In metazoans, fertilization triggers global de novo chromatin reorganization into heterochromatin and euchromatin. The clustering of pericentromeric heterochromatin and the folding of chromosome arms lead to a highly regular Rabl configuration during zygotic genome activation (ZGA)3,4. Concomitantly, active and inactive chromatin regions start to associate to form the A- and B-compartments, respectively2,5,6,7,8,9. The molecular determinants of compartmental forces remain unknown.

Constitutive heterochromatin is enriched for histone 3 lysine 9 di- and trimethylation (H3K9me2/3) and is important for chromatin structure10,11. Members of the heterochromatin protein family bind to constitutive heterochromatin and perform related functions in all eukaryotes12. All family members contain a chromodomain13, which binds to H3K9me2/3, and a chromoshadow domain, which supports homodimerization and protein–protein interactions14. Drosophila expresses five different heterochromatin protein family members12 termed HP1a–HP1e. HP1a (hereafter termed as HP1, encoded by Su(var)2-5) was discovered in Drosophila15 and is essential for early embryonic development, as is the mammalian protein HP1β16,17. HP1 localizes mainly to H3K9me2/3-rich heterochromatin10,15,18, but also to euchromatic sites along chromosome arms19. HP1 might promote heterochromatin compaction through phase separation20, similar to human HP1α21. Whether HP1 is required to initiate genome reorganization in early embryos is unclear.

To address this question, we performed immunofluorescence of Drosophila embryos before ZGA and the establishment of higher-order chromatin architecture5,6, observing diffuse nuclear localization of HP1 (Fig. 1a, Extended Data Fig. 1a). By ZGA, both HP1 and H3K9me3 were strongly enriched at pericentromeric heterochromatin, which was localized apically (reflecting the Rabl configuration) and overlapped with DAPI-dense regions (Fig. 1b, Extended Data Fig. 1b, c). The HP1 signal was around 30 times higher in these regions (Supplementary Methods).

Fig. 1: Localization of HP1 during early embryonic development.
figure 1

a, Top, schematic of early embryonic development. Bottom, immunofluorescence staining at different stages of early embryonic development. HP1 localizes to chromatin before ZGA and becomes enriched at the pericentromeric heterochromatin at ZGA. Scale bar, 20 μm. b, Close-up view of HP1 localization at ZGA. Top, schematic shows the Rabl configuration of the chromosomes at this developmental stage, with the centromeres localizing on top and the chromosome arms reaching to the bottom of the nucleus. Bottom, the centromeric regions display strong HP1 signals. Images in a and b are representative from four biological replicates. Scale bar, 5 μm. c, Heat maps of HP1 ChIP–seq signal at three different early embryonic developmental time points. The signal is centred on HP1 peaks within chromosome arms called at ZGA and ranked by signal intensity at cycles 9–13. HP1 binding to chromatin is already observed before cycle 9, and becomes more enriched during development. d, Box plots of HP1 peak size distribution within chromosome arms at cycle 9, cycles 9–13 and ZGA. e, Box plots of HP1 peak size distribution within pericentromeric regions at cycle 9, cycles 9–13 and ZGA, showing that HP1 peaks get broader at the pericentromeric regions at ZGA. In all box plots, centre line denotes the median; boxes denote lower and upper quartiles (Q1 and Q3, respectively); whiskers denote 1.5× the interquartile region (IQR) below Q1 and above Q3; points denote outliers.

Source data

To characterize HP1 binding at different developmental stages, we performed HP1 ChIP–seq in precisely hand-staged Drosophila wild-type (control) embryos (Fig. 1c, Extended Data Fig. 1d, e). At ZGA, HP1 localized not only to constitutive heterochromatin, such as pericentromeric and telomeric regions (4,394 peaks, 67%) (Extended Data Fig. 1d), but also within chromosome arms (2,213 peaks, 33%) at repeat sequences (43% of non-pericentromeric peaks, 10% long interspersed nuclear elements (LINEs), 30% long-terminal repeats (LTRs)) and unique sequences (57% of peaks) (Extended Data Fig. 1d–g). Consistent with the immunofluorescence analysis (Fig. 1a), HP1 was bound to chromatin even in totipotent nuclei (Fig. 1c–e), albeit at a lower enrichment (16% of the ZGA enrichments) (Supplementary Methods). Notably, the peak size on chromosome arms did not change markedly (Fig. 1d), whereas HP1 spreading occurred at pericentromeric regions during development (Fig. 1e, Extended Data Fig. 1d, Supplementary Methods).

Next, we generated Hi-C data for control embryos precisely hand-staged at ZGA (Fig. 2a, Extended Data Fig. 2a). Chromosomes were clearly segregated into A- and B-compartments (Fig. 2a, b). HP1 was bound not only within B-compartment but also within A-compartment sequences (Fig. 2c, d, Extended Data Fig. 2b–d, Supplementary Methods). As expected, HP1 binding in B-compartment regions systematically overlapped with H3K9me3, localized around repeats and occasionally extended over several kilobases (median peak size 730 bp) (Fig. 2c). By contrast, we detected two different modes of HP1 binding in A-compartment regions. We found that 46% of HP1 binding sites in the A-compartment were sharply localized and enriched for active chromatin marks, and did not overlap with repeats (Fig. 2d, Extended Data Fig. 2d, cluster 2). A second class of HP1 peaks resembled those in the B-compartment (Extended Data Fig. 2d, cluster 1). These might correspond to short stretches of repetitive repressed DNA that cannot be resolved unequivocally by Hi-C. ChIP–seq analysis thus suggests that HP1 binds (1) within active, H3K9ac-rich chromatin in the A-compartment, and (2) within inactive, constitutive heterochromatic domains of the B-compartment.

Fig. 2: HP1 binds both A- and B-compartment regions at ZGA.
figure 2

a, Hi-C contact map of an 8-Mb region on chromosome 3L (resolution 40 kb). Pooled Hi-C data of seven biological replicates are shown (Extended Data Fig. 2a). b, Compartment scores (first eigenvector of the Hi-C map, resolution: 10 kb), same region as in a (Supplementary Methods). c, Heat maps of HP1, H3K9me3 and H3K9ac ChIP–seq signals as well as repeat positions, ±10 kb centred on HP1 peaks occurring in B-compartment regions. HP1 binding overlaps with broad H3K9me3 peaks, repeats and is devoid of H3K9ac. d, As in c for HP1 peaks in A-compartment regions, showing enrichment in H3K9ac and absence of repeats (Extended Data Fig. 2b–d).

To explore the role of HP1 in establishing 3D chromosome organization, we examined early embryos that were depleted of maternally supplied HP1. Because HP1 is essential in Drosophila15, we performed conditional knockdown22 (Extended Data Fig. 3a, Supplementary Methods).

Complete depletion of HP1 blocked development before ZGA, whereas partial knockdown of HP1 still supported development to ZGA (Extended Data Fig. 3b, c, Supplementary Methods). Therefore, we used the partial HP1-knockdown (HP1-KD) embryos in all subsequent experiments. The embryonic lethality of the partial HP1-KD embryos was rescued with a short hairpin RNA (shRNA)-resistant HP1 (HP1-rescue) (Extended Data Fig. 3d), confirming the specificity. HP1 depletion led to strongly reduced binding of HP1 genome-wide, and to upregulation of the telomeric retroelement Het-A that was rescued in HP1-rescue embryos (Extended Data Figs. 1g, 3e, f).

Hi-C analysis of HP1-KD embryos at ZGA revealed major genome-wide changes in chromosome organization (Fig. 3a, Extended Data Fig. 3g, h); we found perturbed Rabl configuration with decreased contact frequencies within and between pericentromeric regions and reduced inter-arm and inter-chromosomal contacts (Fig. 3a). Unexpectedly, we also observed increased intra-chromosomal contacts and milder decay of contact probabilities within chromosome arms (Fig. 3b–d), which suggests an overall increase in chromosome compaction within arms.

Fig. 3: Depletion of HP1 causes increased intra-chromosome compaction and reduced compartmentalization.
figure 3

a, Differential Hi-C contact map (log2-transformed), highlighting increased contact frequencies within chromosome arms, decreased inter-arm and inter-chromosome contacts, reduced associations within and between pericentromeric regions, and increased interactions of pericentromeric regions with chromosome arms in HP1-KD embryos. Biological replicates were pooled; n = 7 control and n = 5 HP1-KD embryos. b, HP1-KD embryos show a milder decay of contact probabilities above 100 kb. c, Hi-C contact maps of 19 Mb on chromosome 2R in control embryos (resolution: 120 kb). d, As in c, in HP1-KD embryos. e, Differential contact enrichment in HP1-KD versus control embryos, sorted by compartment score (Supplementary Methods), shows decreased B-compartment interactions and increased A/B intermixing. Changes relative to the control. f, Scheme of FISH probe design to quantify inter-arm distance and intra-arm compaction. g, Representative 3D-DNA FISH staining of control and HP1-KD embryos at ZGA. Signals from probes on chromosome 2R and chromosome 3L are shown separately and merged with DAPI staining. Scale bar, 5 μm. h, Quantification of physical distances between FISH signals from chromosome 2R and 3L (mean ± s.d., nuclei: control n = 55, HP1-KD n = 35). i, Quantification of compaction of FISH signals from chromosome 2R (mean ± s.d., nuclei: control n = 63, HP1-KD n = 75). j, Differential Hi-C contact map (log2-transformed), highlighting decreased inter-arm and inter-chromosomal contacts, reduced associations within and between pericentromeric regions, and increased interactions of pericentromeric regions with chromosome arms in H3K9M embryos. Biological replicates were pooled; n = 7 control and n = 2 H3K9M embryos. See Supplementary Methods and Extended Data Fig. 5 for further details. P values were determined by Wilcoxon two-sided test.

Source data

Notably, HP1-KD embryos also showed reduced segregation of A- and B-compartments, with a 20% decrease in B-compartment strength (Fig. 3e, Extended Data Fig. 3i, j). This effect was consistent across replicates, chromosome arms and for inter-arm and inter-chromosome contacts (Extended Data Fig. 3j, k). We found almost no compartment switching (Extended Data Fig. 3l). We also detected decreased insulation across topologically associating domains (TADs) (Extended Data Fig. 3m, n). By excluding short-range contacts (less than 500 kb or 3 Mb), we confirmed that the reduction of the B-compartment signal is independent of the reduction in TAD insulation (Extended Data Fig. 3o). Crucially, all of these phenotypes were rescued in HP1-rescue embryos (Extended Data Fig. 4a–d).

To validate the structural defects observed in HP1-KD embryos by Hi-C analysis, we performed 3D DNA fluorescence in situ hybridization (3D DNA FISH) with oligonucleotide probes spanning several megabases on chromosomes 2R and 3L (Fig. 3f, g). Quantitative image analysis of single cells showed that chromosomes were on average separated by larger distances (around 30% increase) in HP1-KD embryos (Fig. 3h, Supplementary Methods), in line with reduced inter-arm and inter-chromosome interactions observed in Hi-C data (Fig. 3a). In agreement with Hi-C data (Fig. 3b), we also found that the volume of the FISH signals was significantly decreased (around 10% decrease) (Supplementary Methods) in HP1-KD embryos (Fig. 3i), which suggests increased compaction of chromosome arms.

HP1 depletion thus perturbs the overall nuclear structure, with reduced proximity between pericentromeric regions, reduced alignment of chromosome arms and increased intra-chromosomal compaction. These global effects are accompanied by a prominent loss of contacts within B-compartment regions. The structural defects of HP1-KD embryos are notable, given that depletion of HP1 was only partial to allow embryos to reach ZGA. Our findings reveal that HP1 has a key role in establishing the 3D genome structure during development.

Only a small fraction of genes and repeats was misregulated in HP1-KD embryos at ZGA (Extended Data Fig. 4e). The most highly upregulated retroelements were localized at telomeric regions (Het-A, TAHRE and TART retrotransposons) and cannot account for the structural changes that we observed genome-wide (Extended Data Fig. 4e, f). We confirmed that HP1-KD embryos did not show defects in the onset of transcription at ZGA, and that both the control and the HP1-KD embryos at ZGA were in interphase (Extended Data Fig. 4g, h).

To investigate the role of HP1 in the establishment versus the maintenance of chromatin structures, we performed Hi-C experiments with differentiated, somatic Drosophila S2 cells. Notably, HP1 depletion did not considerably affect genome architecture (Extended Data Fig. 4i–o), which suggests that HP1 is not required to maintain chromatin structure.

Because HP1 interacts with chromatin by binding to H3K9me2/3, we generated embryos depleted of H3K9me2/3 by overexpressing the histone 3 lysine 9-to-methionine (H3K9M) mutation23 (Extended Data Fig. 5a). Quantitative ChIP–seq for HP1 in precisely hand-staged H3K9M embryos at ZGA showed that HP1 binding was greatly reduced on pericentromeric and repeat regions as well as chromosome arms (Extended Data Fig. 5b–d). However, HP1 was 20% more retained on chromosome arms in H3K9M compared to HP1-KD embryos (Extended Data Fig. 5b, right), which could be due to some residual H3K9me2/3 and/or H3K9me2/3-independent binding of HP1 (Extended Data Fig. 5d, right, cluster 2). ChIP–seq analysis of chromodomain-mutant HP1 (HP1-CD)13 also revealed some residual binding on chromosome arms, further supporting H3K9me2/3-independent binding of HP1 (Extended Data Fig. 5e).

Hi-C maps of H3K9M embryos revealed pericentromeric heterochromatin de-clustering and reduced chromosome arm alignment, but only a mild gain in chromosome arm compaction and mild defects in compartmentalization (Fig. 3j, Extended Data Fig. 5f–j), which could be explained by higher retention of HP1 along chromosome arms in H3K9M embryos (Extended Data Fig. 5b).

Overall, our data indicate that HP1 has a major role in establishing chromatin architecture in early embryos by: (1) mediating the clustering and condensation of constitutive heterochromatin at pericentromeric regions through H3K9me2/3-dependent binding; (2) aiding the overall configuration of chromosome arms; and (3) contributing to the formation of the B-compartment.

Next, we set out to exclude that folding defects observed at chromosome arms in HP1-KD embryos could arise as a mere consequence of the expansion of pericentromeric chromatin. Because it is impossible to completely decouple these effects in vivo, we turned to a genome-wide polymer modelling approach in which chromosomes are represented as chains of three types of 10-kb beads (A, B and C corresponding to A- and B-compartment and pericentromeric/telomeric regions, respectively) confined in a cylindrical nucleus (Fig. 4a, Supplementary Methods). We first optimized a set of interaction energies to reproduce contact probability scaling and compartment strength within arms in control embryos (Extended Data Fig. 6a–c). Next, we mimicked centromere de-clustering by decreasing interactions among C-type beads and their interactions with the nuclear surface (mutant) (Fig. 4b, c). The model recapitulated reduced alignment between chromosome arms (Fig. 4c, right) and increased interactions between pericentromeric regions and chromosome arms (Fig. 4c), but not compaction and compartmentalization defects within arms (Extended Data Fig. 6d, e). These results do not depend on the numbers of centromeric and telomeric beads (Extended Data Fig. 6f–l). This suggests that compartment defects and intra-arm compaction are a consequence of decreased HP1 binding on chromosome arms.

Fig. 4: HP1 establishes de novo chromatin architecture during development via two independent mechanisms.
figure 4

a, Whole-genome polymer model. A- and B-type beads correspond to 10-kb A- and B-compartment regions. C-type beads correspond to pericentromeric and telomeric regions. b, Snapshots of wild-type control (left) and mutant (right) simulations. c, Genome-wide simulated distance maps of control (left) and mutant (centre). Right, differential distance map highlighting increased distances within and between centromeric and telomeric regions and reduced chromosome arm alignment (arrows). d, Polymer model of multi-megabase chromosome arm regions. Interaction energies between 40-kb beads are inferred to reproduce the experimental Hi-C map. e, Experimental and simulated contact maps in control (top) and HP1-KD (bottom) embryos (chr3R 17–20.6 Mb). f, Inferred interaction energies are overall more attractive in the HP1-KD model. P value determined by two-sided Wilcoxon test. Box plots are as in Fig. 1d. g, Left, interaction energies between B-type beads (B–B) become comparatively less attractive in HP1-KD embryos, but more attractive between A-type beads (A–A) and between A and B types (A–B). Right, average interaction energy changes between HP1-KD and control models. B-compartment attractions decrease in the HP1-KD model. Data are mean ± s.e.m., interactions: 990 (A–A), 2,069 (A–B), 1,035 (B–B). h, Chromatin is modelled as a chain of two types (A and B) of interacting 40-kb beads (chr3R 17–20.6 Mb). i, Scaling exponents increase when attractions between all beads are increased by a multiplicative factor, and vice versa. j, Compartment strength (bold line: mean) decreases when attractions between beads are increased, and vice versa. Confidence interval (shaded area) calculated using t-based approximation.

Source data

To understand the cause of compartment defects in HP1-KD embryos and determine whether they might simply arise from increased intra-arm compaction (Fig. 3a–d), we implemented two smaller-scale polymer models designed to uncover the energies driving the folding of chromosome arms.

In the first approach, interaction energies between 40-kb beads were optimized to reproduce experimental Hi-C maps within multi-megabase regions of chromosome arms24,25 (Fig. 4d, Supplementary Methods). For control contact maps (Fig. 4e, top), we found that interaction energies were globally attractive, which accounts for the correct contact probability scaling (Extended Data Fig. 7a), The model predicted that A–A and B–B interactions were on average more attractive than A–B interactions (Extended Data Fig. 7b). For HP1-KD contact maps (Fig. 4e, bottom, Extended Data Fig. 7c, d), we found increased attractions overall between all bead types but comparatively less attractive B–B interactions (Fig. 4f, g). Notably, our findings do not depend on the specific region that is simulated (Extended Data Fig. 7e–l). This suggests that decreased compartmentalization is not a mere consequence of increased compaction after HP1 knockdown (Fig. 3a–d) but instead requires the simultaneous loss of B-specific attractive interactions.

To confirm these findings, we used a more general model that is not designed to reproduce the experimental Hi-C maps but instead describes the behaviour of a polymer when interaction energies between its constituent A- and B-type beads are systematically varied (Fig. 4h, Supplementary Methods). Increasing all A–A, A–B and B–B interaction energies correctly predicted milder scaling of contact probabilities (such as HP1-KD), but led to stronger compartments (Fig. 4i, j, Extended Data Fig. 7m). By contrast, decreasing all interaction energies correctly predicted compartment loss but led to the wrong scaling behaviour (steeper decay) (Fig. 4i, j, Extended Data Fig. 7m). Finally, decreasing only B–B attractions reproduced the observed decrease in compartment strength but resulted in a steeper scaling (Extended Data Fig. 7n). Thus, modifying chromosome compaction alone cannot explain the HP1-KD structural phenotype, which suggests that HP1 depletion perturbs compartmental forces. Notably, these results do not depend on the distribution of A- and B-compartment beads (Extended Data Fig. 7o–r). Analysis of this general polymer model shows that the HP1-KD structural phenotype within arms (increased compaction, lower compartmentalization) arises from two independent mechanisms: decreased specific interactions between B-compartment regions, and increased attraction between all genomic locations.

Our data and modelling approaches suggest that HP1-mediated interactions, which might occur through HP1 oligomerization14 or phase separation20,21, have a major role in establishing 3D genome conformation during embryogenesis. Decreased HP1 binding in pericentromeric heterochromatin led to declustering and decondensation of constitutive heterochromatin and a perturbed Rabl configuration. By contrast, decreased HP1 levels within chromosome arms caused decreased B–B compartment attractions and increased arm compaction, possibly owing to decreased chromatin stiffness. Reduced segregation of B-compartment regions after HP1 knockdown might facilitate interactions between A- and B-type chromatin and allow attractions between active regions to dominate, resulting in globally increased compaction (Extended Data Fig. 7s). This is consistent with quantitative compartment analysis (Fig. 3e, Extended Data Fig. 3i, j) and the overall increase in A–A and A–B interactions in simulations (Fig. 4g). Alternatively, increased attractions could arise from HP1 counteracting condensin II-mediated homologous chromosome pairing or cohesin-mediated loop extrusion.

In the A-compartment, HP1-mediated compartmental forces might be counteracted by surrounding active chromatin modifications such as H3K9ac (Fig. 2d, Extended Data Fig. 7 s). Because the A-compartment is not affected after disruption of the B-compartment (Fig. 3e), we suggest that it is controlled by a distinct driving force independent of HP1.

Our study shows that HP1 is required to establish pericentromeric heterochromatin clustering in early embryos but is dispensable in differentiated cells, consistent with a recent report in mammals26. In differentiated cells, clustering might be driven by other HP1 paralogues or heterochromatin proteins2 favoured by the slower cell cycle, or result from other mechanisms involving solid-like states in heterochromatin condensates27. We also showed that HP1 prevents the collapse of chromosome arms while they elongate to establish the characteristic Rabl configuration. Finally, HP1 is directly involved in the formation of the B- but not the A-compartment region. Because pericentromeric clustering and compartmentalization also occur in mammals, HP1 could have similar functions during mammalian embryogenesis.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.