 signal levels  Fig. , restingels Fig. . The H3K2ac Fig. . AlthougThroughout the genomes of the three cell-types, the H3K27ac signal, but not the H3K27me3 signal centers at the chromatin loci containing either HMGN1 or HMGN2 Fig. . Signifi28. These findings may provide insights into the molecular mechanism whereby HMGNs affect cell-type-specific gene expression.In sum, the ChIP seq data indicate that both HMGN1 and HMGN2 localize to nucleosomes containing the H3K27ac modification, a major epigenetic mark of cell-type-specific chromatin regulatory sitesTo verify that HMGNs bind preferentially to MNH3K27ac and explore the determinants that targets these proteins to the acetylated nucleosomes, we prepared chromatin from purified MEF nuclei, stripped the chromatin binding proteins by salt extraction, digested the salt-extracted chromatin with micrococcal nuclease, fractionated the digest on a sucrose gradient, and isolated chromatin fragments containing either only mononucleosomes (MN) or oligonucleosomes (ON); a mixture of tri-penta nucleosomes Fig. . To the 31  which in a mobility shift assay, migrate slower than MN particles31 Fig. . We reas31 Fig.  have a h31 Fig. . In our 31 Fig. . Dot-blo31 Fig. , indicat31 Fig. . In addiTaken together, the results show that HMGNs preferentially bind to nucleosomes and chromatin fragments containing H3K27ac but show no significant preference for nucleosomes containing other marks of active chromatin such H3K9ac and H3K4me1, or for nucleosomes containing H3K27me3, a mark of transcriptionally silent, compact chromatin.32. To test whether the H3K27ac modification, by itself, is enough to preferentially target HMGN to acetylated MNs, we used commercial recombinant MN (rMN) that were devoid of any modification or contained only the H3K27ac modification (rMNH3K27ac). We end labeled the DNA in rMN with AlexaFluor 488 (green) and the DNA in rMNH3K27ac with AlexaFluor 647 (red) respectively, mixed equal amounts of the red and green fluorescent-labelled particles, performed mobility shift assays at increasing molar ratio of HMGN to the fluorescently labeled rMNs, and scanned the gels to visualize either the green or red signal SAPATGG) spanning the H3K27ac residue  lls Fig. . In agrells Fig. , top. Aglls Fig. , bottom 35, an abundant protein family that binds dynamically to nucleosomes36 and stabilizes chromatin compaction8. Previous ChIP analyses indicated that H1 is evenly distributed throughout the nucleus but shows low chromatin occupancy at TSS38. Our ChIP analyses do not show statistically significant differences between WT and DKO in H1 occupancy at a particular site; however, the aggregate plots indicate a marginally higher occupancy of H1 at enhancers and promoters in DKO, as compared to WT . The preferential location of HMGNs at these sites and the epigenetic changes observed in DKO MEFs, together with the known effect of HMGNs on cellular transcription profiles300 Fig.  or Brd3 300 Fig. . Yet, Ch300 Fig.  and of B300 Fig.  througho300 Fig. . The chr300 Fig. . Similar300 Fig. . In agre300 Fig. .Fig. 4De41, were not affected by the loss of HMGN  that does not serve as a cell-type-specific super enhancer, and does not have a high content of H3K27ac, does not show high HMGN occupancy22. Furthermore, during reprograming of MEFs to induced pluripotent stem cells (iPSC), HMGNs relocate from the acetylated MEFs enhancers regions to the acetylated iPSCs regulatory regions, supporting the notion that HMGN bind preferentially to the regions containing H3K27ac regardless of the underlying DNA sequence22. Now we show a direct correlation between the signal intensity of H3K27ac and HMGN occupancy . These effects are most obvious at enhancers and promoters, regions which are marked by a high level of H3K27ac and high HMGN occupancy. The most highly acetylated promoters, which also show the highest HMGN occupancy in WT cells, show the highest increase in H1 occupancy in DKO cells , anti H3K27me3 (Abcam#ab6002), monoclonal anti H1(Milipore-Sigma #05-457), anti CEBPB (Abcam#ab32358), Anti-Brd3 (Active Motif #61489), Anti-Brd4 (Bethyl Laboratories #A301-985A100), Anti-CEBPB (Abcam #ab32358), Anti-CTCF (EMD Millipore #07-729), Anti-Ets1 (Active Motif #39580), Anti-Ikaros (Active Motif #39355), Anti-Irf8 (Bethyl Laboratories #A304-027A), Anti-Klf4 (Abcam #106629), Anti-Nanog (Active Motif #61419), Anti-Oct4 (Abcam #ab19857), Anti-p300 (Active Motif #61401), Anti-Pax5 (Abcam #183575), Anti-Sox2 (Abcam #97959).The following recombinant mononucleosomes were purchased from Active Motif: unmodified (#81070); H3K27me3 modified (#81834), H3K27ac modified (#81077).55 and resting B cells56 were as previously described. Peptides Histone H3 (23–34) peptide, KAARKSAPATGG and Histone H3K27ac (23–34) peptide, KAAR - K(Ac)—SAPATGG were from AnaSpec, Inc.Wild type and HMGN DKO mouse embryonic fibroblasts, embryonic stem cell lines58. Briefly, purified nuclei were digested by micrococcal nuclease, the chromatin prepared, non-core histone proteins removed by centrifugation in 0.5 M NaCl solutions and cation-exchange chromatography, and the salt-stripped chromatin particles loaded on 5–20% sucrose gradient. Fractions containing either mononucleosomes (MN) and oligonucleosomes  were pooled and dialyzed against 10 mM NaCl, 10 mM Tris-Cl, pH 7.5, 1 mM EDTA. For ChIP-Western analysis the dialyzed nucleosomes were mixed with recombinant HMGN1 or HMGN2 (HMGN:histoneH3 = 50:1 molar ratio) and cross-linked with 1% formaldehyde for 10 minutes at room temperature. After quenching with 0.5 M glycine the HMGN-nucleosome complexes were immunoprecipitated using ChIP-IT Express kit  with either anti-HMGN1, anti-HMGN2 antibodies, or normal rabbit IgG as control. The immunoprecipitated nucleosomes and 2% of the input material were de-cross-linked by heating to 95o for 45 min, and histone H3 isolated by HPLC (Agilent Technologies 1200 series) using a Luna 5 µm CN 100 Å HPLC column . Equal amounts of H3, as determined by OD220, from bound and input material were blotted on Immobilon PVDF membranes , using Schleicher & Schuell Minifold Spot-Blot System and the blots subjected to western analyses.Mono and oligo nucleosomes devoid of protein  were prepared from mouse embryonic fibroblasts (MEFs) derived in our laboratory and from chicken erythrocytes (Rockland R401-0050) as describedFor in vitro binding studies, commercial recombinant mononucleosomes (rMN), or rMN containing H3K27 modifications (Active Motif) were first end-labelled with aminoallyl dUTP using recombinant Terminal Deoxynucleotidyl Transferase (TdT), and then concentrated by spin-dialysis . The dialyzed rMNs were then labeled with either Alexa Fluor 488 (Green-Fluorescent) or Alexa Fluor 647 (Red-Fluorescent) with succinimidyl ester labeling kits . The fluorescent labelled rMN were concentrated by spin-dialysis.59 at ionic strength conditions that lead to binding of 2 molecules of HMGN per nucleosome. Binding reactions contained 100 nM of chromatin particles (25 ng/µl) in 10 μl binding buffer . HMGN1, HMGN2 proteins were added to chromatin particles to generate the molar ratio to nucleosomes listed in the figure legends. The mixtures were incubated at 4 °C for 10 min and loaded onto non-denaturing 5% polyacrylamide gels in 2× TBE  and run at 4 °C. The gel was scanned with ChemiDoc MP (BioRad) using duplex fluorescence detection mode (for Alexa Fluor 488 and Alexa Fluor 647). The images were analyzed by Image Lab Touch Software and QuantityOne (BioRad). The ratios between nonshifted nucleosomes  and shifted (HMGN-bound nucleosomes) were calculated for every titration point.Mobility shift assays were performed as described7 cultured cells were fixed in medium with 1% formaldehyde (v/v) for 10 min at room temperature on a rocking platform, followed by quenching with 125 mM glycine. Crosslinked cells were washed and incubated in 1 ml Chromatin Prep Buffer containing 1 μl proteinase inhibitor cocktail (PIC) and 1 μl of 100 mM PMSF for 10 min on ice followed by centrifugation at 1250× g for 3 min at 4 °C. The pellets were resuspended in 250 μl ChIP Buffer with 2.5 μl PIC and 2.5 μl 100 mM PMSF and sonicated for 10 cycles with Bioruptor (30 s on/ 30 s off). Aliquots of 25 μl of sonicated chromatin were used to generate the input DNA. 5–10 μg of affinity-pure ChIP-grade antibodies) were then added to the rest of the chromatin samples and incubated overnight at 4 °C with rotation. Following incubation 30 µl of protein G agarose beads or Magnetic Beads were added to each reaction and the mixtures were further incubated for 3 h or O/N at 4 °C. The beads were washed five times with Wash Buffer AM1 (Active Motif). ChIP DNA was eluted in 100 μl Elution Buffer AM4 (Active buffer). Cross-links were reversed at 65 °C overnight in the presence of 3 μl of 10% SDS and 5 μl of proteinase K (20 mg/ml). The DNA samples were eluted in 21 μl of elution buffer using MiniElute kit (Qiagen). ChIP-seq library was prepared following the manufacturer’s instructions (Illumina). Briefly, immunoprecipitated and input DNA were blunt ended, ligated to adapters, amplified with PCR and size selected. The ChIP templates were sequenced at 75 bp single read length with Illumina NextSeq 500 system or 101 bp paired-end length with HiSeq 2000 by the NIH CCR sequencing facility (for details see data submission file). For the various samples the number of trimmed reads, successfully mapped to the mouse genome, ranged from 12 to 74 million per sample, with an average of 27 million reads with over 80% of trimmed, non-duplicated reads mapped to the genome. Sequence reads were aligned to the Build 37 assembly of the National Center for Biotechnology Information mouse genome data (NCBI37/mm9). Super-enhancers and enhancers were identified as described before (21). Data for the following histone marks and HMGN were downloaded from GEO archive (accession numbers are indicated in brackets) and processed in the same way as described in the methods section: ES cells: H3K9ac (GSM2417092), H3K4me1 (GSM2629668), H3K27ac (SRP154652) H3K122ac (SRR3144856), HMGNs (SRP154652), H3K27me3 (SRP068453);MEF cells: H3K9ac (GSM1979773), H3K4me1 (GSM3272827), HMGNs(SRP154652); rB cells-H3K27ac(SRP154652), HMGNs(SRP154652). H3K27me3(GSM2184272) All of the rest of the data, were generated in our laboratory.The ChIP-Seq procedure was performed as recommended by Active Motif  Instruction Manuals for ChIP-IT High Sensitivity, ChIP-IT Express, and Chromatin IP purification kits. Briefly, about 10Chromatin binding peaks were initially selected for analysis based on a q-value cutoff 0.01 for broad and 0.05 for narrow regions as reported by the MACS2 peak-calling algorithm (broad or narrow). Peaks were identified as significantly differentially bound using the default threshold of FDR < 0.05. Differentially expressed genes between WT and DKO cells were binned according to the average expression of 3 WT and DKO samples, with 2794 genes assigned to each group. Statistical analyses were performed within the R (ver. 3.6) computing environment and visualized with IGV. Details of statistical analyses can be found in figure legends.https://www.bioinformatics.babraham.ac.uk/projects/fastq_screen) applications. Reads were mapped to UCSC mm9 reference genome using (10.1093/bioinformatics/btp324). Duplicate reads were marked with Picard v. 2.17.11. Reads mapped to blacklisted regions (10.1038/nature11247) were removed from further analysis. For all samples, peak detection of enriched binding regions was performed using either MACS2 v. 2.1.1.20160309 with the default settings, or SICER v. 1.1 with the following parameters: window size 300, gap size 600, FDR < 1e-2, effective genome size 0.75. BigWig files were used for visualization. Correlation heatmaps showing sample relations, scatterplots and peak profiles were generated using DeepTools v. 3.0.1 toolset. Differential binding sites were identified using DiffBind package. To calculate Pearson or Spearman correlation coefficients between different histone mark profiles, the genome was split into bins (bin size =5000 bp) followed by counting numbers of aligned reads within each bin for each sample and calculating a correlation between these sets.Adapter trimming was performed using CutAdapt v.1.16. Sequence quality before and after trimming was checked with FastQC 0.11.5 tool. Sequences were checked for contamination with Kraken v1.1 (10.1186/gb-2014-15-3-r46) and FastQscreen v.0.9.3 (56. Super Enhancer regions were downloaded from SuperDB Database as described22.RNA expression levels were determined by RNA-seq as describedFurther information on research design is available in the Supplemental InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early  Summary: Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression, but morphogens can also induce a graded response within such domains. Morphogen gradients manifest positional information by graded activation of signaling pathways that culminate in the activation of transcription factors. This activation leads to the induction of target genes that establish the fate of the tissue. The most common outcome of morphogen gradients is the division of a target tissue into distinct zones of gene expression, as originally proposed by the ‘French flag’ model . The genDrosophila embryo, where both aspects are operating.Although the morphogen dictates the division of a naive field into distinct uniform zones, there may be instances in which additional patterning within these domains is necessary. For example, in response to the morphogen, coordinated cell movement and tissue morphogenesis may require manifestation of the graded positional information. This output is inherently distinct, in that cells within a given zone would respond differently according to the level of the morphogen. To examine whether a morphogen gradient can also elicit patterning within a designated zone, we have investigated zygotic patterning along the dorsoventral (DV) axis of the Drosophila embryo is established by the Spaetzle/Toll signaling pathway  14, in parallel with embryo cellularization, during which all ∼6000 cortical nuclei are enclosed in individual cells. Live measurements have shown that graded Dorsal nuclear localization is re-established within 10-15 min of the onset of NC 14  (twist (twi) and snail (sna) invaginate by actomyosin-mediated constriction of their apical surfaces to form the ventral furrow (VF). This constriction is driven by activation of the small GTPase Rho1, which leads to recruitment of the actin nucleator Diaphanous (Dia), as well as Rho kinase , which activates myosin II  . After cyosin II A. VF foryosin II . In mutafolded gastrulation (fog), mist  and T48, are involved in myosin recruitment. They trigger the key initiating event, activation of Rho1, via its activator RhoGEF2 .Most of the proteins comprising the contractile machinery are provided maternally and, therefore, are ubiquitous and uniformly distributed within the early embryo. Thus, the ability of the Dorsal-nuclear gradient to shape the spatial pattern of mesoderm internalization relies on directing the patterned expression of zygotic target genes that will drive actomyosin contractility. Three key zygotic target genes that are expressed in the ventral part of the embryo,  RhoGEF2 . Fog is  RhoGEF2 . In addi RhoGEF2 , where mT48 and mist  that define the ventral mesodermal zone downstream of the Dorsal gradient and subsequently drive the expression of a plethora of tissue-specific genes. To monitor the expression of twi quantitatively, we used single-molecule fluorescence in situ hybridization (smFISH), which allows visualization of the active transcription sites (TSs) in the nucleus, as well as the accumulation of individual mRNA molecules in the cytoplasm. Given that the analysis is carried out on fixed embryos, each embryo represents a single time point, and the combined analysis of such ‘snapshots’ of multiple staged embryos allows for the reconstruction of the dynamic expression profile.twi expression displays a switch-like behavior, manifesting a sharp on/off threshold in response to Dorsal-nuclear localization levels. Active twi TSs appeared simultaneously throughout the entire mesoderm region. The spatial distribution of these sites within the mesoderm was uniform along the DV axis  and even appearance of Twi throughout the mesoderm during NC 14  pioneer factor, which clears the regulatory region from nucleosomes . Moreover, binding of Zld to the twi regulatory region is initiated as early as nuclear cycles 8 to 9 . However, whereas twi expression encompassed the entire mesoderm from the outset, the expression of T48 was initially observed only in the central  part of the mesoderm .Twi is necessary for activation of  embryos . However13 to 14    . The iniT48 TSs was measured along the ventral domain of this embryo. This value was then used to derive the activation probability per minute at different positions along the DV axis  indicates that the probability of TS activation declined linearly with the reduction in Dorsal levels. Such a monotonic response to Dorsal is in sharp contrast to the nonlinear response of the twi enhancer to Dorsal, which generates the sharp borders of twi transcription. The activation probability of twi expression was indeed less correlated with Dorsal nuclear levels  two T48 TSs were observed   should give rise to a graded distribution of 92±0.02) A-C. GiveT48 TS activation . The fraction of activated TSs increases over time in a graded manner. If every activated site is transcribing at a similar rate, this would still lead to the graded accumulation of mRNA, because the ventral TSs were activated earlier . The mist enhancer also displayed paused Pol II  , we assumed that transcription had reached a steady-state on all TSs in older embryos. mist mRNA accumulated in the cytoplasm in a graded manner .Expression of enhancer   . Graded Fig. S3) A,B. A grapparent A′,B′. Ofd manner A″,B″. Mod manner C. Finallmist loci, the intensity of TSs and single mRNA spots was compared , necessitating a delay. An indication that regulation of mature mRNA production may contribute to this timing mechanism was observed when comparing the cytoplasmic mRNA accumulation patterns of twi and T48. As reported above  . Given that twi is a short gene of 2.2 kb, no delay in completion of transcription is expected to take place. In contrast, T48 is 29 kb long, because of the presence of large introns encompassing ∼25 kb. This organization is exceptional for early zygotic genes, which are typically intron-less or have only short introns    in the detection of TSs. The onset of T48 5′ transcription was observed in the ventral-most nuclei by 7 min into NC 14, with no apparent detection of TSs displaying the 3′ probe. The positive nuclei predominantly displayed a single 5′ TS . By 24 min, 5′-probe-displaying TSs expanded laterally and more nuclei displaying two spots appeared. TSs that also showed the 3′ probe began to appear in the ventral-most nuclei . The ∼17 min delay between the two panels provides an estimate of the time required for Pol II to complete T48 transcription at 20°C. By 38 min, TSs displaying both 5′ and 3′ probes were detected throughout the mesoderm, with most nuclei showing two spots . T48 mRNA was then observed by both probes in the cytoplasm.If the intron prolongs T48 intron, then transcription of intron-less T48 within a given nucleus should be completed earlier than that of the endogenous locus. To test this prediction, we fused a T48 cDNA construct to its 5′ regulatory region  domain of the mesoderm, expanding laterally over time. This expansion was consistent with a TS activation response to the Dorsal nuclear gradient, which is maximal at the ventral midline. Distribution of the number of activated TSs per nucleus indicated that the probability of activation is independent for each allele. Thus, two alleles within the same nucleus that experience similar levels of nuclear Dorsal may be switched on at different times. The probability of TS activation was linearly correlated with the levels of nuclear Dorsal.We found two distinct Dorsal-dependent mechanisms that generate graded mRNA accumulation for T48 and mist TSs is not known. Transcription of early zygotic genes in the embryo is facilitated by binding of the pioneer factor Zld, which removes nucleosomes from the regulatory regions  in the Drosophila embryo  embryos.The bulk of experiments were performed using wild-type -GFP embryo (BDSC line B-51564).For ectopic Toll activation, we analyzed embryos generated by females carrying 4 driver . Intronlpromoter . The conTable S1). Three hours after egg lay (AEL), embryos were fixed for 25 min in 4% formaldehyde, washed in methanol and kept at −20°C. The following day, embryos were washed in methanol and then in ethanol, rocked in 90% xylene:10% ethanol for 1 h, post-fixed in 4% paraformaldehyde/PBS/0.05 M EGTA for 25 min, washed three times (10 min each), then incubated for 6 min with 10 µg/ml Proteinase K recombinant, PCR Grade  and post-fixed again. Embryos were transferred gradually to 10% Formamide Deionized  in 2× SSC+10 μg/ml ssDNA preheated to 37°C and prehybridized for 30 min at 37°C. Hybridization buffer included 10% FA, 10% dextran, 2 mg/ml bovine serum albumin , ribonucleoside vanadyl complex  and ssDNA+ tRNA in 2× SSC, containing the probe set (1 ng/μl) (Probe sets were designed by Stellaris Probe Designer and purchased from LGC Biosearch Technologies (see 1 ng/μl) . Hybridi1 ng/μl) .z-axis and the TS spots in each channel were then detected using the method of z-projected images were processed with a log-filter , and the detection threshold was defined as the first threshold in the environment of which the number of detected TSs stayed relatively constant [change (in log) smaller than half the maximal relative change across all thresholds (in log)]. Then, eight-connected pixels above the threshold were defined as distinct TSs and their intensity defined as the sum of their pixel intensities. For each embryo, the midline was then defined according to twi fluorescence (or T48 if twi was not available) and the distance (in pixels) between each TS and the midline was calculated. To convert the pixel measurement into nuclei columns, the DAPI image was used to determine the average column distance for each embryo and all distances were converted accordingly. For comparisons with the Dorsal nuclear level, Dorsal levels in each nuclear column were taken from https://github.com/barkailab).After acquisition, raw images were processed in MATLAB (MathWorks). First, and for each channel, the image stack was maximally projected along the After acquisition, raw image stacks were processed in MATLAB. For each fluorescence channel, the ‘local’ background fluorescence (median fluorescence in a moving sphere) was first determined and subtracted from the raw signal. The background-corrected image stacks were processed with a three-dimensional log-filter, and the spots on the processed image stack detected with the mRNAcount function from T*p), with T being the time in NC 14 and p being the activation probability. This formula was used to calculate the activation probabilities in Based on our results, TS activation was assumed to be a stochastic, independent process and, thus, the activated promoter fraction (at a certain nuclear column) can be described by 1−exp(−To calculate the correlation between nuclear dorsal level and the activated fraction or activation probability D-F, we f


High density of Pol I per gene imposes topological constraints that establish a defined pattern of polymerase distribution along the gene, with a persistent spacing between transcribing enzymes. RNA folding during transcription directly acts as an anti-pausing mechanism, implying that proper folding of the nascent rRNA favors elongation in vivo. Defects in co-transcriptional folding of rRNA are likely to induce Pol I pausing. We propose that premature termination of transcription, at defined positions, can control rRNA production in vivo.Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35S/47S in yeast/human) is achieved by up to hundreds of RNA polymerase I (Pol I) enzymes simultaneously transcribing a single rRNA gene. In this review, we present recent advances in understanding the coupling between rRNA production and nascent rRNA folding. Mapping of the distribution of Pol I along ribosomal DNA at nucleotide resolution, using either  Yeast haploid cells contain between 150 and 200 copies of tandemly repeated rRNA genes while the diploid human genome contains around 400 copies. Although present at a high copy number in the genomes, not all rRNA genes are actively transcribed. In budding yeast, only about 50% of the genes on average are transcribed in exponentially growing cells. Each ribosomal gene unit spreads over 9.1 kb of DNA and contains two transcribed regions encoding the 35S pre-rRNA, transcribed by RNA Polymerase I (Pol I), and the 5S rRNA, transcribed by Pol III . These tSaccharomyces cerevisiae Pol I revealed the interactions occurring between its 14 subunits: the two large subunits Rpa190 and Rpa135 organize the enzyme in two modules of similar mass  including two large subunits, Rpa190 and Rpa135, jointly forming the active site of the enzyme  Figure . Crystallar mass . The Pollar mass .upstream activating sequence (UAS) and the core element (CE) (upstream activating factor (UAF), core factor (CF), TATA-binding protein (TBP) and the Rrn3 transcription factor (transcription start site (TSS) and Pol I and Rrn3 are released from the PIC upon transcription initiation. Several structural studies gave new insights into Pol I promoter recognition and melting, and more broadly into transcription initiation by yeast Pol I (Formation of preinitiation complex (PIC) is presented in ent (CE) . Recruitn factor . UAF is n factor . TBP wasn factor . Rrn3 isn factor . Transcrst Pol I . These sD235A) could not suppress the Pol I termination defect observed in absence of Rat1. The absence of both Rat1 and Fob1, bound to the replication fork barrier (RFB) site  site , increasFB) site .rpa49 deletion strain, Rrn3 is recruited less efficiently at the promoter and fails to dissociate from elongating polymerases following transcription initiation  showed reduced Pol I occupancy on the rDNA  method, based on deep sequencing of the 3′ ends of nascent transcripts associated with the polymerase  technique to map the position of Pol I on rDNA during elongation. CRAC consists in crosslinking Pol I to its associated nascent rRNAs during elongation in vivo, followed by complex purification, reverse transcription of associated rRNAs and sequencing of cDNAs . Pol I stalling is known to increase premature termination of the paused Pol I and possibly also of the downstream polymerases (premature termination of transcription (PTT) for Pol II  of the largest Pol II subunit . It is pi.e. the ability of Pol I to carry out continuous RNA synthesis on the DNA template without premature termination. This hypothesis is based on several experimental evidences. First, Miller spreads showed that the amounts of Pol I engaged in transcription are comparable in wild-type (WT) and mutant cells, meaning that the increased production of rRNA is not due to a major enhancement of Pol I initiation rate (in vitro promoter-dependent transcription assays confirmed that transcription initiation rate is similar between WT and SuperPol I. On the other hand, a tailed template assay, measuring elongation rate in vitro, revealed an increased rRNA production by the SuperPol I, likely due to a higher processivity (i.e. a lower production of abortive rRNAs.In order to better understand transcription regulation, Pol I mutants are of particular interest. We have recently identified in a genetic screen a super-active Pol I mutant, bearing a single substitution on the second largest subunit: Rpa135-F301S allele, hereafter named SuperPol I. This mutant induces an increase of rRNA production in yeast . The mecion rate . Moreoveessivity . Taken tMethods allowing to map at nucleotide resolution Pol I pausing sites during elongation revealed a key interplay between RNA folding and elongation rate: formation of rRNA secondary structures prevents backtracking, hence enhances elongation rate. With a large amount of co-transcriptional folding of rRNA, we are now able to study how processing events are affecting Pol I elongation rate. So far limited to budding yeast, there is no doubt that some Pol I regulatory mechanisms are evolutionary conserved, as Pol I elongation rate is limiting for rRNA synthesis in metazoan cells . The und


Triticum aestivum) is an allohexaploid that is generated by two subsequent allopolyploidization events. The large genome size (16 Gb) and polyploid complexity impede our understanding of how regulatory elements and their interactions shape chromatin structure and gene expression in wheat. The open chromatin enrichment and network Hi-C (OCEAN-C) is a powerful antibody-independent method to detect chromatin interactions between open chromatin regions throughout the genome.Bread wheat . The genes linked by HOCIs represent higher expression level and lower coefficient expression variance than the genes linked by other loops, which suggests HOCIs may coordinate co-expression of linked genes. Thousands of interchromosomal loops are identified, while limited interchromosomal loops (0.4%) are identified between homoeologous genes in hexaploid wheat. Moreover, we find structure variations contribute to chromatin interaction divergence of homoeologs and chromatin topology changes between different wheat species. The genes with discrepant chromatin interactions show expression alteration in hexaploid wheat compared with its tetraploid and diploid relatives.Our results reveal open chromatin interactions in different wheat species, which provide new insights into the role of open chromatin interactions in gene expression during the evolution of polyploid wheat.The online version contains supplementary material available at 10.1186/s13059-022-02611-3. Actiresponse . Severalresponse , 5. Genen glumes . The thrn glumes , 8. Recen glumes , 10 and n glumes –14. Exten glumes . The dynr plants . In maizpression , 18.Triticum aestivum) is a widely cultivated allohexaploid crop and evolved through two rounds of interspecific hybridization and polyploidization. The first allotetraploidization occurred 0.36 to 0.50 million years ago and involved hybridization between Triticum urartu (AA) and an undiscovered or extinct species closely related to the Aegilops speltoides (SS) [Triticum turgidum L., AABB) and a goatgrass species  ~ 8000–10,000 years ago resulted in the formation of bread wheat [Polyploidy is widespread in plants and more than 70% of angiosperms are considered to be polyploids . Polyplodes (SS) . The secad wheat .Recent researches about wheat chromosome architecture using Hi-C and HiChIP represented the presence of subgenome-specific territories and highly coordinated expression of genes involved in RNA polymerase II-associated loops , 24. YetIn this study, we generated chromatin interaction maps for hexaploid wheat and its tetraploid and diploid relatives using open chromatin enrichment and network Hi-C (OCEAN-C), which could capture global chromatin interactions between open chromatin regions without relying on specific antibodies . By inteT. aestivum cv. Chinese Spring (AABBDD). A total of 276 million valid interaction pairs were obtained and high reproducibility was observed between two biological replicates (Pearson correlation = 0.97)  of ATAC-seq peaks were overlapped with OCEAN-C peaks  and the peaks located more than 3 kb away from genes were annotated as distal peaks (D). The intrachromosomal loops contained 154,393 (43.2%) P-P interactions (PPI), 152,358 (42.7%) P-D interactions (PDI), and 50,133 (14.0%) D-D interactions (DDI) , which may be due to higher chromatin accessibility in chromatin ends of D subgenome     interacted with a same HOCI in chromosome 3A showed lower CV than the homoeologous genes in chromosome 3B and 3D that were not looped by a specific region  Fig. b. Strikist) Fig. c. For exFurthermore, we identified 1301 chromatin interaction networks (ChINs) containing at least three chromatin loop anchors  among subgenomes Fig. c. For exP < 0.01, Wilcoxon rank-sum test)  Fig. d, suggesst) Fig. e. To furst) Fig. f,g. The st) Fig. f,g.Fig.T. turgidum ssp. durum (AABB) and diploid Ae. tauschii (DD) in addition to hexaploid T. aestivum. OCEAN-C reads of diploid and tetraploid wheat were firstly mapped to published genome sequences of diploid and tetraploid wheat, respectively. However, we found low mapping rates of OCEAN-C data and many anomalous chromatin interaction structures in the OCEAN-C interaction matrix for Ae. tauschii  in AB (T. aestivum vs T. durum) and D subgenomes (T. aestivum vs Ae. tauschii), respectively   and D subgenomes (T. aestivum vs Ae. tauschii), respectively. It is worth noting that up-regulated genes were significantly enriched in genes with more interactions in T. aestivum  has a large genome (16 Gb), only about 1% of which consists of protein-coding sequences [The interactions among ronments . The hexequences . It is iequences , 24, a he genome 6 Gb, onlMore chromatin interactions were observed in the D subgenome compared with the A and B subgenomes Fig. b, which Arabidopsis thaliana [Binding of transcription factors in regulatory elements could recruit RNA polymerase and the basal transcriptional machinery to regulate gene expression , 43. We thaliana . Indeed,Although divergence of transcription and chromatin interactions between homoeologous genes was observed in hexaploid wheat Fig. b, we didIn previous studies , 24, thePolyploidization induces rapid morphologic changes in wheat . ChangesIn summary, we investigate chromatin interaction changes between hexaploid wheat and its tetraploid and diploid relatives using OCEAN-C with low cost and high resolution. Our results show that the genomic structural variations contribute to chromatin interaction divergence among homoeologous genes. The chromatin topology changes mediate expression alteration of genes during polyploidization and evolution of wheat.Triticum aestivum) cultivar “Chinese Spring”, natural allotetraploid wheat , and Aegilops tauschii subsp. strangulata  were used in this study. The seeds were germinated in water for 3 days at 22 °C, then transferred to soil and grown under 18 °C/16 °C in day/night. Leaves in five-leaf stage were harvested and used for the construction of Hi-C, OCEAN-C, ChIP-seq, ATAC-seq, and RNA-seq libraries.The bread wheat  and 0.1% 2-mercaptoethanol). After mixing thoroughly, the slurry was kept on ice for 30 min and filtered by a 70-μm strainer. After centrifugation (3000×g for 5 min), the nuclei pellet was resuspended in 200 μl of 0.5% sodium dodecyl sulfate (SDS). Then the suspension was split into four tubes and incubated at 65 °C for 10 min. After adding 145 μl water and 25 μl 10% TritonX-100, each tube was incubated at 37 °C for 15 min to quench the SDS. The chromatin was digested by adding 25 μl 10× NEBuffer 3 and 50 U DpnII  at 37 °C overnight. On the following day, after incubating at 62 °C for 20 min to inactivate DpnII, the restriction fragments were filled by adding tagging buffer  and 25 μl ddH2O) and incubated at 22 °C for 4 h. Next, filled fragments were proximally ligated by adding ligation buffer ) and kept at 22 °C for 4 h. The mixture was centrifuged at 1000g for 3 min. The pellet was resuspended with 500 μl nuclei lysis buffer  and was kept on ice for 30 min with occasional stirring. The mixture was sonicated to an average DNA fragment size of 300–400 bp (Instrument: Covaris LE220 (Covaris), Duty Cycle: 20, PIP: 50, Cycles/Burst: 200, Time: 100 s). Add 500 μl phenol-chloroform-isoamylalcohol  and vortex for 30 s. After centrifugation at 13,000 g for 5 min at room temperature, the supernatants were extracted and purified by phenol-chloroform-isoamylalcohol  again. The aqueous layer was transferred to a new 1.5-ml tube. RNase A (5 μl) was added following 30 min of incubation at 37 °C. Proteinase K (10 μl) was added and the tubes were kept at 55 °C for 1 h and then at 65 °C for 2 h. DNA was purified using QIAquick PCR Purification Kit  and then pulled down by Dynabeads MyOne Streptavidin T1 beads . Libraries were constructed using NEBNext Ultra II DNA Library Prep Kit  and sequenced on NovaSeq platform (Illumina) for 150 bp paired-end reads.Hi-C libraries were constructed according to a published protocol . About 0The library construction of ChIP-seq with two biological replicates was performed as previous described . Chromat2, 0.1% Triton X-100, 2 mM Spermine, 1 mM PMSF, 1× Cocktail, 10 mM 2-Me). The mixture was filtered by 40 μm strainer and washed by 1 ml nuclei extraction buffer 2 , 10 mM 2-Me). Following resuspension by 300 μl nuclei extraction buffer 3 , the nuclei suspension was loaded on the surface of 600 μl nuclei extraction buffer 3 and centrifuged at 2400g at 4 °C for 20 min. The purified nuclei were resuspended by 1 ml nuclei extraction buffer 1. The small fraction of nuclei were stained with 4,6-diamidino-2-phenylindole and loaded into a hemocytometer to calculate nuclei density. About 50,000 nuclei were incubated with Tn5 transposase  at 37 °C for 30 min. DNA was recovered by MinElute PCR Purification Kit  and amplified for 11-13 cycles. In addition, genomic DNA was used for library construction as input control. Libraries were sequenced on NovaSeq platform (Illumina) for 150 bp paired-end reads.The ATAC-seq libraries were constructed according to the previous study with some modifications . About 0Triticum aestivum were mapped to the genome sequence of Triticum aestivum cv. Chinese Spring (IWGSC RefSeq v1.0) using bowtie2 (version 2.2.9) with default setting. The concordantly mapped reads (MAPQ> 10) were kept and PCR duplication was further removed with Picard. Correlation analysis between the two biological replicates of each mark was performed using deepTools2 (3.1.0.). Peaks of ChIP-seq libraries were detected using MACS2 (2.1.2) with the parameter “-f BAM --nomodel --bw 300 --SPMR -q 0.05”. The peaks of ATAC-seq were identified using MACS2 with the parameter “-q 0.01 -f BAM --nomodel --extsize 200 --shift 100”. The genomic DNA libraries were used as controls. The number of reads in each window was normalized against the total number of reads . Peak-associated genes were defined as genes with a peak within or near the gene body (±3 kb).Sequencing reads of H3K4me3, H3K27me3, and H3K9ac of Chinese Spring were obtained from a previous study [Triticum aestivum cv. Chinese Spring (IWGSC RefSeq v1.0) using HISAT2 with default parameters. Only uniquely mapped reads were kept. RNA-seq reads were normalized to FPKM. The reads from two compared groups were normalized by their respective size factors, which were analyzed by DESeq package with the parameter “estimateSizeFactors.” Fold change (> 2) and P value < 0.05 were used to identify differentially expressed genes (DEGs).RNA-seq reads were mapped to genome sequence of Triticum aestivum, T. durum, and Ae. tauschii were mapped to the reference genome (Chinese Spring (IWGSC RefSeq v1.0) for T. aestivum, AB subgenomes of Chinese Spring (IWGSC RefSeq v1.0) for T. durum, D subgenome of Chinese Spring (IWGSC RefSeq v1.0) for Ae. tauschii ) using HiC-Pro (version 2.11.1) [T. aestivum, T. durum, and Ae. tauschii were used as controls. The peaks of T. aestivum were used as an anchor to identify open chromatin interactions by hichipper [OCEAN-C reads of  2.11.1)  with theichipper . To idenHigh-confidence gene models from the IWGSC (version 1.0) were used for defining triad genes. Homoeologous genes between each pair of A, B, and D subgenomes were identified as previously described . The homA previously described ternary plot-based method was applied for defining bias of histone modifications, ATAC-seq, gene expression, and interaction patterns in triads . EuclideE value less than 0.01 were defined as enriched motifs.To detect enriched transcription factor-binding motifs in the proximal and distal regions, we first detected the proximal (3 kb upstream of the nearest TSS) and distal regions that interacted with gene loci. The regions interacting with more than 5 loci were considered as HOCIs. The motifs were then scanned against the proximal and distal regions using MEME (version 5.3.2)  softwareThe igraph library in R software was used to construct chromatin network components (ChINs)  and calculate the degree of each node (parameter: degree). The nodes and edges of each ChIN present the peaks and chromatin interactions. The Cytoscape  softwareT. durum [Ae. tauschii [The genomes of bread wheat , T. duruT. durum  and Ae. tauschii , were cotauschii . Then thT. aestivum supported by resequencing data of T. durum and Ae. tauschii.Presence/absence variations (PAVs) were identified using the published methods . We extrT. aestivum, T. durum, and Ae. tauschii were merged as a master peak list. The master peak list was used to call open chromatin loops using OCEAN-C reads by hichipper (--min-dist 10000) for each species [OCEAN-C peaks obtained from  species . The int species .T. aestivum, T. durum, and Ae. tauschii. The differentially interacted genes in T. aestivum compared with T. durum and Ae. tauschii were identified using edgeR with FDR < 0.05 and fold-change > 1.5 [The chromatin interaction intensity of each gene was normalized as counts per million mapped reads (CPM) for each biological replicate of ge > 1.5 .2O for 10 min, the slides were mounted in the Vectashield antifade solution . The distance of signals was checked from 50 nuclei using a laser scanning confocal microscope Zeiss LSM780 with OLYMPUS cellSens Standard software.3D DNA fluorescence in situ hybridization (3D-FISH) was performed according to previous study  with somAdditional file 1: Supplementary tables S1-S3 and S7.Additional file 2: Supplementary Figure S1-S6.Additional file 3: Table S4 The intrachromosomal open chromatin loops.Additional file 4: Table S5 The triads with chromatin interaction bias.Additional file 5: Table S6 The interchromosomal open chromatin loops.Additional file 6: Table S8 The differentially interacted intrachromosomal loops between bread wheat and T. durum.Additional file 7: Table S9 The differentially interacted intrachromosomal loops between bread wheat and Ae. tauschii.Additional file 8. Review history.


Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal the order of chromatin states in establishing developmental gene poising in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with ‘Placeholder’ nucleosomes , and DNA hypomethylation. Silencing is initiated by the recruitment of polycomb repressive complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 – establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos. Early vertebrate embryos initiate embryonic/zygotic transcription, termed zygotic genome activation (ZGA), and must distinguish active housekeeping genes from developmental genes, which must be temporarily silenced, but kept available for future activation . DevelopWe addressed these issues further in zebrafish, which conduct full ZGA at the tenth synchronous cell cycle of cleavage stage (~3.5 hr post fertilization (hpf), ~ 2000 cells) . Prior tFirst, we sought a repressive histone modification that might explain how developmental genes are silenced at ZGA. We examined zebrafish embryos at preZGA , ZGA (3.5 hpf ~2000 cells), and postZGA  – and confirmed very low-absent H3K27me3  and H3K9To localize H2Aub1 we conducted chromatin immunoprecipitation (ChIP) experiments at preZGA, ZGA, and postZGA (replicate structures for hoxaa) or narrow solo/dispersed developmental TFs  (prdx2) or narrow  H3K4me3 and H3K27ac occupancy at postZGA , bear marking similar to housekeeping genes, and the robustly transcribed mir430 locus appears markedly enriched in H3K27ac at preZGA  to H2A and H2A.Z . Rnf2 Chg. vsx2) . In coun postZGA . Notablyt preZGA . Finallyt preZGA .We find robust H3K27me3 deposition occurring during postZGA at promoters marked during preZGA with high Rnf2 and H2Aub1, specifically at clusters 1 and 2 . FurtherTo identify candidate TFs that might bind selectively at the promoters of particular clusters, we analyzed the DNA sequences flanking the transcription start site (TSS) (500 bp) at each cluster using the motif finding program, HOMER . LargelyAnalysis of enhancer regions revealed features that were similar to those at developmental promoters . SpecifiCuriously, enhancer cluster 5  was uniqDrosophila and in mammalian embryonic stem (ES) cell cultures, with the Aebp2 and Jarid2 protein components of ncPRC2 recognizing H2Aub1 and both targeting and facilitating H3K27me3 addition  mode of recruitment, involving ncPRC1 action (H2Aub1 addition) followed by the recruitment of PRC2 (ncPRC2) – via H2A/Zub1 recognition – to deposit H3K27me3. This mode and order of recruitment has precedent in addition . We thenaddition , withoutt levels . Aebp2 Ct levels  and at et levels . Taken tTo investigate the possible additional contribution of Jarid2 to ‘reading/binding’ of H2Aub1 (leading to H3K27me3 deposition), we performed ChIP-seq of Jarid2 during postZGA . Here, pDrosophila nuclei bearing H3K27me3-marked regions in all three replicates per condition. Finally, new ectopic H3K27me3 peaks were very rare in the PRT4165 treatment, and none of these loci were bound by Aebp2  where the effect was moderate, as well as non-clustered/solo formats where the effect of PRT4165 was more pronounced  on 4 hpf embryos that were either vehicle-treated (DMSO) or PRT4165-treated from the one-cell stage onward . Here, wonounced .Having observed precocious developmental gene upregulation in response to PRT4165 treatment during postZGA (4 hpf), we were curious whether their upregulation could also be observed during the preZGA and ZGA stages. To test, we repeated our PRT4165 treatment regimen and isolated embryos at preZGA (2.5 hpf) and ZGA (3.5 hpf) stages, and performed RNAseq , coincident with the loss of rnf2 RNA. Notably, loss of rnf2 at 3 dpf was associated with partial upregulation of certain developmental genes, but H3K27me3 remained fully present, showing that Rnf2 is not required for H3K27me3 maintenance  progresses relatively normally, except for the upregulation of a cohort of developmental genes which were formerly H2Aub1 marked. This provides a measure of confidence that the upregulation of developmental genes is largely a direct consequence of H2Aub1 loss in the early embryo. However, as PRT4165 was the only approach we employed that successfully removed the vast majority of H2Aub1, future studies involving orthogonal approaches to elicit Rnf2 loss in zebrafish will be needed to further validate the consequences of H2Aub1 loss in early zebrafish embryos.Pcgf1/6) or through overexpression of an H2A de-ubiquitinase (PR-DUB). Here, both approaches led to precocious transcription of a moderate subset of developmental genes at or shortly after ZGA. Thus, these sets of results align well with our observations in zebrafish.While our work was in review, work on H2Aub1 and H3K27me3 dynamics in the preimplantation mouse embryo was reported by two separate groups . Both stPcgf1/6, which encode subunits of noncanonical PRC1. Here, Pcgf1/6 mutant oocytes were fertilized with wild-type sperm to produce maternal-null Pcgf1/6 embryos. In contrast to PR-DUB overexpression, maternal-null Pcgf1/6 embryos displayed global reductions in both H2Aub1 and H3K27me3 during cleavage stages – a finding that aligns well with our findings. Notably, the reductions of H2Aub1 and H3K27me3 from maternal Pcgf1/6 loss largely returned to wild-type levels by the morula stage, possibly due to zygotically  expressed Pcgf1/6. Here, we postulate that the more severe loss of H3K27me3 from Pcgf1/6 deletion may stem from the temporal difference of disrupting H2Aub1 in the female germline vs. the early mouse embryo, or potentially the developmental stage in which H3K27me3 was assayed ( two cell vs. four cell). A major issue not addressed in these recent papers in mouse is how mechanistically H2Aub1 directs H3K27me3. Notably, our study addresses this mechanism by demonstrating that non-canonical PRC2 complex containing the H2Aub1 ‘reader’ Aebp2 occupies all loci with high H2Aub1 during postZGA, which then receive H3K27me3.Notably, each method of H2Aub1 perturbation in mice was unique (with attendant advantages and caveats) and conferred different impacts on embryogenesis and establishment of H3K27me3 at developmental loci. In mice, zygotic overexpression of PR-DUB resulted in rapid and significant erasure of H2Aub1, precocious transcription of certain developmental genes, and growth arrest at the four-cell stage. However, reduction of H2Aub1 by zygotic PR-DUB overexpression had minimal impact on H3K27me3, likely due to the fact that H3K27me3 is very low at the four-cell stage. In contrast, Mei et al. disrupted H2Aub1 via maternal genetic ablation  of two genes, Interestingly, Chen et al. and Mei et al. utilized hybrid mouse strains to trace parental asymmetries of H3K27me3 and H2Aub1 in the early embryo. Here, both groups uncovered rapid erasure of H2Aub1 from the paternal genome shortly after fertilization which is re-established by the two-cell stage. Our data also demonstrate that the paternal genome is initially devoid of H2Aub1, and rapidly acquires H2Aub1 shortly after fertilization. However, zebrafish sperm are entirely devoid of H2Aub1 , whereasHere, we observe that developmental or housekeeping gene promoters attract either a high- or low-level PRC1 binding, respectively. How ncPRC1 is recruited to CpG islands in ES cells is partially understood, as the ncPRC1 subunit Kdm2b helps recruit ncPRC1 to CpG islands of developmental genes. In this context, Kdm2b binds to hypomethylated CpGs via the CxxC motif . HoweverOne curiosity arising from our work is why zebrafish utilize the non-canonical PRC1 complex, which adds monoubiquitination to H2A/H2A.Z(FV), rather than canonical PRC1 complex, which compacts chromatin for initial developmental gene silencing. Here, we speculate that the rapid (~16 min) cell cycles that characterize the preZGA cleavage state – coupled to the need for continual DNA replication during cleavage stage – are not compatible with the compaction conferred by canonical PRC1 . FurtherWild-type Tübingen zebrafish were maintained as described . All expg for 5 min at 4°C. Pelleted nuclei were washed twice with cold Mild Cell Lysis Buffer. Nuclei were resuspended to a final volume of 800 μl in cold Mild Cell Lysis Buffer and supplemented with 10 μl of sulfuric acid (18.4 M). Samples were sonicated for 10 s  at 30% output using a Branson sonicator. Proteins were extracted for 30 min at 4°C on a rotator, and 160 μl of 100% trichloroacetic acid was added and proteins were allowed to precipitate for 30 min on ice. Samples were centrifuged at 13,000 rpm for 5 min at 4°C. Protein pellets were washed with 800 μl of cold acidified acetone, and centrifuged again at 13,000 rpm for 5 min at 4°C. Protein pellets were washed with 800 μl of cold acetone, and centrifuged again at 13,000 rpm for 5 min at 4°C. Supernatant was discarded and pellets were dried at 37°C for 5 min. Dried protein pellets were resuspended in 2× Laemmli sample buffer and boiled for 8 min. Samples were then used for western blotting. Bands were quantified in ImageJ  was applied to embryos and incubated on ice for 5 min. Embryos were homogenized by passing through a 20-gauge syringe several times. Samples were briefly centrifuged to bring down chorions. Supernatants were transferred to fresh tubes and centrifuged at 1300× n ImageJ .Standard protocol for immunohistochemistry was followed as described . Three bImages were acquired on a Leica SP8 White Light laser confocal microscope. Image processing was completed using Nikon NIS-Elements multi-platform acquisition software with a 40×/1.10 Water objective. Fiji  was utilized to color DAPI channel to cyan, GFP color remained green. Confocal images are max projections of Z stacks taken 0.5 μm apart for a total of the embryo ~7–12 μm.AB_10891618), anti-Rnf2 , anti-H2A.Z , anti-H3K4me1 , anti-Aebp2 , anti-H3K27me3 , anti-H3 , and anti-Jarid2 .The following antibodies were utilized in for the present study: anti-H2Aub1 . Chorions were removed enzymatically by treatment with pronase (1.25 mg/ml in PBS). Dechorionated embryos were gently washed twice with PBS to remove pronase. Samples were fixed with 1% formaldehyde  for 10 min at room temperature with end over end rotation. Fixation was quenched with 130 mM glycine for 5 min at room temperature. Samples were centrifuged for 5 min at 500× g for 5 min at 4°C. Supernatant was discarded and nuclei pellets were resuspended in 1 ml Nuclei Wash Buffer  and rotated at room temperature for 10 min. Samples were centrifuged at 1300× g for 5 min at 4°C to pellet nuclei. Supernatant was discarded and nuclei pellets were resuspended in 100 µl of Nuclei Lysis Buffer . Samples were incubated on ice for 10 min; 900 µl of IP Dilution Buffer  was added to samples.One ml of Mild Cell Lysis Buffer  was applied to cell pellets from 1000 embryos and rotated at 4°C for 10 min. Samples were centrifuged at 1300× Nuclear lysates were sonicated with a Branson Digital Sonifier with the following settings: 10 s duration , 30% amplitude. Seven sonication cycles were performed. Samples were placed in an ice bath for at least 1 min between each sonication cycle. Sonicated samples were centrifuged at 14,000 rpm, 4°C, for 10 min to pellet insoluble material. Supernatants were transferred to new tubes. A portion of the sample was set aside to confirm optimal chromatin shearing by agarose gel electrophoresis.Twenty µl of Dynabeads (Invitrogen) were blocked with 0.5 mg/ml BSA in PBS. Blocked Dynabeads were subsequently applied to each sonicated sample and rotated for 1 hr at 4°C. Samples were placed on a magnet stand for 1 min and precleared supernatant was transferred to a new tube; 5% of the sample was removed and stored at –80°C as input. Antibody and fresh 1× protease inhibitors were added to each sample. Samples were rotated overnight at 4°C.Samples were centrifuged at 14,000 rpm, 4°C, for 5 min to pellet insoluble material. Supernatants were transferred to new tubes; 50 µl of Dynabeads (Invitrogen) were blocked with BSA 5 mg/ml in PBS. Blocked Dynabeads were subsequently applied to each sample and rotated for 6 hr at 4°C.All wash buffers were kept ice cold during stringency washes. Samples were washed eight times with RIPA Buffer , two times with LiCl Buffer , two times with TE Buffer .One-hundred µl of Elution Buffer  was added to beads. Two µl of RNase A  was added to each ChIP and input sample and incubated at 37°C for 30 min with gentle agitation. Ten µl of Proteinase K  was added to each sample and incubated at 37°C for 1 hr with gentle agitation. Crosslinks were reversed overnight at 65°C with gentle agitation. ChIP DNA was purified with a Qiagen MinElute PCR Purification kit (Cat#28004).ChIP-seq libraries were prepared using NEBNext ChIP-Seq Library Prep Reagent Set for Illumina . High-throughput sequencing was performed on Illumina HiSeq 2500 for single-end 50 bp reads or Illumina NovaSeq 6000 for paired-end 50 bp reads.Drosophila melanogaster S2 cells (ATCC Cat# CRL-1963) were spiked into resuspended zebrafish embryo pellets at a ratio of 5:1 (zebrafish cells: S2 cells). ChIP-Rx was subsequently performed in the same way as described above.ChIP-Rx was adapted from ChIP in zebrafish sperm was conducted as described .qPCR was carried out using 2× SsoAdvanced Universal SYBR Green Supermix (Biorad Cat# 1725270) and a Biorad CFX real-time thermal cycler.See free DNA removal kit (Cat# AM1906) was subsequently used to remove contaminating DNA from RNA samples. Intact poly(A) RNA was purified from total RNA samples (100–500 ng) with oligo(dT) magnetic beads and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded mRNA Library Preparation Kit . Purified library quality was assessed on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (Cat# 5067–5582 and 5067–5583). The molarity of adapter-modified molecules was defined by qPCR using the Kapa Biosystems Kapa Library Quant Kit (Cat# KK4824). Individual libraries were normalized to 5 nM and equal volumes were pooled in preparation for Illumina sequence analysis. High-throughput sequencing for RNAseq was performed on an Illumina HiSeq 2500. RNA-seq data displayed in http://www.ebiac.uk/gxa/experiments/E-ERAD-475. We would like to thank the Busch-Nentwich lab for providing RNA-seq data used in Total RNA was harvested from zebrafish embryos with a Qiagen Allprep kit (Cat# 80204). The Invitrogen DNA-Multi-Replica Macs ChIPSeq Wrapper. Called peaks were annotated in R with the ChIPseeker package  data for promoter regions (±1 Kb from TSS) of interest was collected from processed bigwig files by utilizing the program Bio-ToolBox ‘analysis  were perHOMER was utilized for identifying putative TF-binding motifs present at promoters and enhancers . The folMulti-Replica Macs ChIPSeq Wrapper.Analysis of ChIP-seq experiments involving drug treatments was performed by utilizing the AGATCGGAAGAGCACACGTCTGAACTCCAGTCA. The resulting sorted BAM files were subsequently indexed using Samtools , postZGA H3K4me1 ChIP-seq data (PreZGA H2Az and preZGA H3K4me1 ChIP-seq data  (GEO: GSseq data  (GEO: GSseq data  (GEO: GSseq data  (GEO: GSseq data  (GEO: GSWhole genome bisulfite sequencing (WGBS) from All sequencing datasets generated in this study have been deposited at the Gene Expression Omnibus under the accession number GSE168362.PRT4165 (Tocris Cat#5047) was dissolved in DMSO at a concentration of 50 mM. PRT4165 was further diluted to a working concentration of 150 µM in embryo water and mixed vigorously. Zebrafish embryos were collected and immediately placed in embryo water containing 150 µM PRT4165 (or DMSO) and allowed to develop to the desired developmental stage. This manuscript uses genomics tools and pharmacological treatment to study the chromatin landscape change in early-stage Zebrafish embryos, at the critical stage from using maternally deposited transcripts to actively turning on embryotic gene expression. In particular, this work addresses how key chromatin factors coordinate in regulating distinct groups of genes during early vertebrate development and should be of interest to researchers in chromatin biology and developmental biology fields. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard White as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Charles K Kaufman (Reviewer #3).Thank you for submitting your article "Establishment of Developmental Gene Silencing by Ordered Polycomb Complex Recruitment in Early Zebrafish Embryos" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. This study first reported chromatin landscape changes in early zebrafish embryos at three distinct stages at preZGA, ZGA and postZGA stages, using a series of ChIP-seq experiments. This part of the results is of high quality, rigorously analyzed and clearly presented. The study then moved on and used a single drug to inhibit the enzyme with the hope to reduce ubH2A/Z mark. However, using the drug could lead to pleiotropic effect and cause general or secondary phenotypes. For example, this treatment leads to developmental arrest of embryogenesis at very early stage with low to no H3K27me3 mark, according to other data included in this work. Therefore, based on current results, it is hard to draw the conclusion for the causal connection from inhibiting RNF2 enzymatic function to the lack of ubH2A/Z, and subsequently to the lack of H3K27me3. In addition, due to the close interactions between these two Polycomb complexes (PRC1 and PRC2), the actual order could be reversed. Without a genetic experiment or more specific disruption assay, the conclusions should be carefully revised to be more balanced and reflect the caveats with the current results.2. This sequential function of PRC1 and PRC2 has been reported in several other systems, including X inactivation and early embryogenesis in mice. The conceptual advance of this study could be extending this finding to another vertebrate system. However, due to the complications to draw conclusions (see #1 point), the Abstract and Discussion should be revised. Comparisons among these systems and research strategies should be further discussed.3. H3K27me3 pattern should also be analyzed before ZGA. The authors detected a low level of H3K27me3 before ZGA using immunostaining. However, previous ChIP-seq analysis demonstrated that H3K27me3 are present on promoters before ZGA in zebrafish (e.g. PMID: 22137762).4. It is unclear whether Aebp2 is the sole subunit for the recruitment of PRC2 to ubH2A/Z-marked regions. Paralleled analysis of the changes for Aebp2 and H3K27me3 upon RNF2 inhibitor treatment should be performed. And Aebp2-dependent and -independent regions should be separately classified for data analysis.5. The role of PRC1 on the temporal regulation of gene expression needs to be better elucidated. The authors only analyzed the RNA-seq results for RNF2 inhibitor treated embryos postZGA. Therefore, it is currently not clear if the role of PRC1 in transcriptional repression is restricted to this stage. RNA-seq analysis of RNF2 inhibitor treated embryos on other stages are also warranted.Reviewer #2 (Recommendations for the authors):Some specific points:Page 8, line 159-169. I'm confused by the authors' description of the enhancer cluster 5. By visual inspection, this cluster apparently only shared very moderate H3K4me1 and H3K27ac levels. The authors should provide a quantitative analysis similar to Figure 1S2J.Figure 1 and 2. In Figure 1A and 2A. WGBS data of the preZGA stage embryo was shown. Yet in Figure 1B and 2B, the WGBS data was designated to postZGA embryo. Please confirm that the labeling is correct.Page 10, line 209. Only a representative genomic region is shown in Figure 4S2B. Global analysis of the distribution changes for H3K27me3 needs to be performed in parallel with Aebp2 ChIP-seq analysis in Figure 4C.Figure 3 Only colocalization was observed, yet functional links are not established in this study. Therefore, it is justified to claim that Aebp2 mediates H3K27me3 deposition at the current stage. Overall, the contribution of Jarid2 was not examined in the current stage.Reviewer #3 (Recommendations for the authors):To push the approach further, and not required for the current study, I wonder if another perturbation to test the model could be to delete key example genomic loci  to test for loss of proper Placeholder histone deposition. Or, engineer transgenic fish with developmental enhancer/promoter sequences in an ectopic location (or profile an existing transgenic around the time of ZGA) to show that they can drive establishment of the set of Placeholder chromatin/DNA marks that, in their model, delineate developmental gene loci that are silenced at the ZGA but will later be expressed. Essential revisions:1. This study first reported chromatin landscape changes in early zebrafish embryos at three distinct stages at preZGA, ZGA and postZGA stages, using a series of ChIP-seq experiments. This part of the results is of high quality, rigorously analyzed and clearly presented. The study then moved on and used a single drug to inhibit the enzyme with the hope to reduce ubH2A/Z mark. However, using the drug could lead to pleiotropic effect and cause general or secondary phenotypes. For example, this treatment leads to developmental arrest of embryogenesis at very early stage with low to no H3K27me3 mark, according to other data included in this work. Therefore, based on current results, it is hard to draw the conclusion for the causal connection from inhibiting RNF2 enzymatic function to the lack of ubH2A/Z, and subsequently to the lack of H3K27me3. In addition, due to the close interactions between these two Polycomb complexes (PRC1 and PRC2), the actual order could be reversed. Without a genetic experiment or more specific disruption assay, the conclusions should be carefully revised to be more balanced and reflect the caveats with the current results.We thank the reviewers for their overall positive view of the work and examination of the chromatin landscape changes via ChIP approaches. The issues raised here include: 1) whether the inhibition of Rnf2 is appropriate functional evidence, and 2) whether we are confident in the order of H2Aub1 (PRC1 action) preceding H3K27me3 (PRC2 action).Nature Genetics , each used one approach to lower H2Aub1. We agree that our approach could have off target effects, and now state that in the Discussion. We note, however, that the other two mouse papers likewise each took one approach to lower H2Aub1: one employed BAP overexpression, which arrested embryos at the 4-cell stage (when H3K27me3 is extremely low) and therefore could not assess whether H3K27me3 was impaired when H2Aub1 was lowered. The second employed maternal, but not paternal, genetic disruption of PRC1, which showed a transient lowering of H2Aub1 and H3K27me3, but restoration by the morula stage. Therefore, each approach has its advantages and limitations, which we now discuss in the revised Discussion. However, together, all three papers provide complementary evidence for the causal relationship.First, regarding our inhibition experiments – our work, like the other two papers published in We respectfully disagree that current evidence supports the notion that “the actual order (of PRC1/2 complexes) could be reversed”. Our evidence of PRC1/H2Aub1 preceding PRC2/H3K27me3 does not rely solely on the PRT4165 inhibitor experiment – it is fundamentally supported by the temporal relationships of the modifications. In preZGA embryos we clearly detect H2Aub1 by western and IF approaches, and importantly detect robust ChIP specifically at thousands of developmental genes. In contrast, we and almost all other investigators detect no H3K27me3 by Western or IF, nor can we and almost all others detect it at any genes. As detailed in point #3 below – the one paper that has claimed preZGA H3K27me3  showed low H3K27me3 at only 83 genes during preZGA through ZGA, not at the thousands of developmental genes that bear H2Aub1 during preZGA through ZGA. Thus, the vast majority of the evidence points strongly to H3K27me3 being virtually absent during preZGA, whereas we show the clear presence of H2Aub1, and its residence at large numbers of developmental genes during preZGA.ezh2 null mutations. Thus, H3K27me3 appears to play only minor roles until late stages of development, where it acts in a canonical manner.We will note here (and briefly in the Discussion) that the lack of H3K27me3 has no measurable effect on the progression of zebrafish embryos through gastrulation, and through 24hpf, at which they display a normal body plan and organogenesis (though fail by 48hrs). This was determined in work published by L. Kamminga and colleagues  using maternal zygotic bap1 and asxl2, to disrupt H2A/Zub1. As a negative control we utilized a point mutant of bap1 that abolishes catalytic activity. Injected embryos were allowed to develop to 4hpf and subsequently subjected to western blot analysis to assay H2Aub1 abundance. Unfortunately, this approach yielded no appreciable change in bulk levels of H2Aub1 compared to control, despite attempting multiple different doses of the bap1 and asxl2 mRNAs. Importantly, we validated  by Western analysis that the proteins were each expressed. Here, we reason that additional factors (beyond the two we expressed) might be needed to create a fully active (or properly targeted) BAP/ASXL2 complex and/or that inhibitors of BAP1 might be present that prevented enzymatic activity. Regardless, the inability to greatly lower H2A/Zub1 precluded further analyses.We agree with the reviewers that a complementary approach would help to validate our results with the inhibitor, and we expended significant effort on this issue during revision. Regarding alternative inhibitors, PRT4165 is currently the only commercially available inhibitor of Rnf2. In the absence of alternative inhibitors, we attempted genetic approaches, beginning with the expression of a minimal version of the H2A deubiquitinase complex, PR-DUB in early zebrafish embryos. Here, single-cell stage zebrafish embryos were microinjected with mRNA encoding the two essential subunits of PR-DUB, h2af/z mRNA encoding and form of H2A.Z that cannot be ubiquitinated. We also injected wild-type h2af/z as a negative control. Unfortunately, our Western analyses showed that we were not able to arrive at protein levels derived from the injected species that approached the large stores of WT H2A.Z protein present in the embryo, so competition was not achieved, and H2A/Zub1 was not lowered.As a tertiary approach to lower H2A/Zub1, we micro-injected single-cell stage zebrafish embryos with a mutant Taken together, our work parallels the work earlier this year from two groups who each took one effective approach to lower H2AUb1: ours with PRT4165 and theirs with BAP1/ASXL1 overexpression or a PRC1 maternal-specific mutant approach. Each approach has advantages and caveats, and the reviewer rightly points out that our approach can have off target affects. Therefore, we have included this caveat and how it may affect our interpretations accordingly in the revised paper . Specifically, in the Discussion of the revised manuscript we state:“However, it is important to note that PRT4165 may have off-target effects aside from inhibition of Rnf2 which may contribute to our observations.” and“However, as PRT4165 was the only approach we employed that successfully removed the vast majority of H2Aub1, future studies involving orthogonal approaches to elicit Rnf2 loss in zebrafish will be needed to further validate the consequences of H2Aub1 loss in early zebrafish embryos” (page 16 and line #342-345).2. This sequential function of PRC1 and PRC2 has been reported in several other systems, including X inactivation and early embryogenesis in mice. The conceptual advance of this study could be extending this finding to another vertebrate system. However, due to the complications to draw conclusions (see #1 point), the Abstract and Discussion should be revised. Comparisons among these systems and research strategies should be further discussed.Nature Genetics that determines the order for the X chromosome and developmental genes in mouse embryos is now discussed in the revised version . However, at the time when we submitted our work, the order of polycomb complex action at developmental genes was not known. Our manuscript was submitted and progressed to full review at eLife (and was posted on the BioRxiv) prior to the work in mice being published in Nature Genetics. Therefore, we hope that the novelty and conceptual advance of the work will be assessed based on our date of submission.There is indeed precedent for that order in X chromosome inactivation in ES cells, and we have cited that work in the revision. In addition, the work published earlier this year in Nature Genetics) on H2AUb inhibition in mice strongly supports our work in zebrafish, and we have now included those papers in the Discussion of our revised paper . Indeed, their methods for reducing H2AUb1 in mouse embryos led to upregulation of developmental genes, in keeping with our observations. Regarding novelty, our work goes much further, as only our paper addresses the mechanism by which H2AUb1 is ‘read’ in the early embryo by a non-canonical PRC2 complex (bearing Aebp2) to place H3K27me3 on developmental genes. Therefore, we believe our work has conceptual advances both in the areas of overlap, and also in unique aspects related to PRC2 targeting.The recent mouse work .Genome Research 2014; Zhang et al., Molecular Cell, 2018), agree that there are >3500 developmental genes marked by H3K27me3 in postZGA. However, the Lindeman data claims that only 201 genes are marked by H3K27me3 in preZGA embryos . Also, one expects that if the purpose of that H3K27me3 involves gene poising, it should be stable and last into postZGA – and when one queries their data and asks how many genes bear H3K27me3 from preZGA through postZGA, the number drops to 83 genes. These 83 genes are extremely few compared to the >3,500 genes marked in postZGA embryos by H3K27me3 in the Lindeman data and Zhang data. Also, these 83 H3K27me3 genes show very low median ChIP signal during preZGA (less than 2-fold enrichment) and only show clear (>4-fold enrichment) during postZGA  is an interesting and influential paper in the field, however some of the interpretations have required revisiting. The Lindeman paper does claim that H3K27me3 can be found on developmental promotes during preZGA. However, the key questions are – how many promoters, what fraction of promoters, and how robust is the marking? The Lindeman paper and follow-up work by us and others , Wei Xie’s lab  and our lab  did not reveal any peaks for H3K27me3 during preZGA. Furthermore, both our IF data  and Zhang et al., 2018  fail to show H3K27me3 signal preZGA. Taken together, work from several labs supports the view that there is extremely low H3K27me3 in the genome at preZGA, which has resulted either in a lack of ChIP peaks preZGA  or detection of very low H3K27me3 at a very small number of loci . However, all groups agree that after ZGA, H3K27me3 increases dramatically, enabling clear detection at >3500 genes. Therefore, the much larger questions – addressed in our work – are how >3500 developmental genes are silenced at ZGA , and how those >3500 obtain high H3K27me3 .Importantly, we emphasize that H3K27me3 ChIP experiments from the Schier lab , indicates that Aebp2 is not the sole subunit involved in recruitment of PRC2 to H2A/Zub1. Rather, Aebp2 and Jarid2 subunits of ncPRC2 both contribute to recruitment of PRC2 via direct contacts with H2A/Zub1. However, we note that the PRC2 complex can be isolated from cells or produced in a recombinant form with Aebp2, but lacking Jarid2  – suggesting that these proteins are not together in all PRC2 complexes, and that Aebp2 assembles well into PRC2 without Jarid2. Accordingly, we have modified the text of our manuscript to clarify that Aebp2 is not the sole PRC2 subunit involved in recruitment to H2A/Zub1+ nucleosomes.We thank the reviewers for raising these important points, which we have addressed. Previous data including the very nice recent cryo-EM structure of the non-canonical PRC2 complex bound to H2Aub1+ nucleosomes from Eva Nogales’ group  also lose H3K27me3. We believe this revised way of presenting the data conveys the result much clearer than the previous version of Figure 4.5. The role of PRC1 on the temporal regulation of gene expression needs to be better elucidated. The authors only analyzed the RNA-seq results for RNF2 inhibitor treated embryos postZGA. Therefore, it is currently not clear if the role of PRC1 in transcriptional repression is restricted to this stage. RNA-seq analysis of RNF2 inhibitor treated embryos on other stages are also warranted.The reviewers raise an interesting question, which also addresses whether the removal of H2AUb from developmental genes might enable their activation during preZGA, which would confer to developmental genes the status/privilege of ‘minor wave’ genes. To address this question, we performed RNA-seq at preZGA and ZGA timepoints with embryos treated with the RNF2 inhibitor (PRT4165) or vehicle control (DMSO) and examined whether precocious transcription of developmental genes occurred prior to post ZGA. This work has been incorporated into our revised manuscript in Figure 4 —figure supplement 3. GO-term analysis of genes meeting our threshold criteria  uncovered no enrichment of developmental gene activation upon RNF2 inhibition prior to postZGA. Instead, enriched GO-terms of upregulated transcripts correspond to RNA-binding and ribosomal protein genes. Thus, chromatin de-repression  does not cause precocious transcriptional activation of developmental genes until postZGA (4hpf).Here, we reason that the observed precocious transcription of developmental genes at postZGA in embryos treated with PRT4165 relies on binding of opportunistic transcriptional activators. However, these activators may be temporally constrained during preZGA by: 1) translation of mRNA encoding these factor(s) and/or 2) chromatin remodeling events needed to expose binding sites for opportunistic activators.Reviewer #2 (Recommendations for the authors):Some specific points:Page 8, line 159-169. I'm confused by the authors' description of the enhancer cluster 5. By visual inspection, this cluster apparently only shared very moderate H3K4me1 and H3K27ac levels. The authors should provide a quantitative analysis similar to Figure 1S2J.We apologize for the confusion, which also raised a comment from Reviewer #1. There are two points of clarification to make here. First, we now clarify in the manuscript that DNAme patterns are essentially identical/static from 2.5hpf (preZGA) to 4.3hpf (postZGA). On Page 5, line 100 we now state:“For our comparisons to DNA methylation (DNAme), we note that DNAme patterns are reprogrammed between fertilization and the preZGA (2.5hpf) timepoint , but remain static in zebrafish embryos from 2.5 hpf (preZGA) to 4.3 hpf (postZGA). Therefore, for brevity we chose to display only a single timepoint for DNAme data in subsequent figures, which is representative of all developmental stages examined by the genomics approaches in this work”.Second, our reference to H3K27ac being very high refers specifically to enhancer cluster 5 in the postZGA state. Thus, H3K27me3 is very high, and these loci are DNA methylated .Figure 1 and 2. In Figure 1Aand2A. WGBS data of the preZGA stage embryo was shown. Yet in Figure 1B and 2B, the WGBS data was designated to postZGA embryo. Please confirm that the labeling is correct.We thank the reviewer for their comment. The labeling is correct. As noted above, the DNA methylation state remains constant at enhancers and promoters from preZGA to postZGA.Page 10, line 209. Only a representative genomic region is shown in Figure 4S2B. Global analysis of the distribution changes for H3K27me3 needs to be performed in parallel with Aebp2 ChIP-seq analysis in Figure 4C.We agree and have addressed: Figure 4 has been largely revised to examine the changes in localization of Aebp2 and H3K27me3 in parallel, as also requested in the list of unified comments.Figure 3 Only colocalization was observed, yet functional links are not established in this study. Therefore, it is justified to claim that Aebp2 mediates H3K27me3 deposition at the current stage. Overall, the contribution of Jarid2 was not examined in the current stage.Here, our functional link is that when H2Aub1 is prevented (via PRT4165), Aebp2 fails to occupy all sites that formerly had H2Aub1, and H3K27me3 is not deposited at those same loci – which is now much more clearly displayed through the revised analyses present in Figure 4.The reviewer raises an interesting issue in their request to examine Jarid2. Here, although the Jarid2 antibody and resulting ChIP signal was not as robust as with our potent Aebp2 antibody (limiting highly quantitative conclusions) the majority of Jarid2 targets were also Aebp2 targets – consistent with Aebp2 and Jarid2 working together for H2Aub1 recognition .Reviewer #3 (Recommendations for the authors):To push the approach further, and not required for the current study, I wonder if another perturbation to test the model could be to delete key example genomic loci  to test for loss of proper Placeholder histone deposition. Or, engineer transgenic fish with developmental enhancer/promoter sequences in an ectopic location (or profile an existing transgenic around the time of ZGA) to show that they can drive establishment of the set of Placeholder chromatin/DNA marks that, in their model, delineate developmental gene loci that are silenced at the ZGA but will later be expressed.We appreciate the reviewer’s insightful comments. In the future we will use Crispr-CAS9 mediated targeting of transcription factors and histone modifications to test these interesting questions.


Drosophila but also regulates other genes promoting epithelial robustness. We base our hypothesis on both existing literature and our experimental evidence and suggest that such feedback between the cell surface and the nucleus presents a powerful paradigm for epithelial resilience.Epithelial tissues rely on the adhesion between participating cells to retain their integrity. The transmembrane protein E-cadherin is the major protein that mediates homophilic adhesion between neighbouring cells and is, therefore, one of the critical components for epithelial integrity. E-cadherin downregulation has been described extensively as a prerequisite for epithelial-to-mesenchymal transition and is a hallmark in many types of cancer. Due to this clinical importance, research has been mostly focused on understanding the mechanisms leading to transcriptional repression of this adhesion molecule. However, in recent years it has become apparent that re-expression of E-cadherin is a major step in the progression of many cancers during metastasis. Here, we review the currently known molecular mechanisms of E-cadherin transcriptional activation and inhibition and highlight complex interactions between individual mechanisms. We then propose an additional mechanism, whereby the competition between adhesion complexes and heterochromatin protein-1 for binding to STAT92E fine-tunes the levels of E-cadherin expression in  Cells in epithelia adhere to both a common substrate and neighbouring cells . The molThe strength of adhesion correlates with the number of E-cad molecules on cell surfaces . ConsequDrosophila and mammalian E-cad differ in their extracellular domains and are products of independent evolution from a common N-cadherin-like ancestor, transcription of the Drosophila E-cad (encoded by the shotgun gene) is also inhibited by the Snail and Twist TFs  including SNAIL, SLUG , ZEB1/2, and Twist1/2 , all of wist TFs . This co midguts , potenti midguts .Drosophila  to the E-cad promoter, increasing its expression and driving re-epithelization of breast cancer cells   .Drosophila E-cad are not known, another TF – Grhl2 (Grainyhead in Drosophila) – activates E-cad expression in both mammalian and invertebrate cells. In the former, intron-bound Grhl2 promotes E-cad transcription through chromatin looping, playing an essential role in determining an epithelial phenotype  and c-Myc also specifically activate the expression of E-cad promoter through an interaction with the TF AP-2, thus maintaining the epithelial phenotype in mammalian cells . Rb intehenotype . Similarturation .Drosophila, although Kaiso is not present, p120-catenin is nevertheless involved in transcriptional regulation  in triggering changes in gene expression . For exagulation . As categulation . Furthergulation . This raDrosophila) is an evolutionarily conserved protein recruited to E-cad adhesion in both mammals and Drosophila  protein family, STAT92E , necessary to produce the red pigment pteridine. Overexpression of E-cad increased the normalised eye pigmentation with pteridine to 104% from 69% in the control . This peak is consistent with two potential mechanisms for HP1 recruitment – via Su(var)3–9 or one of the two fly Rb-family proteins. Therefore, the modulation of HP1 function downstream of E-cad and non-canonical STAT signalling might alter the E-cad expression.Next, we sought to determine what genes might be regulated through our hypothetical mechanism and examined the HP1 localisation using publicly available ChIP-seq datasets generated using third instar larvae , which iY704F), which acts only in the non-canonical signalling  can be found below: NAB and MRM performed the experiments and wrote the manuscript. NAB and PAS did the bioinformatics analyses. All authors contributed to the article and approved the submitted version.PAS was a shareholder and the managing director of the company SmartImmune Ltd. The remaining 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.