Initiation of diverse epigenetic states during nuclear programming of the

Ann Boijaa and Mattias Mannervika,1

aDepartment of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691 Stockholm, Sweden

Edited by Ruth Lehmann, New York University Medical Center, New York, NY, and approved June 17, 2016 (received for review August 18, 2015) Epigenetic patterns of histone modifications contribute to the main- and B). Comparing these embryos with naïve cells, we found that tenance of tissue-specific expression. Here, we show that such the pioneer factor, Zelda, establishes poised RNA polymerase modifications also accompany the specification of cell identities by at Dorsal target before zygotic activation. Upon Dorsal- the NF-κB Dorsal in the precellular Drosophila mediated cell specification, three distinct histone signatures are embryo. We provide evidence that the maternal pioneer factor, produced, including two modes of transcriptional repression. Our Zelda, is responsible for establishing poised RNA polymerase at Dor- results show how transcription factors coordinate gene activity sal target genes before Dorsal-mediated zygotic activation. At the with initial establishment of distinct epigenetic patterns of histone onset of cell specification, Dorsal recruits the CBP/p300 coactivator modifications. to the regulatory regions of defined target genes in the presump- tive neuroectoderm, resulting in their histone acetylation and tran- Results and Discussion scriptional activation. These genes are inactive in the mesoderm due To investigate the mechanisms by which the Dorsal gradient to transcriptional quenching by the Snail repressor, which precludes generates tissue-specific and what chromatin recruitment of CBP and prevents histone acetylation. By contrast, states that follow, we examined the Dorsal-target genes brinker inactivation of the same enhancers in the dorsal ectoderm is asso- (brk)andshort-gastrulation (sog) whose expression is restricted to ciated with Polycomb-repressed H3K27me3 chromatin. Thus, the the presumptive neuroectoderm (Tollrm9/rm10)instage5embryos Dorsal morphogen gradient produces three distinct histone signa- (Fig. 1 B and C). Expression of brk and sog is repressed in the tures including two modes of transcriptional repression, active re- dorsal ectoderm (gd7) and mesoderm (Toll10B), and transcription pression (hypoacetylation), and inactivity (H3K27me3). Whereas of brk and sog has not yet initiated in naïve cells obtained from DEVELOPMENTAL histone hypoacetylation is associated with a poised polymerase, 1- to 1.5-h-old wild-type (WT) stage 3 embryos (Fig. 1 B and C and H3K27me3 displaces polymerase from chromatin. Our results link Fig. S1). We designed primers across the brk and sog loci, in- different modes of RNA polymerase regulation to separate epige- cluding key features such as the embryonic and shadow enhancers, netic patterns and demonstrate that developmental determinants intergenic regions, several primers in the promoter region, as well orchestrate differential chromatin states, providing new insights in- as primers in the gene body. Occupancy of factors and histone to the link between and developmental patterning. modifications were examined by ChIP followed by quantitative PCR (qPCR). We plotted occupancy in all graphs as fold enrich- epigenetics | cell specification | Drosophila embryo | Dorsal morphogen | ment over two intergenic loci devoid of known factors and Zelda histone modifications. We began by examining the occupancy of RNA polymerase II eneration of many specialized cell types from an identical DNA (pol II) using an antibody against the Rpb3 subunit and found Gsequence is a remarkable property of genomes in multicellular high levels at the promoters of brk and sog in the neuroectoderm organisms. Cell fate is specified by transcription factors through the (Fig. 1D), the tissue where these genes are expressed (Fig. 1 B initiation of differential gene expression patterns, but maintenance and C). Much lower levels of pol II are found over the gene of cell-type–specific gene expression programs often relies on epi- genetic mechanisms (reviewd in ref. 1). Epigenetic events such as Significance Polycomb-mediated repression are therefore important for main- tenance of specific cell identities and have been implicated in hu- Cellular memory is used to transmit information about gene man disease (reviewed in refs. 2 and 3), but how differences in activity to daughter cells and relies on epigenetic mechanisms epigenetic information between cell types arise is poorly understood. such as feedback loops and chemical modifications to DNA and The repressive nature of chromatin limits access of proteins to chromatin. Although epigenetic modifications have been sam- DNA. Pioneer factors facilitate chromatin opening, allowing ad- pled in many cell types, how epigenetic differences between ditional proteins to bind DNA (reviewed in ref. 4). In the Dro- cells arise is poorly understood. We show that a gene regulatory sophila embryo, zygotic genome activation occurs with the help of network controlling is responsible for Zelda (5), a transcription factor with many features of a pioneer orchestrating differences in epigenetic patterns between cell factor. Zelda associates with target genes before their activation (6, types. We further discovered that different modes of RNA po- – 7) and increases chromatin accessibility (8 11). Zelda facilitates lymerase regulation, including poising and displacement, are DNA binding of other transcription factors, including Dorsal, a linked to these distinct chromatin states. Our results provide κ Rel-family transcription factor related to mammalian NF- B(9). novel insights into how transcription regulation and epigenetics Formation of the three germ layers, mesoderm, neuroectoderm, are coordinated during cell specification and explain how gene and dorsal ectoderm, in Drosophila embryos involves establish- regulatory networks can shape the epigenetic landscape. ment of differential gene expression patterns by the Dorsal mor- phogen (reviewed in refs. 12 and 13) (Fig. 1A). Dorsal forms an Author contributions: A.B. and M.M. designed research; A.B. performed research; A.B. intranuclear concentration gradient in response to signaling by the and M.M. analyzed data; and A.B. and M.M. wrote the paper. transmembrane Toll (reviewed in ref. 14). Because many The authors declare no conflict of interest. components of the Toll pathway are strictly maternal, it is possible This article is a PNAS Direct Submission. to obtain mothers that generate a homogeneous population of 1To whom correspondence should be addressed. Email: [email protected]. 10B embryos that consist entirely of presumptive mesoderm (Toll ), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rm9/rm10 7 neuroectoderm (Toll ), or dorsal ectoderm (gd ) (Fig. 1 A 1073/pnas.1516450113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1516450113 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 OFF A dpp x gd7 dorsal ectoderm brk, sog

ON Dl Tollrm9/rm10 sog sog neurogenic brk, sog brk brk ectoderm OFF mesoderm Dl x Toll10B Dorsal nuclear gradient sna Sna brk, sog Dorsal gradient BC 4 brk

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brk 1 sog sna Relative expression 0 evïan dorsal neuroectoderm mredosem wild-type ectoderm (Toll rm9/rm10) (Toll10B) (gd7) Naïve cells Mesoderm

Dorsal ectodermNeuroectoderm D Pol II (Rpb3) ChIP 35 35 Naïve cells Dorsal ectoderm (gd7) 30 30 Neuroectoderm (Tollrm9/rm10) 10B 25 25 Mesoderm (Toll )

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0 0 E Pol II (8WG16) ChIP 16 16 14 14 Dorsal ectoderm (gd7) 12 12 Mesoderm (Toll10B) 10 10 8 8 6 6 4 4 2

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F Pol II (Rpb3) ChIP on hand-sorted naïve embryos 6 7 6 wt naïve 5 zld RNAi naïve 5 4 4 3 3 2 2 1 1

Fold over background 0 0 -11kb -6kb -74bp 215bp““ 2kb 3kb 8kb 13kb -20kb -9k -7kb -93bp 103bp““ 2kb 15kb 20kb Intergenic Promoter Exon 3’UTR Intergenic Shadow Shadow Intergenic Promoter Enhancer Intron Exon Enhancer Enhancer

brk sog

Fig. 1. Zelda establishes a poised polymerase in naïve cells that becomes evicted in dorsal ectoderm, repressed in mesoderm, and transcriptionally engaged in neuroectoderm. (A) Schematic drawings of gene regulation in early Drosophila embryos. Cross-section showing Dorsal gradient and differential gene ex- pression (Left), and regulation of brinker (brk) and short-gastrulation (sog) expression by Dorsal (Dl) and Snail (Sna) in mesoderm, neuroectoderm, and dorsal ectoderm (Right). (B) Wild-type (WT) 1- to 1.5-h (naïve) embryos, 2- to 4-h embryos derived from gd7 (dorsal ectoderm), Tollrm9/rm10 (neuroectoderm), or Toll10B (mesoderm) mutant mothers, and 2- to 4-h WT embryos were hybridized with digoxigenin-labeled brk, sog, and sna probes. Embryos are oriented with anterior to the left and dorsal up. (C) RT-qPCR showing brk and sog expression in embryos consisting of naïve cells, entirely dorsal ectoderm (gd7), neuro- ectoderm (Tollrm9/rm10), or mesoderm (Toll10B) normalized to β-tubulin and plotted relative WT expression, n = 3–4. Error bars represents SEM. (D) ChIP-qPCR of pol II using an Rpb3 antibody in extracts from 2- to 4-h embryos derived from gd7 (dorsal ectoderm), Tollrm9/rm10 (neuroectoderm), or Toll10B (mesoderm) mutant mothers. Values from amplicons located in enhancer, intergenic, promoter-proximal, exon, intron, and 3′ UTR regions are plotted as fold over two intergenic control regions devoid of known factors and histone modifications. Enhancers are shaded gray and promoter-proximal regions in light blue. (E) ChIP-qPCR using a different pol II antibody (8WG16) in embryo extracts from gd7 (dorsal ectoderm) and Toll10B (mesoderm) mutant mothers. (F) Rpb3 an- tibody pol II ChIP-qPCR from hand-sorted naïve cell embryo extracts (nuclear cycle 7–9) derived from WT or Zld RNAi females. Gene models are depicted at the Bottom, but are not drawn to scale. Values from promoter-proximal amplicons as a group differ significantly between mesoderm and dorsal ectoderm (D and E) and between WT and Zelda RNAi cells (F) (two-tailed t test, P < 0.05, n = 2–3). Error bars show SD.

bodies of brk and sog despite expression in this tissue, consistent II antibodies that preferentially recognize the unphosphorylated with earlier reports demonstrating that brk and sog contain a (8WG16) and Ser5-phosphorylated forms of the pol II C-terminal promoter-proximal paused pol II (reviewed in ref. 15). In the domain (CTD) (Fig. 1E and Fig. S2). A previous study found low levels mesoderm, where brk and sog are not expressed, pol II is paused as of pol II at these genes in gd7 (dorsal ectoderm) embryos (15), but our well, although occupancy is much lower than in the neuroectoderm results show that significantly more pol II associates with the brk and (Fig. 1D). By contrast, in the dorsal ectoderm, very little pol II can sog promoter regions in the mesoderm compared with the dorsal be found at the brk and sog loci (Fig. 1D). A difference in pol II ectoderm (P < 0.05, Fig. S2). occupancy at brk and sog betweenthetwotranscriptionally silent We found that pol II occupies the brk and sog promoters at tissues, dorsal ectoderm and mesoderm, was also detected using pol levels equivalent to those found in the mesoderm already in naïve

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1516450113 Boija and Mannervik Downloaded by guest on September 25, 2021 cells where transcription of brk and sog has not yet initiated (Fig. Consistent with this idea, studies in Caenorhabditis elegans have 1D). Association of pol II with the brk and sog promoters in shown that the pioneer factor PHA-4 mediates the recruitment of transcriptionally inactive mesoderm and naïve cells demonstrates poised pol II to foregut genes at an embryonic stage where ex- that pol II is in a poised state and occurs without stable binding of pression has not yet begun (20). It will be interesting to investigate if the transcription factor IID (TFIID) complex and does not result this is a general feature of pioneer factors in the conserved process in histone 3 lysine 4 (H3K4) trimethylation (Fig. S3). A previous of genome activation during animal development. report also noted the absence of H3K4me3 at poised Drosophila Our data show that pol II is displaced from the brk and sog promoters (16). Low levels of Dorsal protein can be detected in promoters as the cells transit from a naïve state to presumptive embryonic nuclei already at nuclear cycle 10, but does not reach dorsal ectoderm (P < 0.05, Fig. 1D). Thus, a change in accessi- the threshold concentration needed for robust activation of sog bility accompanies the transition of naïve cells to dorsal ecto- until cycle 12 (17–19). Our results therefore indicate that a poised derm fate. Taken together, our results demonstrate that the pol II is established at brk and sog before stable Dorsal binding. To mechanism by which brk and sog are transcriptionally silenced rule out that the presence of pol II is due to nuclear cycle 10 differ between the mesoderm and dorsal ectoderm. There is little embryos with low levels of Dorsal or to contaminating older em- occupancy of pol II at these genes in the dorsal ectoderm, bryos in our collections, we hand-sorted naïve embryos in nuclear whereas the pol II that is recruited fails to engage in transcrip- cycles 7–9, based on the number of nuclei and on morphology tion in the mesoderm. (Fig. S1). Pol II ChIP with the Rpb3 antibody showed equivalent The Snail repressor prevents brk and sog from being expressed pol II enrichment at the brk and sog promoters in hand-sorted and in the presumptive mesoderm (21, 22) (Fig. 1A). However, the bulk collected naïve embryos (Fig. 1 D and F and Fig. S4). This mechanism by which Snail represses brk and sog expression in the result demonstrates that pol II occupies Dorsal-target genes in mesoderm is not known. One possibility is that Snail prevents naïve cells before the presence of Dorsal in these nuclei. Dorsal binding to the brk and sog enhancers in the mesoderm. Because the pioneer transcription factor Zelda is required We therefore performed Dorsal ChIP in embryos derived from for the maternal-to-zygotic transition and occupies many genes, Toll mutant flies and found that Dorsal occupies the primary and including brk and sog, before zygotic transcription commences shadow enhancers of brk and sog in both the neuroectoderm and (5–7), we tested whether Zelda is responsible for pol II occu- mesoderm (Fig. 2A). Therefore, binding of Snail to the brk and sog pancy at brk and sog in naïve cells. In chromatin from cycle 7–9 enhancers in the mesoderm does not eliminate Dorsal occupancy, hand-sorted embryos devoid of Zelda, derived from females but prevents Dorsal from activating transcription. BIOLOGY where the maternal contribution of Zelda was knocked down by Next, we investigated whether Snail influences the association of DEVELOPMENTAL RNAi, pol II occupancy at both brk and sog was reduced com- a Dorsal coactivator with these loci. Dorsal and the CBP/p300 pared with WT naïve cells (P < 0.05, Fig. 1F). Similar results coactivator (nejire) preferentially co-occupy genomic DNA genome- were obtained with bulk-collected naïve chromatin obtained wide, genetically interact, and bind each other in vitro and in vivo from females with zelda germ-line clones (Fig. S4). Thus, Zelda (23, 24). Interestingly, ChIP of the CBP coactivator showed high but not Dorsal contributes to the initial recruitment of pol II to occupancy of the brk and sog enhancers in the neuroectoderm, but brk and sog. This suggests that pioneer factors such as Zelda only occupancy at background levels in the mesoderm (P < 0.05, can recruit poised pol II to target genes before their activation. Fig. 2B). This result suggests that Snail quenches Dorsal activity by

Dorsal ChIP A 16 9 Naïve cells 14 8 Dorsal ectoderm 12 7 Neuroectoderm 6 10 Mesoderm 5 8 4 6 3 4 2

Fold over background 2 1 0 0 CBP ChIP B 35 12 Naïve cells 30 10 Dorsal ectoderm Neuroectoderm 25 8 Mesoderm 20 6 15 4 10 2

Fold over background 5 0 0 H3K27ac ChIP C18 14 Naïve cells Dorsal ectoderm 16 12 14 Neuroectoderm 10 12 Mesoderm 10 8 8 6 6 4 4 Fold over background 2 2 0 0 -11kb -6kb -74bp“ 215bp“ 2kb 3kb 8kb 13kb -20kb -9k -7kb -93bp“ 103bp“ 2kb 15kb 20kb Enhancer Intergenic Promoter Exon 3’UTR Intergenic Shadow Shadow Intergenic Promoter Enhancer Intron Exon Enhancer Enhancer

brk sog

Fig. 2. Snail quenches Dorsal activity in the mesoderm by precluding CBP occupancy and histone acetylation. (A–C) ChIP-qPCR in extracts from 1- to 1.5-h embryos (naïve)or2-to4-hembryosfromgd7 (dorsal ectoderm), Tollrm9/rm10 (neuroectoderm), or Toll10B (mesoderm) mutant mothers. Values from amplicons along the brk and sog loci are plotted as fold over one (Dorsal) or two intergenic regions, and H3K27ac further normalized to H3 occupancy. (A) Dorsal. (B)CBP.(C) H3K27ac. Values from enhancers differ significantly between neuroectoderm and mesoderm for CBP but not for Dorsal (two-tailed t test, P < 0.05, n = 2–4), and H3K27ac is significantly different between neuroectoderm and all other conditions (calculated over the entire locus using two-tailed t test, P < 0.05, n = 3–4). Error bars show SD.

Boija and Mannervik PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 preventing recruitment of the CBP coactivator to the brk and sog the dorsal ectoderm but virtually absent in the mesoderm (P < 0.05, enhancers in the mesoderm. Fig. 3A). Another histone modification that is associated with gene Because CBP is a histone acetyltransferase that acetylates silencing is H3K9me3 (reviewed in ref. 27). Levels of H3K9me3 at H3K18 and H3K27 in vivo (25, 26), we performed ChIP with brk and sog are low and do not show consistent differences between H3K18ac and H3K27ac antibodies and plotted occupancy as fold the three tissues (Fig. S6). We conclude that H3K27me3, but not enrichment over the two intergenic control regions, normalized H3K9me3, is associated with silencing of brk and sog expression in to total histone H3. As expected, H3K18ac and H3K27ac are the dorsal ectoderm. high in the presumptive neuroectoderm, with a peak over the brk We then examined whether the presence of H3K27me3 in the and sog promoters (Fig. 2C and Fig. S5). Only low levels of dorsal ectoderm results in recruitment of PRC1. We found that H3K18ac and H3K27ac are found at brk and sog in the dorsal Polycomb protein associates with the brk and sog loci in the dorsal < ectoderm and mesoderm (P 0.05), tissues where CBP fails to ectoderm, but to a much smaller extent in the neuroectoderm and occupy these loci. This shows that CBP and histone acetylation mesoderm (P < 0.05,Fig.3B). Together, this finding lends further are found at brk and sog in the tissue where they are activated by support to the idea that the mechanisms by which brk and sog are Dorsal. We expressed Snail ubiquitously in early embryos by silenced in the mesoderm and dorsal ectoderm differ. In the me- crossing UAS-sna males to females containing a Gal4 driver active soderm, Snail quenches Dorsal activity by preventing CBP occu- in the female germ line (α-tubulin–Gal4VP16). As expected, the presence of Snail prevents brk and sog from being expressed in the pancy and histone acetylation, whereas in the dorsal ectoderm, neuroectoderm (Fig. S5). The presence of Snail in the neuroectoderm Polycomb and H3K27 methylation may contribute to repression. also caused a reduction in H3K27ac at brk and sog (P < 0.05, Fig. We next investigated whether histone modifications are ac- S5), consistent with a quenching mechanism where Snail pre- quired in the three germ layers in response to the Dorsal gra- vents CBP occupancy at these loci. dient or if they are present at brk and sog already in naïve cells. Whereas H3K18ac levels are similar in the mesoderm and We found that H3K27ac is only present at low levels at brk and dorsal ectoderm (Fig. S5), there is less H3K27ac in the dorsal sog in naïve cells, comparable to levels found in the mesoderm ectoderm than in the mesoderm (P < 0.05, Fig. 2C). We there- (Fig. 2C). Therefore, H3K27 hyperacetylation only occurs in fore examined the mutually exclusive mark H3K27me3. This response to Dorsal in the neuroectoderm (P < 0.05). Similarly, histone modification is deposited by the Polycomb repressive H3K27me3 is absent in naïve cells (Fig. 3A), but then acquired complex 2 (PRC2) and is associated with gene repression (reviewed specifically in the dorsal ectoderm (P < 0.05), presumably by a in ref. 2). Interestingly, H3K27me3 levels at brk and sog are high in factor restricted to this cell type.

A H3K27me3 ChIP 25 25 Naïve cells Dorsal ectoderm 20 20 Neuroectoderm 15 15 Mesoderm

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0 0 B Pc ChIP 8 7 Naïve cells 7 6 Dorsal ectoderm 6 5 Neuroectoderm 5 4 Mesoderm 4 3 3 2 2

Fold over background 1 1 0 0 -11kb -6kb -74bp““ 215bp 2kb 3kb 8kb 13kb -20kb -9k -7kb -93bp““ 103bp 2kb 15kb 20kb Enhancer Intergenic Promoter Exon 3’UTR Intergenic Shadow Shadow Intergenic Promoter Enhancer Intron Exon Enhancer Enhancer

brk sog C me me Pc X Pc dorsal ectoderm ? brk, sog

naïve cells pol II pol II ac CBP ac Zld X Dl brk, sog neuroectoderm brk, sog

pol II X mesoderm Dl Sna brk, sog

Fig. 3. The chromatin state at brk and sog differs between dorsal ectoderm, neuroectoderm, and mesoderm. (A and B) ChIP-qPCR as in Fig. 2 with (A) H3K27me3 and (B) Polycomb (Pc) antibodies. H3K27me3 and Pc values from the loci differ significantly between dorsal ectoderm and all other tissues (two-tailed t test, P < 0.05, n = 2–5). (C) Schematic drawing illustrating the chromatin states at brk and sog in naïve and specified embryonic cells. Zelda (Zld) establishes a poised pol II in naïve cells without histone modifications. In dorsal ectoderm, pol II is evicted and H3K27me3-mediated Pc silencing is established. Dorsal protein (Dl) recruits CBP in the neuroectoderm, resulting in histone acetylation (ac) and release of pol II into elongation. Snail (Sna) quenches Dorsal activity by precluding CBP occupancy in the mesoderm, leading to histone hypoacetylation and failure of pol II to engage in transcription.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1516450113 Boija and Mannervik Downloaded by guest on September 25, 2021 Together, these experiments show that low levels of histone 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% sodium deoxycholate, 0.1% SDS), modifications are present in naïve cells, but accumulate in re- WashA (as sonication buffer, but with 500 mM NaCl), WashB (20 mM Tris pH 8, sponse to the Dorsal morphogen (Fig. 3C). Dorsal recruits CBP to 1 mM EDTA, 250 mM LiCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate), and TE. Beads were transferred to new tubes and resuspended in elution establish histone acetylation in the neuroectoderm. In the meso- μ derm, Dorsal activates expression of the Snail repressor, which in buffer (50 mM Tris pH 8, 50 mM NaCl, 2 mM EDTA, 0.75% SDS, 20 g/mL RNase A, 20 μg/mL glycogen). A 4-h incubation at 68 °C was used to reverse turn prevents CBP occupancy and histone acetylation at brk and the cross-linking followed by proteinase K treatment at 37 °C for 2 h. DNA sog. Dorsal may also restrict expression of an unidentified factor to was purified using phenol-chloroform extraction and ethanol precipita- the dorsal ectoderm, which results in further histone deacetylation tion. ChIP samples were eluted in 160 μL0.1× TE pH 8 and duplicates with 2 μL and formation of repressive H3K27me3-chromatin in this germ DNA each analyzed by quantitative PCR using a Bio-Rad CFX96 machine layer. We also find that the different chromatin states are reflected and HOT FIREPol EvaGreen master mix (Solis BioDyne). Primers are listed in in the mode of pol II regulation at these genes. In naïve cells and in Dataset S1. From each IP, the percent of input signal was obtained using the the mesoderm, pol II occupies the promoters in a poised, inactive average of the two duplicates, and occupancy was calculated by nor- state, whereas histones are hypoacetylated. In dorsal ectoderm, malizing the percent input signal against two intergenic regions. Total most pol II is evicted from chromatin that is methylated at H3K27. amount of histone H3 was used to further normalize IPs performed with histone antibodies. Student’s unpaired t test for two-sample equal variance In the neuroectoderm, pol II becomes transcriptionally engaged < and histones hyperacetylated. Thus, transcription and pol II activity was used to find statistically significant differences (P 0.05) between samples. Between two and five biological replicates were generated for are linked to the chromatin state in these germ layers (Fig. 3C). each condition (Dataset S2). Through the efforts of large-scale projects such as ENCODE, the epigenomes of a large number of cell types have been deter- ChIP of Hand-Sorted Naïve Embryos. Embryos 60- to 80-min old were dechor- mined (28, 29). However, how differences between epigenomes ionated in bleach and fixed according to ref. 33. Briefly, the embryos were are initially established during embryo development remains an transferred to V-bottom glass vials containing 1× PBS/0.5% Triton X-100 and 3× outstanding question. We now show that the Dorsal morphogen, heptane and fixed in 1.8% formaldehyde for 15 min in total, including 30 s of which is responsible for specification of different germ layers by vortex, 10 min of shaking at 120 rpm, and centrifugation at 500 × g for 1 min. generating differential gene expression patterns in the Drosophila The reaction was stopped by the addition of PBS/0.5% Triton X-100/125 mM embryo, also directs differences in patterns of histone modifica- glycine. Embryos were washed three times in cold PBS/0.5% Triton X-100 fol- tions (Fig. S7). This demonstrates how gene regulatory networks lowed by vigorous shaking for 10 min at 4 °C in 1× MeOH and 1× heptane. Embryos were washed twice in MeOH and stored at −20 °C until further use. for cell specification may shape the overlaying epigenetic landscape BIOLOGY Before sorting, the embryos were rehydrated into PBS/0.5% Triton X-100

to orchestrate embryo development. DEVELOPMENTAL in a methanol gradient followed by DAPI staining for 30 min. The embryos Materials and Methods were washed three times in PBS/0.5% Triton X-100 for 10 min before sorting under dissecting and fluorescent microscopes on agar plates with a layer of Flystocks and Embryo Collection. w1118 flies were used to collect 60- to PBS/0.5% Triton X-100. Out-of-stage embryos were removed, based on the 80-min (hand-sorted naïve), 1- to 1.5-h (naïve), as well as 2- to 4-h-old wild- number of nuclei and morphology (the presence of pole cells and nuclei type (WT) embryos. The first two collections each day were discarded to positioned at the periphery). Sorted embryos were harvested from the agar remove older embryos retained by females during the overnight period. plate, washed twice in PBS/0.5% Triton X-100, and snap frozen in liquid ni- Mutants in the Toll signaling pathway were used to generate a ho- trogen for −80 °C storage. mogenous population of embryos representing the three germ layers. Tollrm9/TM3 and Tollrm10/TM3 flies(30)wereakindgiftfromAngelaSta- Frozen embryos were placed on ice and RIPA buffer (150 mM NaCl, 1.0% thopoulos, California Institute of Technology, Pasadena, CA, the Toll10B/TM3 IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, proteinase Sb Ser/OR60 stock (30) was from Ylva Engström, Stockholm University, Stock- inhibitor tablets; Roche) was added immediately. The sample was homog- holm, and the v1 gd7/FM3 stock (31) was obtained from the Bloomington enized using a pellet pestle followed by a Dounce homogenizer. The sample Drosophila Stock Center. The 2- to 4-h-old embryos were collected from gd7 was spun at full speed at 4 °C for 5 min and resuspended in RIPA buffer. homozygous mothers, Tollrm9/Tollrm10 transheterozygous females, and Toll10B Sonication was performed in a Bioruptor (Diagenode) using the high power heterozygous females were obtained directly from the balanced stock. The setting at intervals of 30 s of burst/pause for a total time of 12.5 min. Debris w*;P{w[+mC]=matalpha4-GAL-VP16}V2H strain was used as maternal Gal4 was removed by centrifugation and the supernatant was recovered and used driver and crossed to w1118 or UAS-sna males (32) (provided by Justin Kumar, for IP as described above. Indiana University, Bloomington, IN). Embryos where the maternal Zelda contribution has been knocked down were obtained from w*; Antibodies. The following antibodies were used for histone modifications: H3 P{w[+mC]=matalpha4-GAL-VP16}V2H/+; UAS-shRNA-zld/+ females (Abcam, ab1791); H3K4me3 (Millipore, 07-473); H3K9me3 (Abcam, ab8898); crossed with males of the same genotype. Chris Rushlow, New York H3K18ac (Abcam, ab1191); H3K27ac (Abcam, ab4729); H3K27me3 (Abcam, University, New York, provided UAS-shRNA-zld flies, described in ref. 10. Germ- ab6002); basal transcription machinery, Rpb3 (gift from John Lis, Cornell University, line clones of zelda were generated as described (5), with zld294 FRT19A and Ithaca, NY); 8WG16 (Abcam, ab817); PolII S5 (Abcam, ab5131); TBP (Robert Tjian, ovoD FRT19A, hsFLP stocks also obtained from Chris Rushlow. University of California, Berkeley); transcription factor, Dorsal (Christos Samakovlis, Stockholm University, Stockholm); and chromatin regulators, CBP (described in ref. ChIP. Embryos were dechorinated in bleach and nuclei isolated using a 24) and Polycomb (Yuri Schwartz, Umeå University, Umeå, Sweden). Dounce homogenizer in Buffer A1 (60 mM KCl, 15 mM NaCl, 15 mM Hepes pH 7.9, 4 mM MgCl2, 0.5 M DTT, 0.5% Triton X-100, with proteinase inhibitor Quantitative RT-PCR. Total RNA was isolated from 1.5-h-old and 2- to 4-h-old tablets added; Roche). Formaldehyde was added to a final concentration of embryos using TRIzol (Invitrogen) according to the manufacturer’s protocol. A 1.8% for 15 min. The reaction was stopped with 0.225 M glycine on ice. Nuclei total of 1 μg of RNA was DNase I treated (Sigma) and used as template for were washed three times in buffer A1 followed by a single wash in lysis buffer cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit, Applied Bio- (140 mM NaCl, 15 mM Hepes pH 7.9, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium systems). Quantitative PCR was used to analyze the amount of transcript from deoxycholate, 1% Triton X-100, 0.5 M DTT, with proteinase inhibitor tablets brk and sog using exonic primers. The raw gene quantities were calculated added; Roche). Nuclei were resuspended in lysis buffer with 0.1% SDS and using the delta Ct method and the transcript levels for brk and sog were 0.5% N-lauroylsarcosine added. Sonication was performed in a Bioruptor normalized to β-tubulin. Values from 2- to 4-h WT embryos were set to 1. (Diagenode) using high power setting, intervals of 30 s of burst/pause for a total time of 12.5 min. Debris was removed by centrifugation and chromatin Whole Mount in Situ Hybridization. In situ hybridization using digoxigenin- was diluted in equal amounts of lysis buffer before snap freezing in liquid labeled probes was performed essentially as described (34). nitrogen for −80 °C storage. Equal amounts of Protein A and Protein G Dynabeads (Invitrogen) were blocked with 1 mg/mL BSA (Sigma Aldrich) and ACKNOWLEDGMENTS. We thank A. Stathopoulos, Y. Engström, C. Rushlow, 1 mg/mL salmon sperm DNA. Antibody was incubated with the beads for at J. Kumar, J. Lis, R. Tjian, C. Samakovlis, M. Harrison, and Y. Schwartz for least 2 h at 4 °C. Antibody-bead complex was incubated with chromatin reagents and M. Levine and C. Samakovlis for discussions and comments extract corresponding to 30–40 μL of embryos at 4 °C overnight. The beads on the manuscript. The work was supported by Cancerfonden and the Swed- were washed on magnet 5 min each with sonication buffer (50 mM Hepes, ish Research Council.

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