Evidence for Transgenerational Transmission of Epigenetic Tumor Susceptibility in Drosophila

Transgenerational epigenetic inheritance results from incomplete erasure of parental epigenetic marks during epigenetic reprogramming at fertilization. The significance of this phenomenon, and the mechanism by which it occurs, remains obscure. Here, we show that genetic mutations in Drosophila may cause epigenetic alterations that, when inherited, influence tumor susceptibility of the offspring. We found that many of the mutations that affected tumorigenesis induced by a hyperactive JAK kinase, HopTum-l, also modified the tumor phenotype epigenetically, such that the modification persisted even in the offspring that did not inherit the modifier mutation. We analyzed mutations of the transcription repressor Krüppel (Kr), which is one of the hopTum-l enhancers known to affect ftz transcription. We demonstrate that the Kr mutation causes increased DNA methylation in the ftz promoter region, and that the aberrant ftz transcription and promoter methylation are both transgenerationally heritable if HopTum-l is present in the oocyte. These results suggest that genetic mutations may alter epigenetic markings in the form of DNA methylation, which are normally erased early in the next generation, and that JAK overactivation disrupts epigenetic reprogramming and allows inheritance of epimutations that influence tumorigenesis in future generations.


Introduction
Epigenetic regulation of gene expression refers to repression or activation of gene expression via covalent modifications of DNA or histones, such as methylation or acetylation, without changing the DNA sequence of the gene [1][2][3]. Epigenetic modifications are usually stably heritable through subsequent cell divisions, resulting in permanent changes in gene expression profiles, such as those associated with terminal differentiation. However, at critical stages in normal development or disease situations, cells undergo genomewide epigenetic reprogramming, erasing preexisting epigenetic marks and establishing a new set of marks. For instance, major epigenetic reprogramming occurs at fertilization prior to zygotic development, at dedifferentiation that leads to cancer development, and during somatic cell nuclear transfer, a procedure used for cloning or obtaining embryonic stem cells [4][5][6][7].
However, epigenetic marks are not always completely erased from one generation to the next. For instance, genomic imprinting, where clusters of genes or whole chromosomes are preferentially inactivated depending on their parental origin [8,9], can be considered an exception to epigenetic reprogramming, because in this case parental epigenetic markings are retained in the zygote. Loss of imprinting has been shown to increase the likelihood that cancer will develop [10][11][12]. Furthermore, human diseases, such as Prader-Willi and Angelman syndromes [13] and hereditary nonpolyposis colorectal cancer [14], are associated with germline inheritance of epimutations. Though transgenerational epigenetic inheritance has been documented for a variety of eukaryotic organisms ranging from plants to humans [15], the precise mechanisms that regulate epigenetic marking and erasure, as well as those that protect certain epigenetic marks from being reset, are not clear.
We have previously undertaken a genetic approach in order to identify genes that are important for hop Tum-linduced tumorigenesis in Drosophila, and in the process, have found that JAK signaling globally counteracts heterochromatin formation [16]. Further analyses of the identified mutations indicated that a number of those mutations that genetically modify hop Tum-l tumorigenicity also do so epigenetically. In fact, hop Tum-l itself plays an essential role in the maintenance of parental origin epigenetic alterations that subsequently affect tumorigenesis in a transgenerational manner. These results indicate a novel function for the hop Tum-l oncogene: it interferes with the epigenetic reprogramming process.

Paternal-Effect Modification of Tum-l Tumorigenicity
We previously conducted a genetic screen for modifiers of the hop Tum-l hematopoietic tumorigenic phenotype and identified 37 modifier mutations [M(Tum-l)] that dominantly enhanced or suppressed hop Tum-l tumorigenesis in hop Tum-l /þ; M(Tum-l)/þ transheterozygotes [16]. Hematopoietic tumors in hop Tum-l -containing flies were quantified by tumor index (TI) (see Materials and Methods and also [16]). Interestingly, many of the M(Tum-l) mutations (24/37) exhibited paternal-effect modification of hop Tum-l tumorigenicity, such that when hop Tum-l /þ females were mated to males heterozygous for the modifier mutation (M[Tum-l]/þ), tumorigenesis associated with hop Tum-l was modified (enhanced or suppressed) in the F1 generation regardless of the inheritance of M(Tum-l) ( Table  1). The transgenerational effects were confirmed with rebalanced stocks, indicating that they are unlikely to be due to different genetic backgrounds. Since little or no paternal cytoplasmic proteins are carried in the sperm, the observed paternal effects on the zygote suggest an epigenetic mechanism. Possibly, the M(Tum-l) mutations caused epigenetic alterations in the paternal chromosomes and these epigenetic changes were maintained through male meiosis and transmitted to the F1 generation, thereby influencing hop Tum-l tumorigenicity.

Transgenerational Tumor Modification Depends on Maternal hop Tum-l Mutation
To understand the nature of the transgenerational epigenetic modification of hop Tum-l tumorigenicity by the M(Tuml) mutations, we conducted a detailed analysis of Kr, which is one of the first zygotically transcribed ''gap'' genes whose activity is required for the correct segmentation of the embryo [17]. First, we tested two loss-of-function alleles of Kr (Kr 1 and Kr 2 ) and found that they both enhanced hop Tum-l genetically and epigenetically ( Figure 1A; unpublished data), confirming Kr as an E(Tum-l) with epigenetic effects.
To rule out any genetic background effects, we extensively outcrossed a Kr 1 allele, and isogenized and rebalanced it over a CyO balancer chromosome that in previous testing showed no enhancement of hop Tum-l (see Materials and Methods). The new iso-Kr 1 /CyO stock again enhanced hop Tum-l tumorigenicity both genetically and epigenetically, such that when hop Tum-l /þ females were crossed to iso-Kr 1 /CyO males both hop Tum-l /þ; Kr 1 /þ and hop Tum-l /þ; þ/CyO progeny exhibited significantly higher TI ( Figure 1B, columns 2 and 3). Interestingly, when F1 males of þ/Y; þ/CyO, which did not inherit Kr 1 , were backcrossed to hop Tum-l /þ females, we found that the enhancement persisted in the F2 generation in the absence of Kr 1 , but diminished in the F3 ( Figure 1B, columns 4 and 5). Since half and a quarter of the P0 paternal DNA contents (originally exposed Kr 1 ) are inherited in the F2 and F3 generation, respectively, the diluting effect of the enhancement in the absence of the original mutation (Kr 1 ) is consistent with the idea that the modification is epigenetic in nature and is distributed genome wide at multiple loci. To rule out the possibility that Kr 1 induced genome-wide genetic mutations, we conducted the reciprocal cross, mating iso-Kr 1 /CyO females with rare escaper hop Tum-l /Y males. We found that Kr 1 enhanced hop Tum-l only genetically but not epigenetically, such that the TI increased in hop Tum-l /þ; Kr 1 /þ but not in hop Tum-l /þ; þ/CyO female progeny flies ( Figure 1C). The result of the reciprocal cross confirms that the modification is epigenetic in nature, as genetic mutations (changes in DNA sequence) would not be reversible under normal circumstances. However, such a result could also suggest a parentspecific effect of Kr on the hop Tum-l mutation.
To test whether the epigenetic effects of Kr 1 are specific for the male genome, we mated hop Tum-l /þ; Kr 1 /CyO recombinant females to wild-type males. In this cross, the tumor phenotype associated with hop Tum-l was enhanced in both hop Tum-l /þ; Kr 1 /þ and hop Tum-l /þ; CyO/þ progeny flies ( Figure 1D), indicating that the presence of Kr 1 in the female parent can also have epigenetic effects on hop Tum-l tumorigenicity in the F1 generation. Thus, it appeared that Kr 1 was capable of epigenetically altering both male and female genomes, and these alterations could be transmitted through both male and female meioses to the F1. However, the inheritance and/or ability of these parental origin alterations to modify hop Tum-l tumorigenicity epigenetically in the F1 progeny appeared to depend on the presence of hop Tum-l as a maternal mutation.

Transgenerational Epigenetic Effects of Histone Deacetylase Inhibitors on Tum-l Tumorigenicity
To further test the ability of maternal hop Tum-l to maintain parental origin epigenetic changes, we examined the effects of histone deacetylase (HDAC) inhibitors on hop Tum-l tumorigenicity. Since Rpd3, encoding an HDAC, was identified as one of the genes which, when mutated, exhibited both genetic and epigenetic enhancement of hop Tum-l tumorigenicity (Table  1), we reasoned that the epigenetic effect of an Rpd3 mutation on hop Tum-l tumorigenicity might be mimicked by HDAC inhibitors such as tricostatin A (TSA) and sodium butyrate. Indeed, TSA treatment caused increased levels of acetylated histone H3 ( Figure 1E), and increased the tumor index of hop Tum-l /þ flies from 0.38 to 0.96 6 0.06 (p , 0.01). Consistent with a transgenerational epigenetic effect, when wild-type flies that had been treated with TSA were mated with untreated hop Tum-l /þ females and the progeny were raised in the absence of the drug, the TI of hop Tum-l /þ F1 progeny was also significantly increased ( Figure 1F). As with Kr 1 , no

Author Summary
It is well known that many genetic mutations in oncogenes or tumor suppressors can cause or greatly increase a person's susceptibility to cancer. It is generally assumed that persons should feel relieved if they have not inherited the particular ''cancer-causing'' mutation carried by their parents. However, we found that, under certain circumstances, fruit flies carrying tumor suppressor gene mutations can pass the increased tumor risk to all offspring, even those that have not inherited the particular mutation. A likely scenario is that many genetic mutations can lead to epigenetic alterations, that is, changes in the chemical modifications of DNA or the proteins that bind to DNA in the chromosomes, and these changes can have global effects on cell function. Normally, these epigenetic alterations are wiped out and reset in the early embryo, but under certain circumstances such alterations can be inherited. Interestingly, we found evidence that a particular oncoprotein, an overactivated form of a cell-signaling molecule called JAK kinase, can counteract the epigenetic resetting program that normally operates in the early embryo. Thus, the failure of epigenetic reprogramming allows the inheritance of parental epigenetic alterations that affect susceptibility to tumors. epigenetic effect was found in the reciprocal cross ( Figure  1F), suggesting that the presence of hop Tum-l in the early embryo is important for TSA treatment to have a transgenerational epigenetic effect on hop Tum-l tumorigenicity. A similar transgenerational epigenetic effect on hop Tum-l tumorigenicity was also found with another HDAC inhibitor, sodium butyrate (unpublished data).

Maternal hop Tum-l Mutation Maintains Krüppel Mutant Phenotypes in Its Absence
To investigate the maternal hop Tum-l -dependent transgenerational inheritance of epigenetic changes at the level of gene expression, we examined the effects of hop Tum-l on Krdependent expression of the pair-rule gene fushi-tarazu (ftz), which encodes a homeodomain protein required for embryonic patterning [18]. It has been shown that Kr heterozygous embryos exhibit defects in ftz expression [19]. In wild-type embryos, ftz is expressed in seven stripes at the onset of gastrulation ( Figure 2A). In Kr 1 /þ embryos, however, ftz stripe 3 is narrow or weak ( Figure 2B; also see [19]). The same ftz stripe 3 phenotype was found in Kr 2 /þ embryos (unpublished data). We wondered whether the defects in ftz expression might involve epigenetic alterations, and whether these defects could be passed to the next generation in the presence of maternal hop Tum-l mutation. Indeed, we found that the ftz promoter region is differentially methylated in Kr heterozygotes (see below).
We reasoned that if hop Tum-l promotes transmission of parental origin epigenetic alterations to the next generation, then the ftz stripe 3 defect caused by Kr 1 could be retained in embryos from hop Tum-l /þ mothers and Kr 1 /þ fathers that did not inherit Kr 1 . To test this, we examined ftz expression from a ftz-lacZ transgene carried on the CyO balancer chromosome, which contains the Kr þ allele and segregates from Kr 1 in the F1 when Kr 1 /CyO ftz-lacZ flies are used as a parent. In embryos from male and female Kr 1 /CyO ftz-lacZ flies, 70% (n ¼ 61/87) of   Figure S1). Quantification of three independent blots is shown to the right. the b-gal þ embryos exhibited the typical Kr 1 heterozygous defects, characterized by weakened or narrowed stripe 3 expression ( Figure 2B), suggesting that all embryos that are genotypically Kr 1 /CyO ftz-lacZ exhibit the stripe 3 defect. When Kr 1 /CyO ftz-lacZ flies were crossed to wild-type flies, in the F1 embryos, ftz-lacZ was expressed in seven stripes identical to those in the wild-type background, such that these stripes were more or less evenly spaced and similar in intensity (Figure 2A; n ¼ 54). When hop Tum-l females were mated to þ/CyO ftz-lacZ males, we found wild-type ftz-lacZ pattern and no stripe 3 defects similar to those in Kr 1 heterozygotes in the F1 embryos ( Figure 2C; n ¼ 78). Notably, although the JAK/STAT pathway is involved in regulating even-skipped stripe 3 expression [20,21], ftz expression seemed not affected in hop Tum-l mutants. This is consistent with a lack of STAT-binding sites in the ftz promoter region (unpublished data). However, when Kr 1 /CyO ftz-lacZ males were mated to hop Tum-l females, 94% of the F1 b-gal þ embryos retained the stripe 3 defect characteristic of Kr 1 heterozygotes ( Figure 2D; n ¼ 48/51). Since in this mating scheme ftz-lacZ segregated from Kr 1 , embryos that expressed the ftz-lacZ trangene would not inherit Kr 1 and were genotypically þ/þ for the Kr locus. Thus, the presence of hop Tum-l caused retention of the Kr 1specific defective ftz expression pattern in embryos that did not inherit the Kr 1 mutation. These results demonstrate that hop Tum-l can cause transgenerational inheritance of epigenetic changes at a transcriptional level.

Transgenerational Inheritance of Kr-Induced ftz Promoter Methylation
To identify the epigenetic alterations caused by Kr mutations, we examined the DNA methylation status of the 620-bp minimal ftz enhancer in the ftz-lacZ transgene, as the expression of this ftz-lacZ is epigenetically modified by Kr 1 . DNA methylation is the predominant epigenetic modification, and methylation of CpG islands is responsible for gene silencing and genomic imprinting in mammals [5][6][7]. There is evidence for the presence of DNA methylation in Drosophila [22,23]. Drosophila has a Dnmt2-like DNA methyltransferase that mediates methylation of cytosine residues in vivo [24], although the biochemical activity of Drosophila Dnmt2 as a DNA methyltransferase is still to be shown. Methylated cytosines in both CG and CT dinucleotides have been found in many transposons and repetitive sequences in Drosophila genomic DNA [25], and increased promoter DNA methylation is associated with gene silencing [26].
We first assessed the methylation status of the ftz minimal enhancer ( Figure 3A) by digesting total genomic DNA with a methylation-sensitive restriction enzyme BstUI, which cuts unmethylated but not methylated CGCG sequences, followed by quantification of the undigested DNA by PCR. By comparing the time courses of BstUI digestion of genomic DNA samples isolated from Kr þ/À versus wild-type control flies, we concluded that the former is more resistant to BstUI digestion ( Figure 3B and 3C, top panels). Digestion of the same DNA samples with a methylation-insensitive restriction enzyme HaeIII produced no differences between the two samples ( Figure 3B and 3C, bottom panels). These results suggest that the minimal enhancer of ftz-lacZ in Kr þ/À flies is more methylated than in wild-type flies.
We next investigated whether the Kr-dependent differential methylation of the ftz minimal enhancer can be passed to the next generation. We crossed Kr 1 /CyO ftz-lacZ flies to hop Tum-l /þ and wild type females, respectively, and isolated genomic DNA from the F1 flies that inherited the ftz-lacZ transgene. We analyzed the methylation status of the 620-bp minimal ftz enhancer using methylation-sensitive and -insensitive restriction digests as described above. Indeed, we found the ftz enhancer in F1 flies of hop Tum-l /þ females and Kr 1 /CyO ftz-lacZ males was more resistant to a methylationsensitive restriction enzyme than the ftz enhancer in F1 flies of þ/þ females and Kr 1 /CyO ftz-lacZ males ( Figure 3D and 3E), consistent with the idea that hop Tum-l promotes transgenerational inheritance of epigenetic changes.
We employed a second method to confirm that the promoter of the ftz-lacZ transgene has increased DNA methylation in Kr mutants and that this methylation status is transgenerationally inheritable in the presence of hop Tum-l maternal mutation. We isolated total genomic DNA from embryos of different parental genotypes, digested with restriction enzymes, and incubated with antibodies against methylated cytosine. Quantification of immunoprecipitated DNA indicates that that the ftz-lacZ fragment was more methylated in embryos of Kr 1 /CyO ftz-lacZ flies ( Figure 3F) and the higher levels of methylation was maintained in embryos from Kr 1 /CyO ftz-lacZ fathers and hop Tum-l mothers ( Figure 3G).
Finally, to further demonstrate the differential methylation of the ftz minimal enhancer in different genetic backgrounds or pedigrees, we treated the genomic DNA samples with sodium bisulfite, which converts cytosines (C) to thymidines (T), and then cloned and sequenced independent clones for each sample. Sequencing results indicated the presence of two CG (or CT)-rich ''islands'' in the ftz minimal enhancer that are preferentially methylated in Kr þ/À samples or in embryos of Kr 1 /CyO ftz-lacZ father and hop Tum-l mothers ( Figure 4). Thus, Kr mutations indeed induce epigenetic alterations, as exemplified by increased DNA methylation in the ftz minimal enhancer, and such alterations are normally erased, but are transmitted to the next generation if an overactivated JAK kinase is present in the early embryo.

5-Aza-dC Treatment Promotes Tumorigenesis but Inhibits the Epigenetic Effects of Kr on hop Tum-l Tumorigenicity
Since the epigenetic effects of Kr mutations involve DNA methylation, we investigated the effects of inhibiting DNA methylation on the ability of Kr mutations in promoting hop Tum-l tumorigenesis. We raised flies in food containing the  [30]. Arrows above and below the horizontal line represent PCR primers used to amplify a 778-bp fragment, encompassing the 620-bp minimal ftz enhancer (see Figure S2 for sequence). Bent arrow indicates the start of the lacZ sequence. Vertical bars above and below the line represent positions of recognition sequences for restriction enzymes BstUI (CGCG) and HaeIII (GGCC), respectively. (B-E) Time courses of restriction digests of genomic DNA with enzymes sensitive (BstUI) or insensitive (HaeIII) to methylated DNA are shown as agarose gel pictures and quantifications. Genomic DNA was isolated from Kr 1 /CyO ftz-lacZ and þ/CyO ftz-lacZ (wild-type control) adult flies (B, C), or from the F1 progeny flies of Kr 1 /CyO ftz-lacZ males crossed to hop Tum-l/þ females or wild-type females (D, E), and digested with the indicated enzymes for the indicated times (minutes). Digested DNA was amplified with PCR primers shown in (A) and run on an agarose gel. Note that the genomic DNA from Kr þ/À flies or from the F1 progeny of hop Tum-l/þ females and Kr 1 /CyO ftz-lacZ males is more resistant to BstUI digestion than the controls. (F, G) Digested genomic DNA purified from embryos was immunoprecipitated by antibodies to methylated cytosine and amplified by PCR primers shown in (A). Embryos derived from wild-type or Kr þ/À parents (F), or from Kr 1 /ftz-lacZ males crossed to wild-type (top) or Tum-l/þ (bottom) females (G) were used for DNA isolation. Note the presence of higher levels of 5-meC in the ftz-lacZ promoter in Kr þ/À embryos or in those from Tum-l/þ females crossed to Kr 1 /ftz-lacZ males. doi:10.1371/journal.pgen.0030151.g003 DNA methyltransferase inhibitor 5-aza-29-deoxycytidine (5aza-dC) and determined the effects of drug treatment on hop Tum-l -dependent blood tumor formation. When raised at 100 lM 5-aza-dC (a nonlethal dose), hop Tum-l/þ flies exhibited dramatically increased tumors compared with untreated hop Tum-l/þ flies, with TI increased from 0.41 6 0.05 (untreated; n ¼ 116) to 1.27 6 0.15 (treated; n ¼ 68; p , 0.001). Such results are in line with TSA treatment (see above). Similar to the effects of TSA treatment, when wild-type male flies raised in 5-aza-dC were crossed to hop Tum-l/þ females and allowed to produce eggs in the absence of the drug, the F1 flies exhibited increased TIs ( Figure 5), but no TI increase was detected in the reciprocal cross (unpublished data), suggesting a maternal hop Tum-l -dependent transgenerational inheritance. Interestingly, we found that treatment with 5-aza-dC, although by itself promotes hop Tum-l tumorigenesis, abolished the ability of Kr mutations to epigenetically enhance tumors, such that when Kr 1 /CyO male flies raised on 5-aza-dC food were crossed to hop Tum-l/þ females, the epigenetic effects (associated with CyO), but not the genetic effects of Kr, were abolished ( Figure  5). Thus, the DNA methylation methyltransferase inhibitor 5aza-dC both promotes hop Tum-l tumorigenesis and inhibits Kr epigenetic effects. These results suggest that hop Tum-l -induced blood tumors can be both enhanced by a general loss of genomic DNA methylation and suppressed by preventing Kr mutation-induced methylation in specific promoters.

Discussion
We have investigated the effects of genetic and epigenetic mutations on the tumorigenicity of the Drosophila hematopoietic oncogene hop Tum-l , and found that hop Tum-l and its modifier mutations mutually influence each other, both  genetically and epigenetically. In particular, we have shown that a Kr mutation that enhances hop Tum-l tumorigenicity induces ftz promoter methylation, which is associated with repression of ftz stripe 3, and that Tum-l promotes transgenerational inheritance of ftz stripe 3 silencing in the F1 generation in the absence of the Kr mutation.
Taken together, these results suggest that the oncogenic JAK kinase encoded by hop Tum-l is able to antagonize a cellular program that erases epigenetic markings of parental origin, allowing such epigenetic alterations to be maintained in the F1 even in the absence of the original genetic mutation. The epigenetic alterations in turn influence the risk of hop Tum-linduced tumorigenesis in the F1 generation.

Epigenetic Effects of hop Tum-l Modifier Mutations
Many of the M(Tum-l) genes that exhibited paternal-effect modifications encode products with known chromatin remodeling functions. These include HP1, Rpd3, and several Suppressor of variegation (Su[var]) mutations. It is conceivable that flies heterozygous for these mutations have altered chromatin states that could directly influence the epigenetic state of the zygote, leading to paternal effects as shown recently in mice [27]. However, the M(Tum-l) genes that exhibited epigenetic effects on Tum-l tumorigenicity also include those whose functions in chromatin modification are not obvious. These include transcription factors such as Kr and signaling molecules such as the Notch ligand Serrate (Ser). This observation suggests that genetic mutations in genes other than those encoding chromatin remodeling proteins may also cause epigenetic alterations.
Although Kr is expressed only in 20% of the early embryo, lacking Kr causes profound patterning defects, resulting in deletion or defects in over 70% of embryonic segments [28]. As a first zygotically expressed ''gap'' gene, Kr is in the top tier of the regulatory hierarchy that controls pattern formation of the whole organism [28]. Thus, Kr mutations can affect expression of genes that are not directly regulated by Kr. A Kr neomorphic allele (Kr if ) has been shown to affect eye development by an epigenetic mechanism [29]. Our results indicate that the Kr mutation, which likely acts early on, results in the establishment of an epigenetic signature in the genome in the form of methylation of particular promoters, such as the ftz promoter. Repression of certain ''tumor suppressor genes'' may explain the enhancement of the hop Tum-l tumorigenic phenotype by Kr mutations. As an epigenetic modification, DNA methylation is believed to be mitotically stable. In support of this notion, we detected similar methylation patterns in the ftz-lacZ promoter in embryos and adult flies of Kr heterozygotes (Figure 4). Although we have not directly examined germ cells, the transgenerational phenomenon suggests that the Kr-dependent epigenetic signature extends to germ cells, which give rise to sperm and eggs. We envision the possibility that the epigenetic signature of germ cells is established early together with somatic cells, and can be affected by mutations in Kr, which might have a global reach in the early embryo. Alternatively, there is constant communication between germ cells and somatic cells during animal development, such that their epigenetic states will stay in ''sync.'' The precise mechanisms by which germ cells acquire the epigenetic states of somatic cells remain to be investigated.

Requirement of Maternal hop Tum-l for Inheritance of Epigenetic Mutations
When hop Tum-l is inherited from the mother, its product, a hyperactive JAK kinase, is present in the embryo from the very beginning as a maternal contribution. In contrast, when inherited from the father, the hop Tum-l gene product is not present in the early embryo but is expressed as a zygotic gene. Zygotic genes are not transcribed until the midblastula transition or later. The parent-of-origin effect of hop Tum-l on the ability of Kr 1 to modify its tumorigenicity suggests the following scenario. The M(Tum-l) mutations are capable of altering the state of the chromatin, resulting in epigenetic changes in the genome. These ''epigenetic marks'' can be maintained through mitosis and meiosis and transmitted to the F1 progeny, where they are normally erased in the zygote during early embryogenesis. However, the hop Tum-l mutation, if present in the early embryo as a maternal-effect mutation, is able to preserve certain epigenetic alterations of parental origin. In other words, hop Tum-l may play a role in counteracting a mechanism that erases epigenetic marks of parental origin during early embryogenesis.
using the DNeasy Tissue kit (Qiagen) according to the manufacturer's instructions with minor modifications. Thirty 1-2-d-old adult flies or 100 ll of 0-12-h embryos of desired genotypes were homogenized in 180 ll of PBS and 20 ll of proteinase K (1 mg/ml) per manufacturer's protocol. The samples were treated with DNase-free RNase A (Sigma) for 2 h at 37 8C prior to column purification. For restriction digests, 3 lg of genomic DNA was incubated with 10 units of BstUI (New England Biolabs, http://www.neb.com/) or 10 units of HaeIII (New England Biolabs) in 60 ll of total volume at 37 8C. At different time points, an aliquot of the digests was removed and heated at 80 8C to inactivate the restriction enzyme. One microliter of each sample was used for PCR amplification with primers specific to ftz-lacZ (forward: 59-CCCAGGGATCGGACGTAATGTTAT-39; reverse: 59-GGATGTGC TGCAAGGCGATTAAGT-39). Bisulfite treatment was carried out with the EpiTect Bisulfite Kit (Qiagen, http://www1.qiagen.com/) according to the manufacturer's instructions. Genomic DNA (2 lg) was treated in Bisulfite Mix. Treated genomic DNA was amplified with the following strand-specific primers (forward: 59-TTTAGGGATTG GATGTAATGTTAT-39; reverse: 59-AAATATACTACAAAACAATTA AAT-39). The PCR fragments were cloned into pGEM-T vectors (Promega) and independent plasmid DNA isolates were sequenced. Sequencing was carried out by Gene Gateway (http://www. genegateway.com/). For immunoprecipitation, genomic DNA was first digested to completion with EcoRI and BamHI (New England Biolabs). Digested genomic DNA (2 lg) in 200 ll was used for immunoprecipitation with 5 ll of anti-5-meC (Abcam) or control antibody at 4 8C overnight, together with protein-G beads that had been preabsorbed with sonicated single-stranded salmon sperm DNA. The antibody complex was centrifuged and washed and eluted. The presence of ftz-lacZ promoter sequence was quantified by PCR with the above primers.