Significance The genetic properties of heterochromatin can create strikingly random patterns of inherited gene-expression states in otherwise identical cells. The origin of these patterns of clonal expression variation are not understood: How and when in development it is determined how many cells will express, how expression states are inherited through DNA replication, and how often instabilities arise. We developed genetic, mathematical, and molecular–genetic approaches to investigate these questions. Our results indicate that these characteristics are predetermined before chromatin structure is set, and the inheritance displays remarkable instability through development.

Abstract Position effect variegation (PEV) in Drosophila results from new juxtapositions of euchromatic and heterochromatic chromosomal regions, and manifests as striking bimodal patterns of gene expression. The semirandom patterns of PEV, reflecting clonal relationships between cells, have been interpreted as gene-expression states that are set in development and thereafter maintained without change through subsequent cell divisions. The rate of instability of PEV is almost entirely unexplored beyond the final expression of the modified gene; thus the origin of the expressivity and patterns of PEV remain unexplained. Many properties of PEV are not predicted from currently accepted biochemical and theoretical models. In this work we investigate the time at which expressivity of silencing is set, and find that it is determined before heterochromatin exists. We employ a mathematical simulation and a corroborating experimental approach to monitor switching (i.e., gains and losses of silencing) through development. In contrast to current views, we find that gene silencing is incompletely set early in embryogenesis, but nevertheless is repeatedly lost and gained in individual cells throughout development. Our data support an alternative to locus-specific “epigenetic” silencing at variegating gene promoters that more fully accounts for the final patterns of PEV.

Analyses of position effect variegation (PEV) in Drosophila melanogaster and yeasts are the foundation of modern models of epigenetic inheritance. In Drosophila, the normally euchromatic cell-autonomous white+ gene necessary for eye pigmentation is subject to heterochromatin-induced gene silencing through transposition or chromosome rearrangement (Fig. 1A) (1⇓⇓–4). PEV of white+ manifests as clonal patches of contiguous ommatidia that share an expression state (ON or OFF) (Fig. 1 B and C), resulting in hallmark “clonal expression variation.” This developmental clonality of expression has led to concepts of establishment and maintenance. At some point in development an asymmetry in otherwise identical sister cells was randomly established, leading to expression in 1 lineage and gene silencing in the other (5). Thenceforth, expression states were maintained through subsequent cell divisions, including through 1 or many S-phases when all chromosome-bound proteins are removed from chromosomes during replication. This behavior of PEV demanded that the biochemical processes of DNA replication be appended to account for locus-specific regulatory “epigenetic” information being maintained through S-phase in parallel to the replication of DNA sequence. These concepts of epigenetic establishment and maintenance have been definitively demonstrated for some phenomena—X inactivation (6⇓–8), genomic imprinting (9), chromosome imprinting (10, 11), centromere identity (12⇓–14)—but the concept now broadly pervades theories of gene expression in general (15⇓⇓⇓⇓⇓–21), in ecology (15, 22⇓⇓–25), in evolution (26⇓–28), and in disease development (29⇓–31), which imagine many or all gene-expression states to be induced and inherited in much the same way as heterochromatin-induced silencing (cf. refs. 20, 32, and 33).

Fig. 1. PEV expresses a range of phenotypes. (A) Chromosome inversions or transpositions create new heterochromatin–euchromatin breakpoints, and genes near these new breakpoints undergo PEV with different clonal expression patterns. Red circle (“w+”) represents the white+ gene, thick stippled line represents heterochromatin, thin line represents euchromatin, dumbbell shape is transposed DNA, cross-hatching indicates variegation. (B) Patched (or sectored) variegation of Y,10C. (C) Salt-and-pepper variegation of Y,B840.1 (63). (D) Low expressivity of heterochromatin-induced gene silencing resulting in high expression of the variegating white+ gene in In(1)wm4. Left and right eyes of the same individual are shown. (E) High expressivity of silencing, and thus low white+ expression, in an isogenic sibling of the fly in D. (Magnification, B and C, 36×; D and E, 12×.)

While the natural factors that influence establishment have only recently begun to be explored (34, 35), multiple models for maintenance have been proposed, including DNA cytosine methylation and histone modifications stably inherited through S-phase (36⇓⇓–39). The former does not exist in the common yeast model systems, in Caenorhabditis elegans or in Drosophila (40, 41; cf. refs. 42 and 43), and so it is widely accepted that epigenetic gene-regulatory (e.g., heterochromatin-induced silencing) information must be encoded by the histone modifications in these model systems. Histone modifications (and DNA methylation) are part of the mechanisms of activation or repression, although they do not necessarily encode information instructive of gene expression. Histone modifications and DNA methylation patterns are easily changed by introduction of transcriptional repressors or activators (44⇓⇓⇓–48). Intentional modification or recruitment of heterochromatin proteins affect gene expression (49, 50), but it is not clear that modification of histones alone is necessary and sufficient to delimit self-sustaining (i.e., “epigenetic”) heterochromatin. Furthermore, many experimental observations are at odds with the simple model of “establish and maintain” epigenetics, suggesting a need to refine our understanding of these phenomena.

For example, in Drosophila (51) and Saccharomyces cerevisiae (52), excision of a silenced reporter gene from a heterochromatic locus completely derepresses silencing, even in interphase, showing that the silencing information is not maintained on the gene in question. Rather, silencing must be continually imposed by the juxtaposing heterochromatin. Furthermore, derepression is correlated with high-frequency natural changes to ribosomal DNA (rDNA) copy number, which is known to affect heterochromatin function (53, 54), suggesting unmapped genome changes may underlie some of the PEV pattern. The manifest expression versus nonexpression in PEV may therefore not be due to established and maintained silencing information at gene promoters, but rather fluctuations in overall heterochromatin “strength” in the nucleus. A straightforward way to test this possibility would be to determine whether multiple variegating genes fluctuate in concert when independently juxtaposed to different heterochromatic regions. Indeed, when tested at the chromatin of the silent mating-type loci in S. cerevisiae, 2 stochastically inactivated marker genes were perfectly correlated in their expression (55).

In Drosophila, centric or Y-linked heterochromatin often induce large patches (or sectors) of expression (Fig. 1B), whereas chromosome 4 or telomeric heterochromatin often result in “salt-and-pepper” variegation (Fig. 1C), although exceptions occur in both directions. The reasons for these different manifestations are not understood. It was initially imagined that early stochastic establishment of gene silencing by heterochromatin would create large patches, while late establishment would create salt-and-pepper patterns (2, 56⇓–58). The fact that variegating white+ alleles that possess the natural enhancer/promoter of the white+ gene [e.g., In(1)wm4, In(1)wm4h] are not expressed until late pupae (59) would seem prima facie to invalidate these models. However, work by Joel Eissenberg and colleagues (58, 60) refuted the hypothesis of differential establishment of silencing, instead supporting an alternative view where silencing was uniformly established early and stochastically lost at different times in development. These 2 models differ very little, as they both envision clonal patterns as similar errors in establishment or maintenance that differ only in their developmental timing. This timing framework still stands, despite observations by Janice Spofford that the different patterns may differ in genetic properties (2), and by Eissenberg and colleagues, who showed both patterns expressed a remarkably similar appearance throughout development (60).

We sought an alternative, heterochromatin-focused, explanation that could account for the same clonal patterns of PEV. This alternative would need to explain 4 additional features of PEV that current models do not. First, how can expressing and nonexpressing patches be demarcated before the variegated gene promoter is activated (58, 61)? Second, why are the temperature-sensitive periods of PEV limited to very early and very late in development when heterochromatin first forms and later undergoes developmentally induced relaxation (58, 60)? Third, how is maintenance of silencing independent of the cell cycle (60), despite S-phase being the salient critical time for maintenance? Fourth, how can different heterochromatic regions of the genome induce different expressivities of PEV, strongly repressed to near wild-type (62, 63), and how can they induce different clonal expression patterns (2, 60)?

In this work, we address the fundamental question of when expressivity (i.e., the extent of silencing as a fraction of pigmented and nonpigmented ommatidia) of silencing is determined. We find that it is generally set before, at, or around the time of fertilization (likely during oogenesis), which is inconsistent with the currently accepted model of silencing being set early then lost from individual genes through development. This led us to investigate the time of derepression. We created a mathematical simulation of clonal variation to test the accepted model and an alternative model: That the final patterns of PEV can be better understood as a uniformly early event that affects whether a clone of descendent cells manifests relatively stable or unstable silencing (rare or frequent switches both ON-to-OFF and OFF-to-ON). We recapitulated both patched and salt-and-pepper patterns by simulating this alternative model. The simulation also indicated a strongly discriminating test: The accepted model does not predict gains of silencing through development, while the alternative relies on such occurrences. Gains of silencing have not been directly observed in the past, mostly for methodological reasons. We used a variegating gal80 reporter gene in conjunction with a lineage marking system (64) to monitor and observe switches directly in living precursor cells of the variegating eye. Taken together, our data suggest that different expressivities and clonal patterns of PEV result from differences in the early establishment of silencing, and in failures of heterochromatin to continually reestablish silencing continually through development.

Materials and Methods Strains and Husbandry. The main SwiM strain contained variegating GAL80 and a ubiquitous GAL4, was y1 w67c23; Tp(P{w+mC=tubP-GAL80ts}10)PEV.4; P{w+mC=Act5C-gal4}17bF01/TM6B, Tb1. The individual in Fig. 5 was y w/Y; P{tubP-GAL80ts−PEV.4}/+; P{Act5C-gal4}/P{UAS-RFP}, P{UAS-FLP}, P{Ubi-(FRT.STOP.FRT)GFP}, the product of a cross between the SwiM strain and a G-TRACE strain (w*; P{w+mC=UAS-RedStinger}6, P{w+mC=UAS-FLP.Exel}3, P{w+mC=Ubi-p63E(FRT.STOP)Stinger}15F2). In those cell clones in which gal80 is active, the phenotype is RFP− GFP− because of lack of activation of any gal4-dependent transcription. In those cell clones in which gal80 is repressed, the phenotype is RFP+ GFP+, because of direct activation of RFP by Gal4 and FLP-mediated rearrangement and activation of GFP. Full genotypes of other strains, and details on culture conditions, are available in SI Appendix. Dissection and Microscopy. Images of whole flies were taken with a Sony a7iii attached to a Nikon SMZ-1500 microscope, illuminated with a Peak Plus Tactical LED Flashlight. Larvae were dissected in PBS and were visualized with a Zeiss AxioZoom.v16 and images captured with the Zeiss Axiocam 506-mono camera. Photography and quantification are described in SI Appendix. Dissection, Microscopy, and Fluorescence Detection. Adult male flies were killed by exposure to ether. They were rinsed in 70% ethanol and placed in PBS. Wings and legs were removed for better visualization on a a Zeiss AxioZoom.v16 and images captured with the Zeiss Axiocam 506-mono camera. Quantitation for correlation experiments was performed using NIH image. Larvae were dissected in PBS, and were visualized with photography and quantification are described in SI Appendix. Live third-instar larvae were collected into a Petri dish containing PBS and visualized to select male larvae. These males were further sorted based on high and low GFP, excluding the gonads which were uniformly bright between siblings. Individual larvae were imaged as above and returned to individually labeled vials. Adults were then scored for white+ eye pigment and correlated to their GFP fluorescence. Nonfluorescence images of whole flies were taken with a Sony a7iii attached to a Nikon SMZ-1500 microscope, illuminated with a Peak Plus Tactical LED Flashlight. Statistical Analyses. In all cases, α was set to 0.05 prior to the study, and we report whether the null hypotheses (H 0 ) could be rejected based on that criterion. Specific test statistics are reported in the text and figure legends. Justifications for specific tests are described in SI Appendix.

Acknowledgments This work is supported by National Institutes of Health Office of the Director’s Transformative Research Award R01 GM076092. Special support was provided by Dr. Patrick Lyons. Core support was given by the University of Arizona Cancer Center, Grant P30 CA023074, and core services at the University of Arizona provided by the Office of the Vice President for Research. We thank K. G. Golic for suggesting the name “SwiM,” and S. L. Eckert, K. G. Golic, M. Golic, and C. Huckell for support during the finalization of this work.

Footnotes Author contributions: F.B. and K.A.M. designed research; F.B. and K.A.M. performed research; F.B., G.R.H., and K.A.M. contributed new reagents/analytic tools; F.B. and K.A.M. analyzed data; and F.B. and K.A.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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