Next Article in Journal
Heterogeneous Genomic Divergence Landscape in Two Commercially Important European Scallop Species
Next Article in Special Issue
The Stability and Evolution of Genes and Genomes
Previous Article in Journal
ROS Homeostasis Involved in Dose-Dependent Responses of Arabidopsis Seedlings to Copper Toxicity
Previous Article in Special Issue
Mosaic Evolution of Molecular Pathways for Sex Pheromone Communication in a Butterfly
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 14
Issue 1
10.3390/genes14010012
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Epigenetic Silencing of P-Element Reporter Genes Induced by Transcriptionally Active Domains of Constitutive Heterochromatin in Drosophila melanogaster

by
Giovanni Messina
1,
Emanuele Celauro
1,†,
Renè Massimiliano Marsano
2,†,
Yuri Prozzillo
1,† and
Patrizio Dimitri
1,*
1
Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Sapienza Università di Roma, 00185 Roma, Italy
2
Dipartimento di Bioscienze, Biotecnologie e Ambiente, Università di Bari, 70125 Bari, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to the work.
Genes 2023, 14(1), 12; https://doi.org/10.3390/genes14010012
Submission received: 15 November 2022 / Revised: 12 December 2022 / Accepted: 16 December 2022 / Published: 21 December 2022
(This article belongs to the Special Issue The Stability and Evolution of Genes and Genomes)
Browse Figures
Review Reports Versions Notes

Abstract

:
Reporter genes inserted via P-element integration into different locations of the Drosophila melanogaster genome have been routinely used to monitor the functional state of chromatin domains. It is commonly thought that P-element-derived reporter genes are subjected to position effect variegation (PEV) when transposed into constitutive heterochromatin because they acquire heterochromatin-like epigenetic modifications that promote silencing. However, sequencing and annotation of the D. melanogaster genome have shown that constitutive heterochromatin is a genetically and molecularly heterogeneous compartment. In fact, in addition to repetitive DNAs, it harbors hundreds of functional genes, together accounting for a significant fraction of its entire genomic territory. Notably, most of these genes are actively transcribed in different developmental stages and tissues, irrespective of their location in heterochromatin. An open question in the genetic and molecular studies on PEV in D. melanogaster is whether functional heterochromatin domains, i.e., heterochromatin harboring active genes, are able to silence reporter genes therein transposed or, on the contrary, can drive their expression. In this work, we provide experimental evidence showing that strong silencing of the Pw+ reporters is induced even when they are integrated within or near actively transcribed loci in the pericentric regions of chromosome 2. Interestingly, some Pw+ reporters were found insensitive to the action of a known PEV suppressor. Two of them are inserted within Yeti, a gene expressed in the deep heterochromatin of chromosome 2 which carries active chromatin marks. The difference sensitivity to suppressors-exhibited Pw+ reporters supports the view that different epigenetic regulators or mechanisms control different regions of heterochromatin. Together, our results suggest that there may be more complexity regarding the molecular mechanisms underlying PEV.

1. Introduction

Transposable elements (TEs) represent a conspicuous fraction of eukaryotic genomes, varying from 3% in yeast, 15% in D. melanogaster, 45% in humans, over 50% in maize and up to 90% in other plants [1,2,3,4,5].
Based on their structure and transposition mechanism, eukaryotic TEs are classified into two main classes. Class I contains retrotransposons that are mobilized via RNA transposition intermediates, whereas class II elements transpose either using a “cut and paste” mechanism (Subclass I), rolling-circle replication [6], or single strand excision followed by extrachromosomal replication [7].
TEs have shaped eukaryotic genomes [8] including regions of constitutive heterochromatin where a build-up of TEs has been documented in many evolutionarily distant organisms [9,10].
Constitutive heterochromatin has been originally defined based on cytological features, i.e., the compact state during all phases of the cell cycle and C-banding staining. In addition, low gene density, low rate of meiotic recombination, high content of repetitive DNAs and specific epigenetic signatures are hallmarks of the constitutive heterochromatin [11]. However, the lack of functional genes is no more considered a general feature of constitutive heterochromatin of D. melanogaster and other species [11]. Indeed, genome sequencing and annotation have shown that this peculiar genomic compartment contains hundreds of transcriptionally active genes, both unique and repetitive [12,13,14], which together account for a large fraction of the genomic territory of D. melanogaster constitutive heterochromatin [11].
Among the Drosophila TEs that have been extensively characterized both at the genetic and molecular levels, P and I elements are naturally occurring transposons which have been found to insert themselves into the constitutive heterochromatin of D. melanogaster and eventually target the genes therein located [15,16,17,18,19,20,21,22].
Engineered P-elements have been routinely used as DNA integration vectors or to generate collections of single-element insertion lines useful for both forward and reverse genetic studies of gene disruption, enhancer trapping and protein trapping [23,24,25,26,27,28,29,30,31,32].
Beside the above applications, engineered P-elements represent useful tools to monitor the functional state of diverse chromatin domains of D. melanogaster genome. When inserted into regions of constitutive heterochromatin, P-associated reporters undergo strong silencing or expressed following a mosaic pattern, in which some cells fully or partially display the reporter phenotype (known as variegated phenotype), while other cells do not [33,34,35]. The variegated phenotypes produced by transgene insertions into constitutive heterochromatin are very similar to those usually ascribed to position effect variegation (PEV) caused by chromosome rearrangements that relocate a euchromatic gene nearby to constitutive heterochromatin [36]. It is generally believed that the PEV phenotypes displayed by a variety of P-element-reporter genes reflect the silenced state of a given heterochromatin region where the elements are inserted. The variegated phenotype of P-reporter genes inserted in constitutive heterochromatin can be suppressed by known genetic modifiers of PEV [21] and by the Y chromosome, a known suppressor of PEV [37].
P-element-based vectors containing the mini-white gene driven by an hsp70 promoter were also used to characterize the structure of chromatin domains of the D. melanogaster fourth chromosome [38,39]. The results of these works have shown that this chromosome contains heterochromatic domains showing a variegating phenotype alternating with euchromatic domains showing a fully pigmented eye phenotype.
The resistance to exogenous nuclease digestion [40] and to methylation [41] exhibited by P-element reporters inserted within constitutive heterochromatin and their relatively ordered nucleosome arrays [42] together suggested that heterochromatic silencing is due to the loss of sites for binding of transcription factors and/or RNA polymerase.
In conclusion, based on the aforementioned results, P-element reporter genes have been considered a useful tool to investigate the genetic and molecular bases underlying the PEV phenomenon.
Histone modifications have been shown to play a role in the occurrence of epigenetic silencing and thus contribute to PEV [36]. Notably, actively transcribed genes in constitutive heterochromatin must be accessible to RNA pol II and cis-acting transcriptional regulators, although they have regulatory requirements different from those of euchromatic genes [43]. In particular, combinations of negative and active histone modification marks, together with the contribution of key epigenetic regulators, such as the HP1 and SU(VAR)3-9 proteins, are likely to contribute to establishing gene expression in constitutive heterochromatin [11,44,45,46,47,48,49,50,51,52].
Here, we asked whether a given P-element reporter gene inserted into constitutive heterochromatin, within or nearby a transcribed gene, would “sense” the active chromatin state of the surrounding domain, with its expression being driven by the regulatory elements of that region or, on the contrary, would it undergo PEV. To answer this question, we examined 12 lines each containing a mini-white reporter gene carried by a P-element (from here indicated as Pw+ reporters) cytologically and molecularly mapped to the pericentric heterochromatin of chromosome 2 (Table 1 [21,35,53]).
Our results provide new hints on the mechanisms of epigenetic silencing induced by constitutive heterochromatic in D. melanogaster.

2. Materials and Methods

2.1. Drosophila Strains

Fly cultures and crosses were carried out at 25 °C in standard cornmeal yeast medium. The KV lines, each carrying a single SUPor-P element [35], were gifts of Robert Levis. The Su(var)2052 and aubergneQC42 mutants were gifts of Sarah Elgin, while the Su(var)3071 and Su(var)3-91 were gifts of Gunter Reuter. The LP1 mutant was generated by Messina et al. [53].

2.2. Mapping of the P White+ (Pw+) Reporters

The flanking sequences of the KV lines were obtained by inverse PCR (done by Robert Levis) and their locations are reported in FlyBase. The mapping of 19.74.3 and LP1 lines was reported by Messina et al. [53].

2.3. Complementation Test with Df(2Rh)MS41-10

Pw+/SM1, Cy females were crossed to Df(2Rh)MS41-10/SM1, Cy males, and the progeny was scored for the presence of Cy+ and Cy flies. The expected ratio between Cy and Cy+ among the offspring of these crosses was 2:1 because Cy is homozygous lethal. The absence of the Cy+ progeny indicated that a given Pw+ insert was lethal over the deficiency. KV00363 and LP1 were found to be lethal over Df(2Rh)MS41-10 (all progeny showing the Cy phenotype), while for the other inserts the ratio between Cy and Cy+ phenotypes was close to 2:1. For each complementation test, over 100 adults were counted.

2.4. Genetic Crosses with PEV Suppressors

We crossed w/w; Su(var)2052/SM6, CyS; +/+ or w/w; +/+; Su(var)3071/TM3, Sb females to males w/Y; Pw+/SM1,Cy from 10 selected lines carrying a Pw+ reporter mapped to 2R heterochromatin balanced over the SM1,Cy chromosome (Figure 1). For the crosses in Figure 1A, we isolated the F1 male progeny carrying the suppressor (w/Y; Pw+/Suvar) and the control progeny carrying only the variegated Pw+ reporter (w/Y; Pw+/SM1, Cy). Similarly, for the crosses in Figure 1B, we isolated the F1 male progeny carrying the suppressor (w/Y; Pw+/+; Suvar/+), and the control progeny with only the variegated Pw+ reporter (w/Y; Pw+/+; TM3, Sb/+). All the aforementioned male progeny were isolated for eye pigment quantification.

2.5. Eye Pigment Assays

The extraction of red eye pigment was performed according to Ephrussi and Herold [55], as described in Prozzillo et al. [56]. For each genotype, three replicate samples of 10 heads were performed. Absorbance at 480 nm was then measured using a 96-well plate in a VICTOR Multilabel Plate Reader spectrophotometer (PerkinElmer, Waltham, MA, USA). Photographs of representative adult fly eyes were taken using a Nikon SMZ745T stereoscopic microscope (Minato, Tokyo, Japan) equipped with a digital C-mount camera.

2.6. Distribution of Epigenetic Marks

The distribution of the epigenetic marks in the regions of constitutive heterochromatin is available in Genome Browser of FlyBase and was also retrieved from the ModEncode data.

3. Results

3.1. Analysis of P-Element Insertions

First, we asked whether Pw+ reporters inserted into actively transcribed domains of constitutive heterochromatin are subjected to PEV. To answer this question, among a collection of KV lines [21] showing strong mini-white silencing, we selected 10 lines with elements located within the pericentric portions of chromosome 2 (Figure 2). These regions have been extensively characterized at both genetic and molecular levels [11,14,20,43,53,57,58,59,60,61]. Each KV line carries a single SUPor-P element which has two reporter genes, the mini-white gene and the intron-less yellow gene, as well as two “suppressor of hairy wing” [Su(Hw)] binding sites flanking the mini-white gene [62]. This design allows a robust expression of the yellow reporter and, together with the presence of the Y chromosome, facilitates the recovery of SUPor-P pericentric insertions that would be otherwise missed due to the strong silencing of the mini-white reporter [21,63].
The analysis of the insertion sites showed that the SUPor-P element are located within or close to genes that are expressed (Table 1). In particular, as shown in Figure 3, according to FlyBase and FlyAtlas, the genes linked to the inserted elements are expressed during different developmental stages and in the eyes of both male and female. We also examined the LP1 line carrying a Pw+ reporter into the Yeti gene and its progenitor insert of line 19.74.3, which maps about 2 kb downstream Yeti ([53], Table 1). In addition to the mini-white reporter, these elements contain Fab-7 insulator sequences characteristic of the bithorax complex [64].
All the P-insertions mapping to the right arm of chromosome 2 heterochromatin (2Rh) were tested in complementation analyses with Df(2Rh)MS41-10. This deletion lacks almost the entire mitotic heterochromatin of 2Rh, spanning from the deep region h40 to the very distal 46 [58,59,65], together with all the known genes, including rolled, CG40191, CG41265, Yeti, dpr21, Haspin and p120 ctn (See Figure 1, from Marsano et al. [11]). These results showed that, with the exception of LP1 and KV00363 (lethal alleles of Yeti), the analyzed P-inserts in 2Rh were fully viable over Df(2Rh)MS41-10. The insert in line KV00462 which maps to 2Lh was homozygous viable. Thus, it is conceivable that, while the expression of Yeti was clearly affected, the expression of the genes linked to the other insertions was not significantly perturbed. Indeed, in the LP1 homozygotes no Yeti transcripts were detected [53].
Together, from these analyses it emerged that, despite their location in expressed domains of constitutive heterochromatin, all the examined lines exhibited a strong silencing of the Pw+ reporters. The males of some lines (KV00524, KV00158, KV00384, KV00376 and 19.74.3) exhibited a slightly stronger red-eye pigmentation compared to that shown by the females of the same line (Figure 2), an effect that could be due to the PEV suppression exerted by the Y chromosome [36,37].

3.2. Experiments with Genetic Modifiers of PEV

Loss-of-function mutations resulting in dominant suppression of PEV were shown to identify crucial epigenetic regulators, such as the HP1 protein [36]. Thus, first we investigated whether the variegation of Pw+ reporters showed by the lines under investigation can be suppressed by Su(var)205 and Su(var)307, two dominant PEV suppressors [36]. To do this, we crossed w/w; Su(var)2052/SM6, Cy,S; +/+ or w/w; +/+; Su(var)3-71/TM3, Sb females to males of 10 selected Pw+ reporters (Figure 2) balanced over the SM1, Cy chromosome (Figure 1; Section 2).
The results of this analysis are summarized in Figure 4. Su(var)2052 clearly suppressed the variegation of Pw+ reporters in lines KV00158, KV00376, KV00369 and KV00171. However, this was not the case for the remaining five Pw+ reporters in lines KV00249, KV00299, KV00363, KV00524 and LP1, which were quite insensitive to the suppression. Su(var)3071 suppressed most Pw+ reporters, although with a variable efficiency. However, the reporters of LP1 and KV00299 lines showed only a slight suppression effect, while the one of KV00363 was clearly insensitive.
Moreover, mutations in piwi, aubergine or homeless genes, encoding components of the piRNA pathway [66,67], also suppress the silencing of Pw+ inserted in heterochromatin as a result of reduction of H3 Lys9 methylation and delocalization of HP1. Thus, we also tested the effect of the aubergineQC42 mutant allele on the variegation of the Pw+ reporters under investigation (see Section 2 for the scheme of crosses). As shown in Figure 4, the trend of the effect was very similar to that found with Su(var)2052 in that the Pw+ reporters of KV00249, KV00299, KV00363 and LP1 lines were insensitive to suppression, with the exception of KV00524.
The product of the wild-type Su(var)205 gene, the heterochromatin-associated HP1 protein, interacts with the histone methyltransferase (HMTase) encoded by the wild-type Su(var)3-9 gene. The SU(VAR)3-9 methyltransferase selectively methylates histone H3 at lysine 9 (H3-K9), and it is generally accepted that H3K9me stabilizes the binding of HP1a to chromatin [68,69,70]. The Su(var)3-9 mutant alleles are known to be dominant modifiers of PEV [36]. We then tested the effect of the Su(var)3-91 mutation on the variegation of Pw+ reporters from KV00249, KV00299, KV00363, LP1 and 19.74.3 lines. As shown in Figure 5, the variegation of Pw+ reporters in KV00249, KV00299, KV00363 and LP1 lines was insensitive to suppression by Su(var)3-91, whereas the Pw+ variegation in the 19.74.3 was efficiently suppressed. In conclusion, using different PEV suppressors (Su(var)2052, Su(var)3-91 and aubergineQC4), we obtained comparable results with the same Pw+ reporters (Figure 4 and Figure 5).

4. Discussion

It is generally believed that PEV is induced by transcriptionally inactive constitutive heterochromatin. The aim of this work was to further investigate this aspect by studying the heterochromatin-induced silencing on a number of Pw+ reporters inserted into different positions of the pericentromeric regions of chromosome 2, within or nearby a transcribed gene (Table 1; Figure 3 and Figure 6). We found that all the examined reporters subjected to strong PEV (Figure 2) map within or close to genes/domains that support an appreciable level of transcription during development and in the eye (Figure 3). Moreover, some reporters were suppressed by classical dominant PEV suppressors encoding epigenetic factors/regulators required for heterochromatin establishment, while other reporters in lines KV00249, KV00299, KV00363 and LP1 were less sensitive or even insensitive to the action of the suppressors (Figure 4, Figure 5 and Figure 6). In general, there appears to be no significant correlation between the mini-white expression levels for each Pw+ reporter line and the transcription levels of the heterochromatic gene(s) linked to it. For example, CG40191 is on average more expressed than dpr21, and yet the respective inserts have the same behavior towards suppression.
Although the heterogeneity of the genetic background might partially contribute to explaining the different sensitivity to epigenetic regulators tested, these results suggest that the chromatin state characteristic of transcriptionally active heterochromatin may not be sufficient to guarantee the proper expression of a given reporter gene inserted within or nearby. In other words, the regulatory requirements for the expression of the autochthonous genes in heterochromatin seem to be different, or even antipodal, from those needed for adventitious DNA sequences, such as the Pw+ reporters. However, the observation that the silencing of some reporters is abolished by the tested suppressors also suggests that the product of wild-type alleles of some suppressors, on one hand, can induce the silencing of the reporters, but, on the other hand, may be required for, or is compatible with, the expression of the genes located in pericentric heterochromatin. The effect of the product of wild-type alleles of suppressors could be performed directly or indirectly.
Indeed, the expression of light and rolled heterochromatic genes of chromosome 2 was found to be affected when moved away from their native location to euchromatin by chromosome rearrangements [70,71]. Moreover, genetic and molecular studies have shown that the proper expression of light, rolled, and other heterochromatic genes such as Rpl15 and Dbp80 depends on the HP1a protein [72,73,74,75]. In accordance, large-scale mapping experiments carried out in Drosophila Kc cells have shown that HP1a and SU(VAR)3-9 are associated with pericentric genes, such as light and rolled genes [44,46,47].
Together, the available findings support the idea that histone marks, together with HP1a and other epigenetic regulators, are involved to ensure the expression of genes located in constitutive heterochromatin. However, some apparently contradictory results [44,51] indicated that differences in the epigenetic regulation of heterochromatic genes may exist between in vivo and in vitro systems.
The insensitivity to suppressors showed by Pw+ reporters in KV363 and LP1 lines is intriguing for several reasons. Firstly, Yeti is an efficiently expressed gene in the heterochromatin of chromosome 2 (Figure 2 [11,53,76,77,78,79]) and carries active chromatin marks (Table 1). Secondly, the P-insertion of the 19.74.3 line, the progenitor of LP1, maps roughly 2 kb downstream the 5′ of Yeti (Figure 6), but its silencing is efficiently suppressed by Su(var)2052, Su(var)3071, Su(var)3092 and aubergneQC42 (Figure 4 and Figure 5). Thirdly, Su(var)205, Su(var)309 and Su(var)307 are among the most general and effective PEV suppressors and were already found to efficiently erase the silencing of many pericentric reporters [36,80]. Finally, the distribution of HP1 and SU(VAR)309 proteins along the pericentric regions was found to be enriched in the heterochromatin of 2R where Yeti is located [44,46,47].
The molecular bases underpinning insensitivity to suppressors exhibited by Pw+ reporters in KV363 and LP1 lines are currently elusive. A trivial explanation for the apparently strong silencing could be that the Pw+ element in these lines is structurally defective so that the expression of the mini-white is compromised. However, we found that in both lines the element can still transpose in the presence of the Delta-2-3 element, a known source of the P-transposase, giving rise to de novo fully pigmented red-eye insertions (not show). This result excludes the occurrence of significant DNA sequence alterations in the Pw+ element of both KV363 and LP1 lines.
It is possible that the chromatin state of the Yeti gene is regulated by epigenetic regulators/mechanisms different from those controlled by the wild-type alleles of the tested PEV suppressors.
We did not find recurrent repeated sequences flanking the reporters, suggesting the absence of specific relationships between location and sensitivity to suppression. However, two non-coding RNAs, CR44042 and CR44043, were associated with Yeti and l(2)41Ab genes (Figure 6), respectively, with the Pw+ reporter of KV00299 being inserted within CR44043. It could be possible that the transcription of CR44042 and CR44043 may interfere somehow with that of the reporters.
A consistent body of experimental evidence suggested that nascent RNA may play a direct role in transcription and chromatin regulation, in that it may function as regulator of its own expression, giving rise to positive-feedback loops in which active gene expression states can be maintained [81]. This model could explain the resistance to all PEV suppressor exhibited by the Pw+ reporters in LP1 and KV00363 lines. We hypothesize that in wild-type conditions the Yeti-encoded RNA interacts with its own gene at the level of DNA/chromatin, acting as an epigenetic insulator or as a positive effector, giving rise to an active chromatin state which allows a stable expression of Yeti. However, following a P-insertional mutation, a consequent lack of functional Yeti transcripts (or a low level of transcription) would shut-down the expression of the gene disrupting its active chromatin state with the acquisition of a closed chromatin structure. As a consequence, the Pw+ reporters inserted within Yeti would become silenced (Figure 7). Such positive RNA feedback mechanism, if existing, should be insensitive to the action of classical PEV suppressors.

5. Conclusions

In conclusion, we have shown that transcriptionally active domains of constitutive heterochromatin can induce epigenetic silencing of Pw+ reporters. Whatever the genetic bases of this phenomenon are, our results represent a remarkable paradox suggesting that there may be more complexity regarding the molecular mechanisms underlying PEV.

Author Contributions

Conceptualization P.D., E.C., R.M.M., G.M. and Y.P.; Methodology P.D., E.C., R.M.M., G.M. and Y.P.; Validation P.D., E.C., R.M.M., G.M. and Y.P.; Experiments E.C., P.D., R.M.M., Y.P. and G.M.; Writing P.D., G.M. and R.M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from Sapienza University of Rome, Progetti di Ricerca di Ateneo #RM120172B851A176 (P.D.) and the University of Bari, Progetti di Ricerca di Ateneo #00869718Ricat (R.M.M.).

Data Availability Statement

Data are contained within the article.

Acknowledgments

We thank Robert Levis for gift of KV lines; we also thank Sarah Elgin and Gunter Reuter for gift of PEV suppressors. Finally, we are grateful to the two reviewers for their help in improving the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Flavell, R.B. Repetitive DNA and chromosome evolution in plants. Philos. Trans. R. Soc. B Biol. Sci. 1986, 312, 227–242. [Google Scholar] [CrossRef]
  2. SanMiguel, P.; Tikhonov, A.; Jin, Y.-K.; Motchoulskaia, N.; Zakharov, D.; Melake-Berhan, A.; Springer, P.S.; Edwards, K.J.; Lee, M.; Avramova, Z.; et al. Nested Retrotransposons in the Intergenic Regions of the Maize Genome. Science 1996, 274, 765–768. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Kidwell, M.G.; Lisch, D. Transposable elements as sources of variation in animals and plants. Proc. Natl. Acad. Sci.USA 1997, 94, 7704–7711. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Kaminker, J.S.; Bergman, C.; Kronmiller, B.; Carlson, J.; Svirskas, R.; Patel, S.; Frise, E.; A Wheeler, D.; E Lewis, S.; Rubin, G.; et al. The transposable elements of the Drosophila melanogaster euchromatin: A genomics perspective. Genome Biol. 2002, 3, research0084.1. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Wells, J.N.; Feschotte, C. A Field Guide to Eukaryotic Transposable Elements. Annu. Rev. Genet. 2020, 54, 539–561. [Google Scholar] [CrossRef]
  6. Kapitonov, V.V.; Jurka, J. Rolling-circle transposons in eukaryotes. Proc. Natl. Acad. Sci. USA 2001, 98, 8714–8719. [Google Scholar] [CrossRef] [Green Version]
  7. Kapitonov, V.V.; Jurka, J. Self-synthesizing DNA transposons in eukaryotes. Proc. Natl. Acad. Sci. USA 2006, 103, 4540–4545. [Google Scholar] [CrossRef] [Green Version]
  8. Romano, N.C.; Fanti, L. Transposable Elements: Major Players in Shaping Genomic and Evolutionary Patterns. Cells 2022, 11, 1048. [Google Scholar] [CrossRef]
  9. Dimitri, P.; Corradini, N.; Rossi, F.; Mei, E.; Zhimulev, I.; Vernì, F. Transposable elements as artisans of the heterochromatic genome in Drosophila melanogaster. Cytogenet. Genome Res. 2005, 110, 165–172. [Google Scholar] [CrossRef]
  10. Marsano, R.M.; Dimitri, P. Constitutive Heterochromatin in Eukaryotic Genomes: A Mine of Transposable Elements. Cells 2022, 11, 761. [Google Scholar] [CrossRef]
  11. Marsano, R.M.; Giordano, E.; Messina, G.; Dimitri, P. A New Portrait of Constitutive Heterochromatin: Lessons from Drosophila melanogaster. Trends Genet. 2019, 35, 615–631. [Google Scholar] [CrossRef]
  12. A Hoskins, R.; Smith, C.D.; Carlson, J.W.; Carvalho, A.B.; Halpern, A.; Kaminker, J.S.; Kennedy, C.; Mungall, C.J.; A Sullivan, B.; Sutton, G.G.; et al. Heterochromatic sequences in a Drosophila whole-genome shotgun assembly. Genome Biol. 2002, 3, Rresearch0085.1. [Google Scholar] [CrossRef] [Green Version]
  13. Hoskins, R.A.; Carlson, J.W.; Kennedy, C.; Acevedo, D.; Evans-Holm, M.; Frise, E.; Wan, K.H.; Park, S.; Mendez-Lago, M.; Rossi, F.; et al. Sequence Finishing and Mapping of Drosophila melanogaster Heterochromatin. Science 2007, 316, 1625–1628. [Google Scholar] [CrossRef] [Green Version]
  14. Hoskins, R.A.; Carlson, J.W.; Wan, K.H.; Park, S.; Mendez, I.; Galle, S.E.; Booth, B.W.; Pfeiffer, B.D.; George, R.A.; Svirskas, R.; et al. The Release 6 reference sequence of the Drosophila melanogaster genome. Genome Res. 2015, 25, 445–458. [Google Scholar] [CrossRef] [Green Version]
  15. Kidwell, M.G.; Kidwell, J.F.; Sved, J.A. Hybrid Dysgenesis in Drosophila melanogaster: A Syndrome of Aberrant Traits Including Mutation, Sterility and Male Recombination. Genetics 1977, 86, 813–833. [Google Scholar] [CrossRef]
  16. Rubin, G.; Kidwell, M.G.; Bingham, P.M. The molecular basis of P-M hybrid dysgenesis: The nature of induced mutations. Cell 1982, 29, 987–994. [Google Scholar] [CrossRef]
  17. Sang, H.; Pélisson, A.; Bucheton, A.; Finnegan, D. Molecular lesions associated with white gene mutations induced by I-R hybrid dysgenesis in Drosophila melanogaster. EMBO J. 1984, 3, 3079–3085. [Google Scholar] [CrossRef] [Green Version]
  18. Bucheton, A. I transposable elements and I-R hybrid dysgenesis in Drosophila. Trends Genet. 1990, 6, 16–21. [Google Scholar] [CrossRef]
  19. Zhang, P.; Spradling, A.C. Insertional mutagenesis of Drosophila heterochromatin with single P elements. Proc. Natl. Acad. Sci. USA 1994, 91, 3539–3543. [Google Scholar] [CrossRef] [Green Version]
  20. Dimitri, P.; Arcà, B.; Berghella, L.; Mei, E. High genetic instability of heterochromatin after transposition of the LINE-like I factor in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 1997, 94, 8052–8057. [Google Scholar] [CrossRef]
  21. Konev, A.Y.; Yan, C.M.; Acevedo, D.; Kennedy, C.; Ward, E.; Lim, A.; Tickoo, S.; Karpen, G.H. Genetics of P-element transposition into Drosophila melanogaster centric heterochromatin. Genetics 2003, 165, 2039–2053. [Google Scholar] [CrossRef] [PubMed]
  22. Dimitri, P.; Bucheton, A. I element distribution in mitotic heterochromatin of Drosophila melanogaster reactive strains: Identification of a specific site which is correlated with the reactivity levels. Cytogenet. Genome Res. 2005, 110, 160–164. [Google Scholar] [CrossRef] [PubMed]
  23. Rubin, G.M.; Spradling, A.C. Genetic transformation of Drosophila with transposable element vectors. Science 1982, 218, 348–353. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Robertson, H.M.; Preston, C.R.; Phillis, R.W.; Johnson-Schlitz, D.M.; Benz, W.K.; Engels, W.R. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics 1988, 118, 461–470. [Google Scholar] [CrossRef] [PubMed]
  25. Cooley, L.; Kelley, R.; Spradling, A. Insertional Mutagenesis of the Drosophila Genome with Single P Elements. Science 1988, 239, 1121–1128. [Google Scholar] [CrossRef]
  26. Cooley, L.; Berg, C.; Kelley, R.; McKearin, D.; Spradling, A. Identifying and cloning Drosophila genes by single P element insertional mutagenesis. Prog. Nucleic Acid Res. Mol. Biol. 1989, 36, 99–109. [Google Scholar] [CrossRef]
  27. Spradling, A.C.; Stern, D.; Beaton, A.; Rhem, E.J.; Laverty, T.; Mozden, N.; Misra, S.; Rubin, G. The Berkeley Drosophila Genome Project Gene Disruption Project: Single P-Element Insertions Mutating 25% of Vital Drosophila Genes. Genetics 1999, 153, 135–177. [Google Scholar] [CrossRef]
  28. Zhai, R.G.; Hiesinger, P.R.; Koh, T.-W.; Verstreken, P.; Schulze, K.L.; Cao, Y.; Jafar-Nejad, H.; Norga, K.K.; Pan, H.; Bayat, V.; et al. Mapping Drosophila mutations with molecularly defined P element insertions. Proc. Natl. Acad. Sci. USA 2003, 100, 10860–10865. [Google Scholar] [CrossRef] [Green Version]
  29. O’Kane, C.J.; Gehring, W.J. Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. USA 1987, 84, 9123–9127. [Google Scholar] [CrossRef] [Green Version]
  30. Wilson, C.; Pearson, R.K.; Bellen, H.J.; O’Kane, C.J.; Grossniklaus, U.; Gehring, W.J. P-element-mediated enhancer detection: An efficient method for isolating and characterizing developmentally regulated genes in Drosophila. Genes Dev. 1989, 3, 1301–1313. [Google Scholar] [CrossRef]
  31. Hazelrigg, T.; Levis, R.; Rubin, G.M. Transformation of white locus DNA in Drosophila: Dosage compensation, zeste interaction, and position effects. Cell 1984, 36, 469–481. [Google Scholar] [CrossRef]
  32. Bellen, H.; Levis, R.; Liao, G.; He, Y.; Carlson, J.W.; Tsang, G.; Evans-Holm, M.; Hiesinger, P.R.; Schulze, K.L.; Rubin, G.; et al. The BDGP Gene Disruption Project. Genetics 2004, 167, 761–781. [Google Scholar] [CrossRef] [Green Version]
  33. Levis, R.; Hazelrigg, T.; Rubin, G.M. Effects of Genomic Position on the Expression of Transduced Copies of the white Gene of Drosophila. Science 1985, 229, 558–561. [Google Scholar] [CrossRef]
  34. Karpen, G.H.; Spradling, A.C. Analysis of subtelomeric heterochromatin in the Drosophila minichromosome Dp1187 by single P element insertional mutagenesis. Genetics 1992, 132, 737–753. [Google Scholar] [CrossRef]
  35. Roseman, R.; Pirrotta, V.; Geyer, P. The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J. 1993, 12, 435–442. [Google Scholar] [CrossRef]
  36. Elgin, S.C.; Reuter, G. Position-Effect Variegation, Heterochromatin Formation, and Gene Silencing in Drosophila. Cold Spring Harb. Perspect. Biol. 2013, 5, a017780. [Google Scholar] [CrossRef] [Green Version]
  37. Dimitri, P.; Pisano, C. Position effect variegation in Drosophila melanogaster: Relationship between suppression effect and the amount of Y chromosome. Genetics 1989, 122, 793–800. [Google Scholar] [CrossRef]
  38. Sun, F.-L.; Haynes, K.; Simpson, C.L.; Lee, S.D.; Collins, L.; Wuller, J.; Eissenberg, J.C.; Elgin, S.C.R. cis -Acting Determinants of Heterochromatin Formation on Drosophila melanogaster Chromosome Four. Mol. Cell. Biol. 2004, 24, 8210–8220. [Google Scholar] [CrossRef] [Green Version]
  39. Riddle, N.C.; Leung, W.; Haynes, K.A.; Granok, H.; Wuller, J.; Elgin, S.C.R. An Investigation of Heterochromatin Domains on the Fourth Chromosome of Drosophila melanogaster. Genetics 2008, 178, 1177–1191. [Google Scholar] [CrossRef] [Green Version]
  40. Wallrath, L.L.; Elgin, S.C. Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 1995, 9, 1263–1277. [Google Scholar] [CrossRef]
  41. Boivin, A.; Dura, J.-M. In Vivo Chromatin Accessibility Correlates with Gene Silencing in Drosophila. Genetics 1998, 150, 1539–1549. [Google Scholar] [CrossRef] [PubMed]
  42. Sun, F.-L.; Cuaycong, M.H.; Elgin, S.C.R. Long-Range Nucleosome Ordering Is Associated with Gene Silencing in Drosophila melanogaster Pericentric Heterochromatin. Mol. Cell. Biol. 2001, 21, 2867–2879. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Howe, M.; Dimitri, P.; Berloco, M.; Wakimoto, B.T. Cis-effects of heterochromatin on heterochromatic and euchromatic gene activity in Drosophila melanogaster. Genetics 1995, 140, 1033–1045. [Google Scholar] [CrossRef] [PubMed]
  44. Greil, F.; van der Kraan, I.; Delrow, J.; Smothers, J.F.; de Wit, E.; Bussemaker, H.J.; van Driel, R.; Henikoff, S.; van Steensel, B. Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 2003, 17, 2825–2838. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Yasuhara, J.C.; Wakimoto, B.T. Molecular landscape of modified histones in Drosophila heterochromatic genes and euchromatin-heterochromatin transition zones. PLoS Genet. 2008, 4, e16. [Google Scholar] [CrossRef] [Green Version]
  46. de Wit, E.; Greil, F.; van Steensel, B. Genome-wide HP1 binding in Drosophila: Developmental plasticity and genomic targeting signals. Genome Res. 2005, 15, 1265–1273. [Google Scholar] [CrossRef] [Green Version]
  47. De Wit, E.; Greil, F.; Van Steensel, B. High-Resolution Mapping Reveals Links of HP1 with Active and Inactive Chromatin Components. PLoS Genet. 2007, 3, e38. [Google Scholar] [CrossRef] [Green Version]
  48. Fanti, L.; Pimpinelli, S. HP1: A functionally multifaceted protein. Curr. Opin. Genet. Dev. 2008, 18, 169–174. [Google Scholar] [CrossRef]
  49. Riddle, N.C.; Minoda, A.; Kharchenko, P.V.; Alekseyenko, A.A.; Schwartz, Y.B.; Tolstorukov, M.Y.; Gorchakov, A.A.; Jaffe, J.D.; Kennedy, C.; Linder-Basso, D.; et al. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin. Genome Res. 2011, 21, 147–163. [Google Scholar] [CrossRef] [Green Version]
  50. Saha, P.; Sowpati, D.T.; Mishra, R.K. Epigenomic and genomic landscape of Drosophila melanogaster heterochromatic genes. Genomics 2019, 111, 177–185. [Google Scholar] [CrossRef]
  51. Saha, P.; Sowpati, D.T.; Soujanya, M.; Srivastava, I.; Mishra, R.K. Interplay of pericentromeric genome organization and chromatin landscape regulates the expression of Drosophila melanogaster heterochromatic genes. Epigenetics Chromatin 2020, 13, 41. [Google Scholar] [CrossRef]
  52. Wallrath, L.L.; Rodriguez-Tirado, F.; Geyer, P.K. Shining Light on the Dark Side of the Genome. Cells 2022, 11, 330. [Google Scholar] [CrossRef]
  53. Messina, G.; Damia, E.; Fanti, L.; Atterrato, M.T.; Celauro, E.; Mariotti, F.R.; Accardo, M.C.; Walther, M.; Verni, F.; Picchioni, D.; et al. Yeti, an essential Drosophila melanogaster gene, encodes a protein required for chromatin organization. J. Cell Sci. 2014, 127, 2577–2588. [Google Scholar] [CrossRef] [Green Version]
  54. Kharchenko, P.V.; Alekseyenko, A.A.; Schwartz, Y.B.; Minoda, A.; Riddle, N.C.; Ernst, J.; Sabo, P.J.; Larschan, E.; Gorchakov, A.A.; Gu, T.; et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 2011, 471, 480–485. [Google Scholar] [CrossRef] [Green Version]
  55. Ephrussi, B.; Herold, J.L. Studies of Eye Pigments of Drosophila. I. Methods of Extraction and Quantitative Estimation of the Pigment Components. Genetics 1944, 29, 148–175. [Google Scholar] [CrossRef]
  56. Prozzillo, Y.; Cuticone, S.; Ferreri, D.; Fattorini, G.; Messina, G.; Dimitri, P. In Vivo Silencing of Genes Coding for dTip60 Chromatin Remodeling Complex Subunits Affects Polytene Chromosome Organization and Proper Development in Drosophila melanogaster. Int. J. Mol. Sci. 2021, 22, 4525. [Google Scholar] [CrossRef]
  57. Hilliker, A.J. Genetic analysis of the centromeric heterochromatin of chromosome 2 of Drosophila melanogaster: Deficiency mapping of EMS-induced lethal complementation groups. Genetics 1976, 83, 765–782. [Google Scholar] [CrossRef]
  58. Dimitri, P. Cytogenetic analysis of the second chromosome heterochromatin of Drosophila melanogaster. Genetics 1991, 127, 553–564. [Google Scholar] [CrossRef]
  59. Corradini, N.; Rossi, F.; Verni, F.; Dimitri, P. FISH analysis of Drosophila melanogaster heterochromatin using BACs and P elements. Chromosoma 2003, 112, 26–37. [Google Scholar] [CrossRef]
  60. Rossi, F.; Moschetti, R.; Caizzi, R.; Corradini, N.; Dimitri, P. Cytogenetic and molecular characterization of heterochromatin gene models in Drosophila melanogaster. Genetics 2007, 175, 595–607. [Google Scholar] [CrossRef]
  61. Coulthard, A.B.; Alm, C.; Cealiac, I.; Sinclair, D.A.R.S.A.; Honda, B.M.H.M.; Rossi, F.; Dimitri, P.; Hilliker, A.J. Essential Loci in Centromeric Heterochromatin of Drosophila melanogaster. I: The Right Arm of Chromosome 2. Genetics 2010, 185, 479–495. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Roseman, R.R.; Johnson, E.A.; Rodesch, C.K.; Bjerke, M.; Nagoshi, R.N.; Geyer, P.K. A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster. Genetics 1995, 141, 1061–1074. [Google Scholar] [CrossRef]
  63. Yan, C.M.; Dobie, K.W.; Le, H.D.; Konev, A.Y.; Karpen, G.H. Efficient Recovery of Centric Heterochromatin P-Element Insertions in Drosophila melanogaster. Genetics 2002, 161, 217–229. [Google Scholar] [CrossRef] [PubMed]
  64. Hagstrom, K.; Muller, M.; Schedl, P. Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex. Genes Dev. 1996, 10, 3202–3215. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Andreyeva, E.N.; Kolesnikova, T.D.; Demakova, O.V.; Mendez-Lago, M.; Pokholkova, G.V.; Belyaeva, E.S.; Rossi, F.; Dimitri, P.; Villasante, A.; Zhimulev, I.F. High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants. Proc. Natl. Acad. Sci. USA 2007, 104, 12819–12824. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Pal-Bhadra, M.; Leibovitch, B.A.; Gandhi, S.G.; Rao, M.; Bhadra, U.; Birchler, J.A.; Elgin, S.C.R. Heterochromatic Silencing and HP1 Localization in Drosophila Are Dependent on the RNAi Machinery. Science 2004, 303, 669–672. [Google Scholar] [CrossRef] [Green Version]
  67. Nishida, K.M.; Saito, K.; Mori, T.; Kawamura, Y.; Nagami-Okada, T.; Inagaki, S.; Siomi, H.; Siomi, M.C. Gene silencing mechanisms mediated by Aubergine–piRNA complexes in Drosophila male gonad. RNA 2007, 13, 1911–1922. [Google Scholar] [CrossRef] [Green Version]
  68. Bannister, A.J.; Zegerman, P.; Partridge, J.F.; Miska, E.A.; Thomas, J.O.; Allshire, R.C.; Kouzarides, T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 2001, 410, 120–124. [Google Scholar] [CrossRef]
  69. Lachner, M.; O’Carroll, D.; Rea, S.; Mechtler, K.; Jenuwein, T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 2001, 410, 116–120. [Google Scholar] [CrossRef]
  70. Nakayama, J.-I.; Rice, J.C.; Strahl, B.D.; Allis, C.D.; Grewal, S.I.S. Role of Histone H3 Lysine 9 Methylation in Epigenetic Control of Heterochromatin Assembly. Science 2001, 292, 110–113. [Google Scholar] [CrossRef]
  71. Eberl, D.F.; Duyf, B.J.; Hilliker, A.J. The role of heterochromatin in the expression of a heterochromatic gene, the rolled locus of Drosophila melanogaster. Genetics 1993, 134, 277–292. [Google Scholar] [CrossRef]
  72. Hearn, M.G.; Hedrick, A.; Grigliatti, T.A.; Wakimoto, B.T. The effect of modifiers of position-effect variegation on the variegation of heterochromatic genes of Drosophila melanogaster. Genetics 1991, 128, 785–797. [Google Scholar] [CrossRef]
  73. Clegg, N.; Honda, B.; Whitehead, I.; Grigliatti, T.; Wakimoto, B.; Brock, H.; Lloyd, V.; Sinclair, D. Suppressors of position-effect variegation in Drosophila melanogaster affect expression of the heterochromatic gene light in the absence of a chromosome rearrangement. Genome 1998, 41, 495–503. [Google Scholar] [CrossRef]
  74. Lu, B.Y.; Emtage, P.C.R.; Duyf, B.J.; Hilliker, A.J.; Eissenberg, J.C. Heterochromatin Protein 1 Is Required for the Normal Expression of Two Heterochromatin Genes in Drosophila. Genetics 2000, 155, 699–708. [Google Scholar] [CrossRef]
  75. Schulze, S.R.; Sinclair, D.A.R.; Fitzpatrick, K.A.; Honda, B.M. A Genetic and Molecular Characterization of Two Proximal Heterochromatic Genes on Chromosome 3 of Drosophila melanogaster. Genetics 2005, 169, 2165–2177. [Google Scholar] [CrossRef] [Green Version]
  76. Prozzillo, Y.; Monache, F.D.; Ferreri, D.; Cuticone, S.; Dimitri, P.; Messina, G. The True Story of Yeti, the "Abominable" Heterochromatic Gene of Drosophila melanogaster. Front. Physiol. 2019, 10, 1093. [Google Scholar] [CrossRef] [Green Version]
  77. Messina, G.; Atterrato, M.T.; Fanti, L.; Giordano, E.; Dimitri, P. Expression of human Cfdp1 gene in Drosophila reveals new insights into the function of the evolutionarily conserved BCNT protein family. Sci. Rep. 2016, 6, 25511. [Google Scholar] [CrossRef]
  78. Messina, G.; Celauro, E.; Atterrato, M.T.; Giordano, E.; Iwashita, S.; Dimitri, P. The Bucentaur (BCNT) protein family: A long-neglected class of essential proteins required for chromatin/chromosome organization and function. Chromosoma 2014, 124, 153–162. [Google Scholar] [CrossRef]
  79. Messina, G.; Prozzillo, Y.; Bizzochi, G.; Marsano, R.M.; Dimitri, P. The Green Valley of Drosophila melanogaster Constitutive Heterochromatin: Protein-Coding Genes Involved in Cell Division Control. Cells 2022, 11, 3058. [Google Scholar] [CrossRef]
  80. Haynes, K.A.; Gracheva, E.; Elgin, S.C.R. A Distinct Type of Heterochromatin Within Drosophila melanogaster Chromosome 4. Genetics 2007, 175, 1539–1542. [Google Scholar] [CrossRef]
  81. Skalska, L.; Beltran-Nebot, M.; Ule, J.; Jenner, R. Regulatory feedback from nascent RNA to chromatin and transcription. Nat. Rev. Mol. Cell Biol. 2017, 18, 331–337. [Google Scholar] [CrossRef] [PubMed]
Genes 14 00012 g001 550
Figure 1. Scheme of genetic crosses. (A) Crosses with PEV suppressor mapping to chromosome 2 (Su(var)2052 and aubergneQC4); (B) Crosses with PEV suppressor mapping to chromosome 3 (Su(var)3071 and Su(var)3-91). For a detailed explanation of the crosses see Material and Methods.
Figure 1. Scheme of genetic crosses. (A) Crosses with PEV suppressor mapping to chromosome 2 (Su(var)2052 and aubergneQC4); (B) Crosses with PEV suppressor mapping to chromosome 3 (Su(var)3071 and Su(var)3-91). For a detailed explanation of the crosses see Material and Methods.
Genes 14 00012 g001
Genes 14 00012 g002 550
Figure 2. Strong silencing of Pw+ reporters. Phenotypic analysis of the 12 lines carrying a single Pw+ reporter inserted in constitutive heterochromatin of chromosome 2. All the Pw+ reporters are balanced over the SM1, Cy chromosome and exhibited a strong silencing giving rises to extremely variegated eyes which in some cases are almost white. The males of lines KV00462, KV00524, 19.74.3 KV00158 and KV00384 exhibited a stronger red-eye pigmentation compared to that of females, a difference that could be due to the presence of Y chromosome.
Figure 2. Strong silencing of Pw+ reporters. Phenotypic analysis of the 12 lines carrying a single Pw+ reporter inserted in constitutive heterochromatin of chromosome 2. All the Pw+ reporters are balanced over the SM1, Cy chromosome and exhibited a strong silencing giving rises to extremely variegated eyes which in some cases are almost white. The males of lines KV00462, KV00524, 19.74.3 KV00158 and KV00384 exhibited a stronger red-eye pigmentation compared to that of females, a difference that could be due to the presence of Y chromosome.
Genes 14 00012 g002
Genes 14 00012 g003 550
Figure 3. Heatmaps showing expression profile of the heterochromatic genes linked to the examined Pw+ reporters compared to that of white gene. Expression across developmental stages (A) and in the eyes of males and females (B). Shades of color from red to green indicate the expression bin classification from 1 (no/extremely low expression) to 7 (very high expression). The gray color means that no data about CR43266 expression are available. Developmental stage and eye expression data were obtained from FlyBase and FlyAtlas, respectively.
Figure 3. Heatmaps showing expression profile of the heterochromatic genes linked to the examined Pw+ reporters compared to that of white gene. Expression across developmental stages (A) and in the eyes of males and females (B). Shades of color from red to green indicate the expression bin classification from 1 (no/extremely low expression) to 7 (very high expression). The gray color means that no data about CR43266 expression are available. Developmental stage and eye expression data were obtained from FlyBase and FlyAtlas, respectively.
Genes 14 00012 g003
Genes 14 00012 g004 550
Figure 4. Effects of dominance of Su(var)2052, Su(var)3-71 and aubergineQC4 on the silencing of Pw+ reporters. (A) Histograms summarizing the quantification of eye pigment in presence and absence of PEV suppressors; (B) Examples of eye phenotypes of Pw+ reporters in flies with or without suppressors. The silencing of Pw+ reporter in 19.74.3 line was efficiently suppressed by Su(var)2052, Su(var)3-71 and aubergineQC4; silencing in KV00363 and LP1 lines was unaffected by Su(var)2052 and aubergineQC4, while silencing in LP1 was only partially suppressed by Su(var)3-71.
Figure 4. Effects of dominance of Su(var)2052, Su(var)3-71 and aubergineQC4 on the silencing of Pw+ reporters. (A) Histograms summarizing the quantification of eye pigment in presence and absence of PEV suppressors; (B) Examples of eye phenotypes of Pw+ reporters in flies with or without suppressors. The silencing of Pw+ reporter in 19.74.3 line was efficiently suppressed by Su(var)2052, Su(var)3-71 and aubergineQC4; silencing in KV00363 and LP1 lines was unaffected by Su(var)2052 and aubergineQC4, while silencing in LP1 was only partially suppressed by Su(var)3-71.
Genes 14 00012 g004
Genes 14 00012 g005 550
Figure 5. Effects of Su(var)3-91 on silencing of Pw+ reporters. (A) Examples of eye phenotypes of Pw+ reporters in flies with or without Su(var)3-91. (B) Histograms summarizing the quantification of eye pigment in presence and absence of Su(var)3-91. The silencing of the Pw+ reporter in 19.74.3 was efficiently suppressed, while that of KV00363, LP1, KV00299 and KV00249 was unaffected. For the scheme of crosses see Figure 1 and Materials and Methods.
Figure 5. Effects of Su(var)3-91 on silencing of Pw+ reporters. (A) Examples of eye phenotypes of Pw+ reporters in flies with or without Su(var)3-91. (B) Histograms summarizing the quantification of eye pigment in presence and absence of Su(var)3-91. The silencing of the Pw+ reporter in 19.74.3 was efficiently suppressed, while that of KV00363, LP1, KV00299 and KV00249 was unaffected. For the scheme of crosses see Figure 1 and Materials and Methods.
Genes 14 00012 g005
Genes 14 00012 g006 550
Figure 6. Genomic localization of the Pw+ reporters under investigation. (A) Cytogenetic map of the heterochromatin of chromosome 2 showing the location of the genomic regions analyzed in this work. From left to right: 2L:h35, 2R:h40-41, 2R: h44, 2R:h45-46, 2R:h46. The genomic regions in (B–F) show the localization of the Pw+ reporter insertions relative to genes and the distribution of TEs and repetitive sequences. Red triangle = fully suppressed Pw+ reporter; dashed triangle = Pw+ reporter suppressed only in some cases; white triangle = Pw+ reporter insensitive to suppression; grey triangle = Pw+ reporter not tested for suppression.
Figure 6. Genomic localization of the Pw+ reporters under investigation. (A) Cytogenetic map of the heterochromatin of chromosome 2 showing the location of the genomic regions analyzed in this work. From left to right: 2L:h35, 2R:h40-41, 2R: h44, 2R:h45-46, 2R:h46. The genomic regions in (B–F) show the localization of the Pw+ reporter insertions relative to genes and the distribution of TEs and repetitive sequences. Red triangle = fully suppressed Pw+ reporter; dashed triangle = Pw+ reporter suppressed only in some cases; white triangle = Pw+ reporter insensitive to suppression; grey triangle = Pw+ reporter not tested for suppression.
Genes 14 00012 g006
Genes 14 00012 g007 550
Figure 7. Pw+ silencing by perturbation of a positive RNA feedback mechanism. The insertion of a Pw+ reporter within the Yeti gene affects its expression. According to a positive RNA feedback model, the consequent lack of functional Yeti transcripts would induce the formation of a closed chromatin structure of the gene itself. As a result, the mini-white gene is silenced. Colored circles represent different chromatin marks.
Figure 7. Pw+ silencing by perturbation of a positive RNA feedback mechanism. The insertion of a Pw+ reporter within the Yeti gene affects its expression. According to a positive RNA feedback model, the consequent lack of functional Yeti transcripts would induce the formation of a closed chromatin structure of the gene itself. As a result, the mini-white gene is silenced. Colored circles represent different chromatin marks.
Genes 14 00012 g007
Table
Table 1. Features of the insertion lines described in this work.
Table 1. Features of the insertion lines described in this work.
LineP-ElementP-Element
Position		Affected Gene(s)Gene PositionInsert Position
Relative to Nearby Genes		Sensitivity to SuppressorsChromatin State
(Gene)		Chromatin State
(Insertion Site)		Natural TE
Insertions
(+/− 5 kb)
KV00462P{SUPor-P}2L:22839699 [−]lnRNA:CR43266
CG17715		2L:22840517..22843668 [−]
2L:22844220..22854541 [+]		818 bp downstream
4521 bp upstream		Y1
1,2,7		71360
KV00524P{SUPor-P}2R:1260501 [+]CG401912R:1264140..1267747 [+]3639 bp upstreamY/N1,771360
19.74.3P{Fab7-w#un1}2R:1341511 [+]Yeti (CG40218)2R:1343403..1345119 [−]1892 bp upstreamY1NRND
LP1P{Fab7-w#un1}2R:1345084 [+]Yeti (CG40218)2R:1343403..1345119 [−]Start codonN11
KV00363P{SUPor-P}2R:1345097 [+]Yeti (CG40218)2R:1343403..1345119 [−]5′UTRN11
KV00158P{SUPor-P}2R:1358001 [−]l(2)41Ab (CG41265)2R:1349392..1443935 [+]1st intronY/NNRNRinvader1
1360 (3 copies)
KV00299P{SUPor-P}2R:1366708 [+]l(2)41Ab
(CG41265)		2R:1349392..1443935 [+]1st intronNNRNR
KV00249P{SUPor-P}2R:1370175 [+]l(2)41Ab
(CG41265)
lnRNA:CR44043		2R:1349392..1443935 [+]
2R:1368921..1371468 [−]		2nd intron
Gene body		NNR
ND		NR
KV00376P{SUPor-P}2R:1412310 [+]l(2)41Ab
(CG41265)		2R:1349392..1443935 [+]2nd intronY/NNRNR
KV00171P{SUPor-P}2R:3147095 [−]dpr21
(CG42596)		2R:3066499..3191011 [+]Intron 5Y/NNDNDGypsy4
1360
KV00384P{SUPor-P}2R: 4014047 [+]Haspin
(CG40080)		2R:4014111..4049342 [+]64 bp upstreamY1NRF
297
KV00369P{SUPor-P}2R:4593333 [−]p120ctn
(CG17484)		2R:4595288..4609492 [+]1955 bp upstreamY1,2,77Doc3 (2 copies)
Doc
1360 (5 copies)
INE-1 (5 copies)
Diver (2 copies)
Cr1a
Burdock
Gypsy12
Genomic positions are relative to the dm6 genome assembly. [+] and [−] indicate the orientation of genes and inserts relative to the genome assembly. The chromatin state detected in the gene body and at the P-element insertion site are described in Kharchenko, et al. [54] (chromatin state 1: Active promoter/transcription start site region; chromatin state 2: Actively transcribed exon; chromatin state 7: Heterochromatin). 2L: Heterochromatin of the left arm of chromosome 2; 2R: Heterochromatin of the right arm of chromosome 2. NR: Not Reported. ND: None Detected. Y: Suppressed; N: Not Suppressed.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Messina, G.; Celauro, E.; Marsano, R.M.; Prozzillo, Y.; Dimitri, P. Epigenetic Silencing of P-Element Reporter Genes Induced by Transcriptionally Active Domains of Constitutive Heterochromatin in Drosophila melanogaster. Genes 2023, 14, 12. https://doi.org/10.3390/genes14010012

AMA Style

Messina G, Celauro E, Marsano RM, Prozzillo Y, Dimitri P. Epigenetic Silencing of P-Element Reporter Genes Induced by Transcriptionally Active Domains of Constitutive Heterochromatin in Drosophila melanogaster. Genes. 2023; 14(1):12. https://doi.org/10.3390/genes14010012

Chicago/Turabian Style

Messina, Giovanni, Emanuele Celauro, Renè Massimiliano Marsano, Yuri Prozzillo, and Patrizio Dimitri. 2023. "Epigenetic Silencing of P-Element Reporter Genes Induced by Transcriptionally Active Domains of Constitutive Heterochromatin in Drosophila melanogaster" Genes 14, no. 1: 12. https://doi.org/10.3390/genes14010012

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop