Monitoring of Switches in Heterochromatin-Induced Silencing Shows Incomplete Establishment and Developmental Instabilities

Monitoring of switches in heterochromatin-induced silencing shows incomplete establishment and developmental instabilities

Farah Bughioa, Gary R. Huckellb,1, and Keith A. Maggerta,2

aDepartment of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ 85724; and bPrivate address, San Diego, CA 92120

Edited by James A. Birchler, University of Missouri, Columbia, MO, and approved August 28, 2019 (received for review June 6, 2019) Position effect variegation (PEV) in Drosophila results from new While the natural factors that influence establishment have juxtapositions of euchromatic and heterochromatic chromosomal only recently begun to be explored (34, 35), multiple models for regions, and manifests as striking bimodal patterns of gene ex- maintenance have been proposed, including DNA cytosine pression. The semirandom patterns of PEV, reflecting clonal rela- methylation and histone modifications stably inherited through tionships between cells, have been interpreted as gene-expression S-phase (36–39). The former does not exist in the common yeast states that are set in development and thereafter maintained model systems, in Caenorhabditis elegans or in Drosophila (40, 41; without change through subsequent cell divisions. The rate of in- cf. refs. 42 and 43), and so it is widely accepted that epigenetic stability of PEV is almost entirely unexplored beyond the final gene-regulatory (e.g., heterochromatin-induced silencing) infor- expression of the modified gene; thus the origin of the expressiv- mation must be encoded by the histone modifications in these ity and patterns of PEV remain unexplained. Many properties of model systems. Histone modifications (and DNA methylation) PEV are not predicted from currently accepted biochemical and are part of the mechanisms of activation or repression, although theoretical models. In this work we investigate the time at which they do not necessarily encode information instructive of gene expressivity of silencing is set, and find that it is determined be- expression. Histone modifications and DNA methylation pat- fore heterochromatin exists. We employ a mathematical simula- terns are easily changed by introduction of transcriptional re- GENETICS tion and a corroborating experimental approach to monitor pressors or activators (44–48). Intentional modification or switching (i.e., gains and losses of silencing) through develop- recruitment of heterochromatin proteins affect gene expression ment. In contrast to current views, we find that gene silencing is incompletely set early in embryogenesis, but nevertheless is repeat- (49, 50), but it is not clear that modification of histones alone is necessary and sufficient to delimit self-sustaining (i.e., “epige- edly lost and gained in individual cells throughout development. ” Our data support an alternative to locus-specific “epigenetic” silenc- netic ) heterochromatin. Furthermore, many experimental ob- “ ing at variegating gene promoters that more fully accounts for the servations are at odds with the simple model of establish and ” final patterns of PEV. maintain epigenetics, suggesting a need to refine our understanding of these phenomena. heterochromatin | position effect variegation | Drosophila | gene For example, in Drosophila (51) and Saccharomyces cerevisiae silencing | epigenetics (52), excision of a silenced reporter gene from a heterochromatic locus completely derepresses silencing, even in interphase, nalyses of position effect variegation (PEV) in Drosophila showing that the silencing information is not maintained on Amelanogaster and yeasts are the foundation of modern thegeneinquestion.Rather,silencing must be continually models of epigenetic inheritance. In Drosophila, the normally imposed by the juxtaposing heterochromatin. Furthermore, + euchromatic cell-autonomous white gene necessary for eye pigmentation is subject to heterochromatin-induced gene silencing Significance through transposition or chromosome rearrangement (Fig. 1A) + (1–4). PEV of white manifests as clonal patches of contiguous The genetic properties of heterochromatin can create strikingly ommatidia that share an expression state (ON or OFF) (Fig. 1 B random patterns of inherited gene-expression states in other- and C), resulting in hallmark “clonal expression variation.” This wise identical cells. The origin of these patterns of clonal ex- developmental clonality of expression has led to concepts of es- pression variation are not understood: How and when in tablishment and maintenance. At some point in development an development it is determined how many cells will express, how asymmetry in otherwise identical sister cells was randomly estab- expression states are inherited through DNA replication, and lished, leading to expression in 1 lineage and gene silencing in the how often instabilities arise. We developed genetic, mathe- other (5). Thenceforth, expression states were maintained through matical, and molecular–genetic approaches to investigate these subsequent cell divisions, including through 1 or many S-phases questions. Our results indicate that these characteristics are when all chromosome-bound proteins are removed from chro- predetermined before chromatin structure is set, and the in- mosomes during replication. This behavior of PEV demanded that heritance displays remarkable instability through development. the biochemical processes of DNA replication be appended to account for locus-specific regulatory “epigenetic” information Author contributions: F.B. and K.A.M. designed research; F.B. and K.A.M. performed re- being maintained through S-phase in parallel to the replication of search; F.B., G.R.H., and K.A.M. contributed new reagents/analytic tools; F.B. and K.A.M. DNA sequence. These concepts of epigenetic establishment and analyzed data; and F.B. and K.A.M. wrote the paper. maintenance have been definitively demonstrated for some The authors declare no conflict of interest. phenomena—X inactivation (6–8), genomic imprinting (9), chromo- This article is a PNAS Direct Submission. some imprinting (10, 11), centromere identity (12–14)—but the Published under the PNAS license. concept now broadly pervades theories of gene expression in 1Retired. general (15–21), in ecology (15, 22–25), in evolution (26–28), and 2To whom correspondence may be addressed. Email: [email protected]. in disease development (29–31), which imagine many or all gene- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. expression states to be induced and inherited in much the same 1073/pnas.1909724116/-/DCSupplemental. way as heterochromatin-induced silencing (cf. refs. 20, 32, and 33). First published September 16, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1909724116 PNAS | October 1, 2019 | vol. 116 | no. 40 | 20043–20053 Downloaded by guest on September 28, 2021 A w+ 2 - transposition w+ 1 - chromosome inversion

BCD

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×.)

derepression is correlated with high-frequency natural changes showed both patterns expressed a remarkably similar appearance to ribosomal DNA (rDNA) copy number, which is known to throughout development (60). affect heterochromatin function (53, 54), suggesting unmapped We sought an alternative, heterochromatin-focused, explana- genome changes may underlie some of the PEV pattern. The tion that could account for the same clonal patterns of PEV. This manifest expression versus nonexpression in PEV may therefore alternative would need to explain 4 additional features of PEV not be due to established and maintained silencing information that current models do not. First, how can expressing and non- at gene promoters, but rather fluctuations in overall hetero- expressing patches be demarcated before the variegated gene chromatin “strength” in the nucleus. A straightforward way to promoter is activated (58, 61)? Second, why are the temperature- test this possibility would be to determine whether multiple sensitive periods of PEV limited to very early and very late in variegating genes fluctuate in concert when independently jux- development when heterochromatin first forms and later un- taposed to different heterochromatic regions. Indeed, when dergoes developmentally induced relaxation (58, 60)? Third, how tested at the chromatin of the silent mating-type loci in S. cer- is maintenance of silencing independent of the cell cycle (60), evisiae, 2 stochastically inactivated marker genes were perfectly despite S-phase being the salient critical time for maintenance? correlated in their expression (55). Fourth, how can different heterochromatic regions of the ge- In Drosophila, centric or Y-linked heterochromatin often in- nome induce different expressivities of PEV, strongly repressed duce large patches (or sectors) of expression (Fig. 1B), whereas to near wild-type (62, 63), and how can they induce different chromosome 4 or telomeric heterochromatin often result in clonal expression patterns (2, 60)? “salt-and-pepper” variegation (Fig. 1C), although exceptions In this work, we address the fundamental question of when occur in both directions. The reasons for these different mani- expressivity (i.e., the extent of silencing as a fraction of pig- festations are not understood. It was initially imagined that early mented and nonpigmented ommatidia) of silencing is deter- stochastic establishment of gene silencing by heterochromatin mined. We find that it is generally set before, at, or around the would create large patches, while late establishment would cre- time of fertilization (likely during oogenesis), which is inconsis- ate salt-and-pepper patterns (2, 56–58). The fact that variegating tent with the currently accepted model of silencing being set + white alleles that possess the natural enhancer/promoter of the early then lost from individual genes through development. This + white gene [e.g., In(1)wm4, In(1)wm4h] are not expressed until led us to investigate the time of derepression. We created a late pupae (59) would seem prima facie to invalidate these mathematical simulation of clonal variation to test the accepted models. However, work by Joel Eissenberg and colleagues (58, model and an alternative model: That the final patterns of PEV 60) refuted the hypothesis of differential establishment of si- can be better understood as a uniformly early event that affects lencing, instead supporting an alternative view where silencing whether a clone of descendent cells manifests relatively stable or was uniformly established early and stochastically lost at differ- unstable silencing (rare or frequent switches both ON-to-OFF ent times in development. These 2 models differ very little, as and OFF-to-ON). We recapitulated both patched and salt-and- they both envision clonal patterns as similar errors in establish- pepper patterns by simulating this alternative model. The sim- ment or maintenance that differ only in their developmental ulation also indicated a strongly discriminating test: The ac- timing. This timing framework still stands, despite observations cepted model does not predict gains of silencing through by Janice Spofford that the different patterns may differ in ge- development, while the alternative relies on such occurrences. netic properties (2), and by Eissenberg and colleagues, who Gains of silencing have not been directly observed in the past,

20044 | www.pnas.org/cgi/doi/10.1073/pnas.1909724116 Bughio et al. Downloaded by guest on September 28, 2021 mostly for methodological reasons. We used a variegating gal80 A BC 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. Results and Discussion DEF The extent of silencing in the adult eye (i.e., the expressivity of PEV) is a combination of at least 2 things: Variation in the amount of silencing in cell lineages within an individual, and variation in the amount of silencing between isogenic siblings (e.g., the individuals in Fig. 1 C and D are isogenic siblings) (63). The time at which this latter contribution to expressivity is established is not known. G Bilaterally Correlated PEV Shows That Expressivity Is Set Around Fertilization. We reasoned that if the prevailing model for PEV is correct (65–70), then every S-phase represents an independent potential loss of silencing, andsolossesofsilencinginany lineage would be independent of losses in any other lineage. We tested this prediction by analyzing the variegation in both halves of individuals from recently isogenized strains of 3 variegating + − − white reporter genes: whitemottled 4, whitemottled 4h, and Y,10C.

The first 2 are chromosomes bearing inversions that juxtapose GENETICS the natural white enhancer/promoter with centric heterochro- Fig. 2. Clonal variation in left and right eyes is correlated. (A) Categories + matin of the X chromosome. Y,10C is a Y-linked P{white }, (≤25% of the eye pigmented, <50% [but more than 25%], <75%, <100%, + fully wild-type) of pigmentation of left (abscissa) and right (ordinate) eyes which possesses a white transgene controlled by the minimal m4 hs from individual In(1)w male flies. Areas of the red circles represent frac- heat-shock-70 gene promoter (w ) (71, 72) and a GFP gene tion of the total population (n = 180 eyes) falling into those categories. expressed by a ubiquitin promoter (73), both of which variegate Areas of the black circles represent expectation if left and right eyes were (54). We quantified expression categorically (as in ref. 63) and + independent, determined by binomial probabilities of population distribu- found a strong correlation in expressivity of white between left tion derived from population pigmentation data. The observed and expected 2 and right eyes of the same individuals from all 3 strains (Fig. 2 A–C; data were compared using χ analysis. Rejected H0 states that the eyes are see figure legend for statistical analyses). This correlation was independent in their pigment categories, χ2 = 79.21, df = 14. (B)AsinA but clear even when the flies were raised at high (29 °C) or low with In(1)wm4h, n = 168, χ2 = 51.25. (C)AsinA but with Y,10C, n = 194, χ2 = m4 2 (12 °C) temperatures, which suppress and enhance variegation, 48.84. (D)AsinA but with In(1)w raised at 29 °C, n = 152, χ = 80.21. (E)Asin m4 = χ2 = respectively, or were raised on medium containing a chemical A but with In(1)w raised at 12 °C, n 126, 226.95. (F)AsinA but with In(1)wm4 raised in the presence of 50 mM sodium butyrate, n = 102, χ2 = 30.81. suppressor of variegation (74) (Fig. 2 D–F). Rare extreme excep- + (G) Distributions of pigment categories from F2 offspring of flies with high tions to the preponderant range of white expression were addi- degree of silencing (pink, n = 152), medium silencing (red, n = 190), or low tionally informative. Eyes with almost no expression (strongly silencing (dark red, n = 210). P < 0.05 for graphs in A–F. repressed, in some cases merely single ommatidia expressing + white )werealways(n = 10 of 10) found on a fly head opposite a second such rare eye. We confirmed that these exceptions were or placodal determination, before cytological condensation or not the consequence of cryptic polymorphisms by collecting low- heterochromatin formation in nuclear division cycle 14 (78–82), + expressing exceptions, flies with medial ranges of expression, and before the activation of the hsp70 (58) or white promoters (59, high-expressing flies, and mating them pairwise. Even in the sec- 73), before the onset of any zygotic transcription at the mid- ond subsequent generation, these 3 subpopulations each re- blastula transition (83), and before heterochromatin-induced capitulated normal and statistically indistinguishable variation in silencing is first genetically detectable (58, 84). This suggests a expression (Fig. 2G)(H0 states that these 3 populations of pig- common factor that affects expressivity between individuals is set ment categories derive from 1 population, χ2 = 7.15, df = 8, P > down in eggs, sperm, or at fertilization (85), is influential prior to 0.5). We conclude that the variation in expression is not due to and at the time heterochromatin is first formed, and its influence + spontaneous second-site enhancers or suppressors, either domi- persists through development until as late as white activation nant or recessive. in pupae. This correlation between left and right indicates that a com- To determine if this correlation persists throughout develop- mon factor controls expressivity in both halves of the organism. ment, we analyzed third-instar larval males bearing Y,10C Syncytial nuclei, and tissues separated by morphogenetic move- chromosomes and scored for expression of variegating GFP in- ments after cellularization, generally do not cross the left–right dependently in the left and right eye imaginal discs. Silencing + division plane; thus, whatever factor correlates PEV expressivity of the white and GFP transgenes on a normally paternally in adults acts prior to the separation of the presumptive eye inherited Y,10C was near-complete, so we removed the paternal anlagen in embryos. Analysis of patterns of X chromosome loss imprint on the Y (63) by crossing chromosomes through females in gynandromorphs (75–77) indicates that the nuclear division of and analyzing the male progeny of a C(1)DX/Y,10C mother. the embryo that separates left and right imaginal cuticular an- When total fluorescence in dissected imaginal discs was quanti- lagen is usually the very first zygotic division. Thus, we interpret fied, the levels of expression of GFP varied between isogenic the correlations between the left and right eyes of adults to re- individuals but were not statistically different between left and veal a common silencing potential between the first 2 zygotic right halves of the organism (Fig. 3A) (regression coefficient 1.00, nuclei. This occurs well before cellularization, before germ-layer 95% confidence interval 0.71 to 1.30, Pearson’s R2 = 0.734;

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+ Fig. 3. Relationship of clonal variation of 2 linked variegating genes: Ubiquitin-GFP and white .(A) Correlation between GFP fluorescence in left- and right- wing imaginal discs dissected from third-instar males bearing Y,10C. Fluorescence units are arbitrary, but consistent across all individuals (n = 21) of the + experiment. (B) Correlation between different white pigment categories (abscissa) and GFP expression in the thorax and abdomen (ordinate), taken from adult males bearing Y,10C selected on the basis of the former. (C) Illustrative examples for B.(D) Illustrative examples of individual Y,10C-bearing male third- instar larvae, selected on the basis of low GFP expression (Upper) or high GFP expression (Lower) in nongerm tissue, then scored for white+ expression as adults (Right). (Magnification, eyes in C and D,25×; adults in C,12×; and larvae in D,15×.)

rejected H0 states that there is no correlation between fluorescence GFP expression, 17 (26%) were white or nearly so, while 48 + of the left and right discs, specifically that the aforementioned (74%) had high levels of white pigmentation as adults. We + coefficient is not unity, F = 52.44, P < 0.05). These results in- conclude that the adult expression of white is statistically pre- dicate that the influence of an early-acting factor can be detected dicted by larval GFP expression (rejected H0 states that a priori throughout development. selection based on GFP fluorescence would have no bearing on + white pigmentation as adults, χ2 = 48.0305, df = 1, P < 0.05). Covariegation of Linked Genes Confirms the Persistence of This effect is unlikely to be cross-talk between enhancers and Expressivity through Development. Our finding that the propensity promoters of the 2 reporter genes because it has been previously to derepress is set before repression exists challenges current shown that induction of 1 variegating gene does not affect var- concepts for heterochromatin-induced silencing that posit that iegation of a closely linked variegating gene (60, 86). epigenetic information is stored at a gene promoter, and requires These 3 correlations—GFP expression between different tis- + us to consider PEV as a property of heterochromatin strength sues of larvae (Fig. 3A), white expression to GFP expression in (i.e., its ability to act as a transcriptional repressor). This has a adults (Fig. 3 B and C), and larval GFP expression to adult + strong prediction not made by the current model, namely that white expression (Fig. 3D)—all argue that the variation seen in expressivity in 1 part of the body should correlate with expressivity PEV is not reflecting variation in epigenetic information at gene in other parts of the body. To test this prediction, we again ana- loci, but rather is reflecting organism-by-organism variation in lyzed males bearing matroclinous Y,10C chromosomes. We sorted + some common factor, set at the time of fertilization, and gen- adult males with strong and weak silencing of white and then erally maintained in the soma throughout development. quantified the level of GFP expression in thoraces and abdomens of whole animals. Expression of the former was strongly correlated Development of an Alternative Model for PEV. The currently ac- with the latter (Fig. 3 B and C) (Kendall’s τ = 0.695, rejected H0 cepted model for PEV posits that large patches of expression states that there is no correlation between pigmentation category result from early derepression events while smaller clones of salt- and GFP fluorescence, P < 0.05). and-pepper variegation result from late events of derepression. It Finally, we confirmed that expressivity was stable through is implicit in this model that, regardless of when derepression development by hand-sorting male third-instar larvae by cate- occurs, silencing once lost is permanently lost. Although many gorical (“high” and “low”) GFP fluorescence, and returning mathematical modelings of epigenetic silencing have hypotheti- them to food and allowing them to grow to adulthood (Fig. 3D). cal gains and losses with each mitosis (50, 87–90), we are not Of 69 larvae with low GFP expression, 59 (86%) had white or aware of any study that has explained any mechanism for gains of near-white eyes as adults, the remainder (14%) possessed eyes silencing. Thus, the assumption that silencing once lost is never + with many white -expressing ommatidia. Of 65 larvae with high gained has remained almost entirely unchallenged.

20046 | www.pnas.org/cgi/doi/10.1073/pnas.1909724116 Bughio et al. Downloaded by guest on September 28, 2021 We envisioned another possibility for the origin of these 2 indicates that Gal80 has never been absent because even a short distinct patterns of clonal variation that can accommodate gains period of time of Gal4 activity, in the absence of Gal80, is suf- in silencing. We reasoned that it would be possible to create ficient to cause the FLP-dependent irreversible activation of clonal patches or individual salt-and-pepper patterns by leaving GFP expression. fixed the time of “establishment” of heterochromatin-induced The presence of these nonfluorescent patches of cells indicates silencing (i.e., early embryo), and instead alter the stability (the that those cell clones expressing a variegating gene late in de- probability of accurate reestablishment) of silencing in sub- velopment are the result of progenitor cells in which silencing of + sequent divisions. This alternative hypothesis might explain the gal80 was never established. For example, white clones in adult 2 extreme patterns of PEV. Large patches could arise from early eyes may best be understood not as cells in which silencing was establishment/nonestablishment and relatively low frequencies of established and lost, but as cells in which establishment was gains and losses in silencing (decisions once made are faithfully defective. This observation is consistent with the presence of maintained through development). Salt-and-pepper variegation a very early “sensitive” period of heterochromatin-mediated si- could arise from equally early establishment/nonestablishment lencing and our finding that factors controlling expressivity may followed by relatively high frequencies in switching (many sub- act around fertilization. sequent losses and gains of silencing through development). To Green and green-yellow fluorescent cell clones indicate losses of silencing, establish the validity of this hypothesis, we first created a sim- and yellow clones indicate persistent silencing. Cells expressing only plified simulation to determine if stability rather than timing GFP have had Gal4 activity at some point in development, and could create patched and salt-and-pepper patterns of PEV. The then lost it due to derepression of gal80. Gal80 binds and re- −9 simulation and our analyses can be found in SI Appendix. A key presses preformed Gal4-DNA complex with a Kd < 10 , a 10- finding was that the alternative stability hypothesis stated above fold higher affinity than that of Gal4 for DNA (95, 96), so the requires the occurrence of gains of silencing throughout devel- gal4-dependent G-TRACE is very sensitive to gal80 expression opment, while the conventional timing hypothesis does not. regardless of the level of gal4 expression. In solely green fluo- Therefore, we sought to create a live reporter system sensitive to rescent clones (Fig. 5C), derepression must have occurred some such gain events. hours or days before, to provide Gal80 time to repress Gal4- mediated transcription, and Gal4-dependent RFP mRNA and Switch Monitoring to Characterize PEV through Development. In protein time to decay below detection limits. Some clones had

order to monitor switches of heterochromatin-induced silencing low (Fig. 5D) or moderate (Fig. 5E) levels of RFP at the time of GENETICS throughout development, we considered heterochromatin func- analysis, presumably from more-recent losses of silencing and tion as a developmental stage, and adopted the G-TRACE de- incomplete decay of RFP gene products (e.g., a cell shortly after velopmental lineage tracer (64) to monitor it. G-TRACE contains point “3” in Fig. 4C). For the most part, low or moderate levels a UAS-RFP that reports on current gal4 activity, and a paired of RFP occurred in patches of contiguous cells with comparable UAS-FLP and ubiquitin-FRT-STOP-FRT-GFP that reports on RFP fluorescence (Fig. 5A), which we interpret to be loss events historic gal4 activity. To create a system particularly sensitive to in the single progenitor cell to that clone. The sizes of such acquisitions of silencing, we made gal4 expression ubiquitous and clones modally varied from about 15 to 60 cells, indicating losses embedded the Gal4 repressor, gal80, in silencing heterochromatin of silencing 4 to 6 cell divisions prior to analysis. Cell clones that (Fig. 4A). had both high-RFP and high-GFP (Fig. 5F) likely indicate per- In this configuration a red fluorescent cell in a nonfluorescent sistent silencing or losses of silencing very recently. clone would indicate recent gains of silencing of gal80, the point We observed rare contiguous clusters of yellow fluorescent of discrimination between the current and the alternative hy- cells that contained individual green fluorescent cells in their potheses. Yellow fluorescence would indicate persistent silenc- midst (Fig. 5G). We believe these individuals to be cells that lost ing, green fluorescence the loss of silencing, and nonfluorescent silencing individually without passage through the cell cycle. We cells those in which silencing was not established (Fig. 4B). Be- acknowledge that those individual cells are sisters to cells that cause RFP expression precedes GFP expression (Fig. 4C, events might also have lost silencing, perhaps as a product of their ulti- 1 and 2) and because RFP decays once gal4 is repressed (Fig. 4C, mate shared cell division; however, we see no evidence of paired between events 3 and 4), whereas GFP will not, the RFP-to-GFP sister cells with similarly reduced RFP. The half-life of RFP is ratio and size of clones can further be used to infer the timing of reported to be 90 h (97), although it is not known if this is true of gains and losses of silencing. Cells in which red fluorescence is all cell types and in all developmental stages, so loss of silencing matched with submaximal green fluorescence are those in which may have happened much earlier or more recently in develop- silencing has been gained [and Gal80 decayed, with a half-life of ment. Regardless, we believe that green fluorescent cells within ∼2 h (91)], and the degree of green fluorescence will indicate yellow fluorescent patches represent bona fide loss of silencing how long ago that happened (Fig. 4D). Cells in which green events. fluorescence is maximal and red fluorescence is not are those in In terminally differentiated examples of clonal expression + which silencing was lost, and the degree of RFP decay will in- variation, such as white ommatidia in the Drosophila eye or + dicate how long ago derepression occurred. We used this “switch yellow cuticular spots, clones of expressing cells would corre- monitoring” (SwiM) system to detect losses and gains of silenc- spond to a sum of those cells that never established silencing ing through development of living eye-antennal imaginal discs. (i.e., nonfluorescent cells in the SwiM system) and those in which silencing was established and then lost (i.e., green fluorescence Analysis of Eye-Antennal Imaginal Discs Using the SwiM System. or those cells in which green fluorescence is maximal and red Because of the high degree of variation in PEV between individ- fluorescence is dwindling). Application of the SwiM system has uals (Figs. 1 D and E and 2) (92–94) it was impossible for us to allowed us to discriminate between the different histories of meaningfully determine frequencies of these events in populations heterochromatin-induced silencing that these cells experienced of flies. We therefore sought to test the stability hypothesis by through development. Based on our studies, we conclude that close analysis of individual imaginal discs (Fig. 5A). clonal variation is best viewed as originating from 2 different Nonfluorescent cell clones indicate incomplete establishment of silencing. sources: Failure to establish silencing and failure to maintain it. In larval eye-antennal and wing discs we observed clones of cells We view PEV as a “heterochromatin” phenomenon rather than that were nonfluorescent (Fig. 5B). These correspond to cells in a “promoter” phenomenon; therefore, it is not certain that these which the ubiquitous gal4 activity was inhibited by the presence are mechanistically different events (see below in Conclu- of Gal80. Notably, the lack of green fluorescence in these cells sions and Further Thoughts).Ashasbeenreportedearlier

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Fig. 4. Schematic diagram of the SwiM system. (A) Graphical representation of the components of the SwiM system. Cross-hatched GAL80 (purple) represents variegating gal80 gene. Red uRFP (UAS-RFP), blue uFLP (UAS-FLP), aGFP (actin-GFP), and circle with triangle (excised FRT-Terminator) extrachromosomal circle represent components of the G-TRACE lineage tracer system. (B) Fluorescence outcomes of different repression and derepression states for the SwiM system. Asterisks (*) indicate the time of either the loss-of-silencing or gain-of-silencing events. (C) Delays between RFP activation (1) and GFP activation (2), and between RFP inactivation and the beginning of detectable mRNA and protein decay (3), and the final state without detectable RFP (4), allow for comparison of GFP and RFP fluorescence in individual cells to determine whether a cell has undergone gal80 repression (1, 2) or derepression (3, 4). (D) Quick- reference table summarizing (B and C).

(51, 60, 98), we see no evidence that cell division represents a We detected red fluorescent cells appearing in 2 patterns. particularly sensitive point in maintaining heterochromatin- First, they appeared as individual cells within fields of non- induced silencing. fluorescent cells (Fig. 5H). These cells have lost gal80 expression Red cell clones indicate gains of silencing. The stability hypothesis for the first time in development. Such red fluorescent cells will predicted that loss events would be observed throughout devel- mature into yellow fluorescence as GFP accumulates, and close opment, but also that we would detect gains of silencing. The analysis of red and green fluorescence in these cells indicates SwiM system was designed to be sensitive to gains, which are that there is indeed a very low level of green fluorescence in expected to appear as red fluorescent cells with no or low levels of many of them at the time of analysis. GFP expression. The time frame in which RFP accumulates to The presence of these cells supports the hypothesis that the detectability prior to the build-up of FLP, the FLP-catalyzed ge- final expressivity and pattern of clonal variation includes gains as nome rearrangement, and the build-up and maturation of GFP to well as losses of heterochromatin-induced gene silencing. It is detectability is relatively narrow, less than 2 h. We therefore therefore preferable to think of PEV as fluctuations in hetero- expected red fluorescent cells to be rare, but critically informative. chromatin strength through development, perhaps similar to the

20048 | www.pnas.org/cgi/doi/10.1073/pnas.1909724116 Bughio et al. Downloaded by guest on September 28, 2021 A Green We also observed a few GFP-expressing and non-RFP cells that were multiple cell diameters away from any other fluorescent cell (Fig. 5I). These are likely to be cells in which silencing was first lost, then reacquired, all without passage through S-phase. We interpret these cells as direct indicators of cell-autonomous fluctuations of heterochromatin strength. The second pattern we detected was of a few red fluorescent cells occurring in clusters (Fig. 5J). We can imagine 2 possible explanations for this observation. First, they could represent artifacts of the gain of gal80 silencing at some point in the past Red (e.g., 2 cell divisions for clones of 4 cells), and the recent decay of Gal80 to a level incapable of repressing Gal4. Second, they could represent synchronized gains of silencing in sisters, cousins, and second-cousins. This scenario would indicate that cells are pre-

D F determined to gain (or lose) silencing at some time in the future. J

C I Supportive evidence that this is the correct interpretation comes

E G from the preponderance of small green fluorescent clusters in B nonfluorescing clones, suggesting that losses of silencing (which

H are not subject to issues of perdurance of Gal80)arealsopre- determined some divisions before manifestation. The nature of such a predeterminant is unknown. However, these predetermined B CD instabilities may be explained as a consequence of fluctuations in the efficacy of a heterochromatin component in cell clones. For example, if the concentration of some protein necessary for heterochromatin function were to decrease in a single cell, one might expect that the entire clone descended from it may synchronously

EFG lose silencing. GENETICS A different lineage tracer more sensitive to changes. To confirm the dynamism of switching we infer from the G-TRACE components, we combined the gal4 and variegating gal80 transgene components of the SwiM system with I-TRACE (101). This lineage tracer uses a similar UAS-RFP, but also contains a ubiquitously expressed HI J GFP and a UAS-driven short-hairpin RNA directed at GFP. With I-TRACE in place of G-TRACE, red fluorescent cells would be those in which gal80 is currently repressed, green cells indicative of current gal80 expression and consequent repression of gal4,and yellow cells revealing new repression or derepression events Fig. 5. SwiM system analysis of a third-instar eye-antennal imaginal disk. as GFP and RFP fluorescence replace each other (Fig. 6A). (A) Entire disk with red- and green-channel separations. Separations are I-TRACE is therefore more sensitive to changes in expression of presented as inverted black-on-white for clarity, bordered with green and gal80, but unable to resolve the direction of the switch. In imaginal red. In all cases the green separation is on the top and the red on the bot- discs from flies bearing ubiquitously expressed gal4, variegating tom. (Inset) Location of subsequent regions (B–J). (B) Nonfluorescent cells gal80, and I-TRACE, we observe most cells express red or green indicate cells in which silencing was never established. Red and green fluorescence. However, many contiguous or individual cells exhibit channels are enhanced past linearity to establish the lack of either fluores- both red and green fluorescence (Fig. 6B). These are cells expe- cence in the cells. (C) Green fluorescence (within dotted border) indicates riencing new gains and losses (Fig. 6C), confirming the ongoing losses of silencing some time before analysis. (D) Green with a trace of red fluorescence indicates losses of silencing more recently than in C.(E)Red instability of heterochromatin-induced gene silencing. fluorescence in the 2 patches (arrows) are greater than in D and differ from WhilewehavepresentedadetailedanalysisoftheSwiM each other, indicating progressively more-recent losses of silencing. (F) Bright system in a single eye-antennal disk, the same types of cells are (maximal) red and green fluorescence indicate ongoing silencing. (G)Asingle found in all such imaginal discs we observed (SI Appendix, Figs. cell (arrow) expressing green fluorescence but not red fluorescence indicates a S2 and S3), and we believe the SwiM system is revealing common loss of silencing within a clone of cells that are still silencing. (H) Array of cells features of the fluctuating nature of heterochromatin-induced expressing red and not green fluorescence, indicating recent gains of silencing. gene silencing through development. Note that they are individual, but nonetheless cluster within the imaginal disk. (I) Individual green fluorescing cells (arrows) without red fluorescence in the Photoswitchable Fluorescent Proteins Confirm Gains and Losses Both middle of nonfluorescent clones indicate cells that were not silenced, then Exist and Are Postmitotic. Finally, we combined ubiquitously acquired silencing, then lost it again. (J) Contiguous cluster of cells expressing red fluorescence and not green fluorescence (within dotted border) suggest expressed gal4 and variegating gal80 with a gal4-controlled Kaede that all of the cells in this clone gained silencing at the same time, despite photoswitchable protein. Kaede is translated and matures as a being many cell divisions descendent from a common ancestor. (Magnifica- green fluorescing protein, but brief exposure to UV radiation tion, A,150×; Insets indicate magnification of B–J.) converts it to red fluorescence. The former state is relatively unstable (half-life of 6 h), whereas the red form is very stable (half- life greater than 7 d) (102). We photoconverted intact animals, returned them to culture, and allowed them 24 h before dissection idea proposed by Ahmad and Henikoff (98), rather than in locus- and analysis. We once again observed the same types of cell events specific modulation. The existence of such gains of silencing is that we detected using G-TRACE. Yellow fluorescence using inarguable in purely undifferentiated mitotic systems, such as in Kaede occurs in cells in which gal80 silencing is complete, and S. cerevisiae and Schizosaccharomyces pombe, where individual corresponds to yellow fluorescence using G-TRACE. Non- cells with either expression state of reporter genes are capable of fluorescent cells using Kaede corresponds to no or green fluo- recreating colonies with both states (99, 100). rescence with G-TRACE. Red fluorescence indicates losses of

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B E

Fig. 6. Use of I-TRACE and Kaede with variegating gal80 to reveal recent switches in repression state. (A) Graphical representation of I-TRACE components in conjunction with variegating gal80 and ubiquitously expressed gal4. Temporal schematic shows that I-TRACE is sensitive to changes in the repression state of gal80, although it poorly discriminates between gains and losses of silencing. (B) Illustrative image of a third-instar eye-antennal imaginal disk, including region enlarged in C.(C) Highlight of region of active switching (dotted outline). (D) Graphical representation of photoswitchable Kaede in conjunction with variegating gal80 and ubiquitously expressed gal4. Temporal schematic shows that Kaede can detect and discriminate derepression of gal80 prior to the time of photoconversion (purple line) and new repression of gal80 after photoconversion. (E) Illustrative example including region enlarged in F.(F) Highlight of a clone of cells (dotted outline) or individual cells (arrows) exhibiting new gains of gal80 silencing. (Magnification, B, 100×; E, 120×; Insets indicate magnification of C and F.)

gal80 silencing at some point in development prior to UV pho- Conclusions and Further Thoughts. Our results indicate that the toconversion, just as green (or green-yellow) fluorescence does potential strength of heterochromatin is set at or before fertil- when using G-TRACE. With Kaede, strictly green fluorescence ization, incompletely established around gastrulation (60), then indicates gains of silencing at some point after UV photo- lost and gained stochastically and repeatedly during develop- conversion (Fig. 6D). The time between UV treatment and dis- ment. Such fluctuations of heterochromatin strength can occur section was longer than the time between RFP detection and GFP without consequence to the variegating genes, at least until the detection in G-TRACE, and so we expected to see more green genes are “queried” for silencing at the time of gene expression. fluorescent cells when using Kaede. We observed individual and In the case of white or yellow variegation this occurs in late pu- small clones of green fluorescent cells (Fig. 6 E and F), confirming pae, with the final PEV expressivity and patterns reflecting the the presence of gains of silencing in imaginal discs. Individual final outcome of gains and losses through development up until green fluorescent cells are unequivocally gains of silencing without that point. This is the logical way in which fields of various sizes mitosis (especially in those cells immediately behind the mor- (i.e., the amount of the eye to be pigmented, the patched vs. salt- phogenetic furrow and the final cell division in these tissues), just and-pepper patterns) can exist prior to when the gene expression as individual red fluorescent cells are individuals in which silencing that defines them has initiated. In our SwiM system, where the was lost without cell division. Small clones of green fluorescent ubiquitin promoter queries gene expression continuously, we cells no doubt share ancestry and are coordinately acting in their directly observe these fluctuations creating fields of cells, related gain of silencing. Again, this implies that these cells have had some by lineage, whose silencing potential has been recorded through change and are determined to gain silencing some hours (or cell development. The cause of the heterochromatin fluctuation is divisions) hence, suggesting the cause of gains and losses of si- yet unknown, but could conceivably be 1 or more of a number of lencing prefigures the actual gains and losses of silencing. factors. Fluctuations may be influenced by changes at the rDNA,

20050 | www.pnas.org/cgi/doi/10.1073/pnas.1909724116 Bughio et al. Downloaded by guest on September 28, 2021 which are somatically unstable (103), known to impact PEV Materials and Methods (54, 104, 105), and can cause the same phenomenon in yeast (53). Strains and Husbandry. The main SwiM strain contained variegating GAL80 + Loss of other repeat DNAs (106) that alter PEV expressivity in and a ubiquitous GAL4,wasy1 w67c23; Tp(P{w mC=tubP-GAL80ts}10)PEV.4; +mC 1 trans (107, 108) might also affect heterochromatin fluctuations. P{w =Act5C-gal4}17bF01/TM6B, Tb . The individual in Fig. 5 was y ts− PEV.4 + It is also consistent with our data that fluctuations in concen- w/Y; P{tubP-GAL80 }/ ; P{Act5C-gal4}/P{UAS-RFP}, P{UAS-FLP}, P{Ubi-(FRT.STOP.FRT)GFP}, the product of a cross between the SwiM strain + + trations or activities of soluble heterochromatin-establishing fac- and a G-TRACE strain (w*; P{w mC=UAS-RedStinger}6, P{w mC=UAS-FLP.Exel}3, + tors, such as Piwi-interacting RNAs, small-interfering RNAs, or P{w mC=Ubi-p63E(FRT.STOP)Stinger}15F2). In those cell clones in which gal80 is protein components of heterochromatin (e.g., concentrations of 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 maternally deposited or zygotically expressed HP1) could underlie + + the patterns of PEV. Some or all of these factors might have been phenotype is RFP GFP , because of direct activation of RFP by Gal4 and FLP- mediated rearrangement and activation of GFP. identified previously as dose-limiting genetic modifiers of PEV, Full genotypes of other strains, and details on culture conditions, are although a simple mass action model is likely insufficient to fully available in SI Appendix. account for the behaviors of PEV that we and others have described (see ref. 109 for a detailed treatment). Dissection and Microscopy. Images of whole flies were taken with a Sony a7iii Viewing PEV as fluctuations in heterochromatin function attached to a Nikon SMZ-1500 microscope, illuminated with a Peak Plus rather than epigenetic information at gene promoters is a rela- 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 tively simple shift in our view, but is capable of explaining camera. Photography and quantification are described in SI Appendix. heretofore difficult-to-explain PEV phenomena. These include the following: 1) The instability of silencing in G1 (51) despite Dissection, Microscopy, and Fluorescence Detection. Adult male flies were the evident stability through G1 and S, 2) the correlations be- killed by exposure to ether. They were rinsed in 70% ethanol and placed in tween 2 variegating reporters in the same nucleus (55), 3) the PBS. Wings and legs were removed for better visualization on a a Zeiss correlation between PEV and the instability of repeat genes AxioZoom.v16 and images captured with the Zeiss Axiocam 506-mono camera. Quantitation for correlation experiments was performed using (110, 111), 4) why the temperature sensitive periods for PEV NIH image. Larvae were dissected in PBS, and were visualized with pho- suppression/enhancement do not correspond to the times of the tography and quantification are described in SI Appendix. Live third-instar presumed losses of silencing, and 5) our observation here that larvae were collected into a Petri dish containing PBS and visualized to select expressivity precedes activity of a variegating promoter. If this male larvae. These males were further sorted based on high and low GFP, GENETICS model is true, it renders moot the search for biochemical mech- excluding the gonads which were uniformly bright between siblings. Indi- vidual larvae were imaged as above and returned to individually labeled anisms to maintain locus-specific histone modification patterns vials. Adults were then scored for white+ eye pigment and correlated to through S-phase, because this type of maintenance may not exist their GFP fluorescence. for PEV. What appears as maintenance may instead be the suc- Nonfluorescence images of whole flies were taken with a Sony a7iii at- cesses or failures of heterochromatin to persistently reestablish tached to a Nikon SMZ-1500 microscope, illuminated with a Peak Plus Tactical silencing on closely neighboring genes. LED Flashlight. Notably, in a study of mouse PEV (50), ectopic recruitment of Statistical Analyses. In all cases, α was set to 0.05 prior to the study, and we HP1 induced silencing (49) that could be monitored for stability report whether the null hypotheses (H0) could be rejected based on that in the form of self-sustained mitotic maintenance. The empirical criterion. Specific test statistics are reported in the text and figure legends. rates derived from these experiments did not themselves explain Justifications for specific tests are described in SI Appendix. the stability of silencing in either the transgene or natural heterochromatin, and some hypothetical factor was required to ACKNOWLEDGMENTS. This work is supported by National Institutes of Health Office of the Director’s Transformative Research Award R01 properly model self-sustaining heterochromatin-induced gene GM076092. Special support was provided by Dr. Patrick Lyons. Core support silencing (50, 87). We suggest that it is not possible to model was given by the University of Arizona Cancer Center, Grant P30 CA023074, stable heterochromatin-induced gene silencing without a bona 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 fide block of heterochromatin that is constantly reestablishing “SwiM,” and S. L. Eckert, K. G. Golic, M. Golic, and C. Huckell for support silencing (51, 52). during the finalization of this work.

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