Download Recurrent Gene Duplication Diversifies Genome

Recurrent Gene Duplication Diversifies Genome Defense
Repertoire in Drosophila
Mia T. Levine,†,1 Helen M. Vander Wende,‡,1 Emily Hsieh,§,1 Emily-Clare P. Baker,¶,1 and Harmit S. Malik1,2,*
1
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA
Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA
†
Present address: Department of Biology, University of Pennsylvania, Philadelphia, PA
‡
Present address: Department of Molecular and Cellular Biology, University of California Berkeley, CA
§
Present address: Department of Microbiology, Mt. Sinai School of Medicine, New York
¶
Present address: Hittinger Lab, Department of Genetics, University of Wisconsin-Madison, WI
*Corresponding author: E-mail: [email protected]
Associate editor: Nadia Singh
2
Abstract
Transposable elements (TEs) comprise large fractions of many eukaryotic genomes and imperil host genome integrity.
The host genome combats these challenges by encoding proteins that silence TE activity. Both the introduction of new
TEs via horizontal transfer and TE sequence evolution requires constant innovation of host-encoded TE silencing machinery to keep pace with TEs. One form of host innovation is the adaptation of existing, single-copy host genes. Indeed,
host suppressors of TE replication often harbor signatures of positive selection. Such signatures are especially evident in
genes encoding the piwi-interacting-RNA pathway of gene silencing, for example, the female germline-restricted TE
silencer, HP1D/Rhino. Host genomes can also innovate via gene duplication and divergence. However, the importance of
gene family expansions, contractions, and gene turnover to host genome defense has been largely unexplored. Here, we
functionally characterize Oxpecker, a young, tandem duplicate gene of HP1D/rhino. We demonstrate that Oxpecker
supports female fertility in Drosophila melanogaster and silences several TE families that are incompletely silenced by
HP1D/Rhino in the female germline. We further show that, like Oxpecker, at least ten additional, structurally diverse,
HP1D/rhino-derived daughter and “granddaughter” genes emerged during a short 15-million year period of Drosophila
evolution. These young paralogs are transcribed primarily in germline tissues, where the genetic conflict between host
genomes and TEs plays out. Our findings suggest that gene family expansion is an underappreciated yet potent evolutionary mechanism of genome defense diversification.
Key words: gene duplication, heterochromatin, genome defense, transposable elements, HP1 proteins.
pathogen interaction, driving rapid innovation on the part of
both TEs as well as host machinery to curb TE proliferation.
The fate of individual TE copies and their evolutionary
impact on host–TE genetic conflict depends on their site of
insertion in host genomes. For instance, TE insertions into the
gene-rich euchromatic genome compartment have a higher
likelihood of disrupting host genes either directly or by ectopic exchange. Moreover, elevated recombination in the euchromatic compartment allows natural selection to become
more efficient at removing even slightly deleterious TE insertions from the population (Charlesworth and Langley 1989).
In contrast, TE insertions into the gene-poor heterochromatic
compartment enjoy a lower likelihood of disrupting genes
and so have higher likelihood of persistence. These forces all
contribute to TE accumulation in heterochromatin
(Charlesworth et al. 1994; Dimitri and Junakovic 1999;
Blumenstiel et al. 2002; Hoskins et al. 2002; Maside et al. 2005).
Heterochromatin, however, is not exclusively a refuge for
TEs. Instead, heterochromatin can be a potent source of TE
silencing by virtue of its abundant repressive histone marks
ß The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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Mol. Biol. Evol. 33(7):1641–1653 doi:10.1093/molbev/msw053 Advance Access publication March 14, 2016
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Fast Track
Active and inactive copies of transposable elements (TEs)
riddle eukaryotic genomes. TE copies make up 50% of
the human genome (Lander et al. 2001; de Koning et al.
2011) and up to 85% of others (Schnable et al. 2009). TE
activity imperils host fitness in many ways, for example, by
interrupting exons, perturbing transcription, inducing alternative splicing, nucleating heterochromatin, or leading to ectopic recombination (Montgomery et al. 1987; Langley et al.
1988; Kazazian 2004; Slotkin and Martienssen 2007; Lee 2015).
Despite imposing a fitness cost to the host, TE copy number
can increase over evolutionary time if transposition occurs in
the germline (Doolittle and Sapienza 1980; Orgel and Crick
1980). Here, TE copies have direct access to the next generation. On rare occasions, these germline insertion events are
adaptive (Daborn et al. 2002; Schlenke and Begun 2004;
Aminetzach et al. 2005; Feschotte 2008; Gonzalez et al.
2008; Guio et al. 2014; Kapusta and Feschotte 2014; Mateo
et al. 2014). Most often, TE replication results in a fitness cost
to the host. As a result, this relationship is akin to a host–
Article
Introduction
Levine et al. . doi:10.1093/molbev/msw053
and the specialized proteins that propagate them (Elgin and
Grewal 2003). Indeed, TEs embedded in constitutive heterochromatin near centromeres and telomeres are enriched for
histone H3K9 di- and tri-methyl, marks diagnostic of chromatin inaccessible to transcriptional machinery (Filion et al.
2010; Ho et al. 2014). Furthermore, a heterochromatic insertion can have deleterious consequences on the fitness of the
TE family (Grewal and Elgin 2007). For instance, host genomes
can utilize the accumulation of some TEs in heterochromatin
to suppress TE activity. In Drosophila and mice, clusters of
degenerate TE “graveyards” and active transposons can seed
the germline-specialized piRNA (“piwi-interacting-RNA”)
pathway, which targets active TEs transcribed from these
loci (Aravin et al. 2007; Brennecke et al. 2007; Zamore
2007). These “piRNA clusters” are concentrated at euchromatin–heterochromatin boundaries (Yamanaka et al. 2014)
and produce piRNAs through an elaborate series of steps,
which have been reviewed extensively (Iwasaki et al. 2015;
Czech and Hannon 2016; Hirakata and Siomi 2016) and are
briefly summarized below.
In Drosophila melanogaster, most primary piRNAs are generated from precursor transcripts produced from “dualstrand clusters”, in which piRNA precursors are transcribed
off both genomic strands (“uni-strand” piRNA clusters also
exist in Drosophila and produce piRNA precursor transcripts
via a distinct mechanism) (Brennecke et al. 2007). Dual-strand
clusters are epigenetically marked by the H3K9me3 epigenetic
modification (Le Thomas et al. 2014) and recruit HP1D/
Rhino, a female germline-specific HP1 paralog (Klattenhoff
et al. 2009). HP1D/Rhino recruits the Cutoff protein via a
linker protein Deadlock; together, these three proteins mediate transcription, block splicing, inhibit premature transcription termination and degradation of precursor transcripts
and license them for further processing in cytoplasmic processing sites (Klattenhoff et al. 2009; Pane et al. 2011; Mohn
et al. 2014; Sapetschnig and Miska 2014; Zhang et al. 2014).
These piRNA precursors are then exported to the perinuclear
cytoplasm (nuage), where they are cleaved into piRNA intermediates on mitochondrial surfaces and loaded onto PIWI
proteins (Olivieri et al. 2010; Saito et al. 2010; Qi et al. 2011;
Zhang et al. 2012; Murota et al. 2014; Han et al. 2015; Mohn
et al. 2015), followed by trimming and methylation to produce mature PIWI–piRNA complexes. PIWI–piRNA complexes can directly mediate transposon silencing by
heterochromatin nucleation (Klenov et al. 2011; Wang and
Elgin 2011; Sienski et al. 2012; Huang et al. 2013; Le Thomas
et al. 2013). Alternately, Aubergine–piRNA complexes can
enter a feed–forward cycle of amplification to produce secondary piRNAs via the “ping-pong” pathway (Brennecke et al.
2007).
Host silencing mechanisms provide protection against TE
proliferation and yet, evolutionary innovation of TEs recurrently evade this host machinery. Not only does the coding
sequence across TEs within the same family change over time,
but also entirely new TE families can frequently invade naı̈ve
genomes by horizontal transfer (Daniels et al. 1990; Jordan
et al. 1999; Sanchez-Gracia et al. 2005; Bartolome et al. 2009;
Gilbert et al. 2010; Lerat et al. 2011; Kofler et al. 2015).
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Closely related species can, therefore, encode non-overlapping TE families despite recent speciation. TE evasion of
host machinery puts pressure back on the host to keep
pace with TE innovation, and even to evolve novel recognition and silencing mechanisms. Consistent with this idea,
several recent analyses have shown pervasive signatures of
positive selection at host genome defense factors, including
multiple genes that encode piRNA pathway factors (Obbard
et al. 2006, 2011; Kolaczkowski et al. 2011; Bernhardt et al.
2012; Kelleher et al. 2012; Lee and Langley 2012; Simkin et al.
2013). Adaptive evolution at a subset of these genes may be
driven by antagonism with infectious viruses as has been seen
for components of the siRNA production machinery in insects (Marques and Carthew 2007; Obbard et al. 2009; Saleh
et al. 2009; Nayak et al. 2010, 2013; Murray et al. 2013;;
Schnettler et al. 2014; van Mierlo et al. 2014). Even if this
were the case, several rapidly evolving host defense genes
target TEs exclusively (Vermaak et al. 2005; Maheshwari
et al. 2008; Satyaki et al. 2014), supporting the idea that TE
innovation contributes to the directional selection of host
defense factors.
Although directional selection of single-copy host gene
orthologs is a potent mechanism of adaptation against TEs,
it is not the only possible source of evolutionary innovation.
Gene family expansion and turnover can also generate adaptive molecular novelty. Indeed, host genes involved in viral
restriction show abundant evidence of both diversifying selection as well as gene turnover. Recurrent turnover of genes
encoding antimicrobial peptides in Drosophila (Sackton et al.
2007; Lazzaro 2008), or TRIM and APOBEC proteins in mammals (Sawyer et al. 2007; Tareen et al. 2009; Munk et al. 2012)
exemplify frequent lineage-specific expansions of host immune factors. One rare example of gene expansion that
amplifies host defense repertoires against TEs comes from
studies of the KRAB–ZNF family in mammals (Nowick
et al. 2010; Thomas and Schneider 2011). Many of these proteins act as DNA-binding “adaptors” that target heterochromatin silencing machinery to individual TE copies (Wolf and
Goff 2009; Jacobs et al. 2014). The pace of innovation of
KRAB–ZNF proteins parallels the novelty of endogenous retroviruses in mammals (Thomas and Schneider 2011). This
dynamic suggests a “tit-for-tat” cycle of gene duplication
and adaptation to specifically repress newly originated TEs
in mammalian genomes via epigenetic silencing (Jacobs et al.
2014). KRAB–ZNFs thus represent a powerful example of
host gene innovation that is, at least in some cases, unambiguously driven by TE changes.
KRAB–ZNFs are found exclusively in mammals. We expect, however, that host gene family expansions that combat
TE innovation are a common feature of eukaryotic genomes.
We specifically investigated the hypothesis that gene family
expansion is a potent evolutionary mechanism shaping host
genome defense repertoires in Drosophila. We took advantage of the identification of a bona fide TE silencer—
Heterochromatin Protein 1D (HP1D)/Rhino, which supports
piRNA cluster transcription in D. melanogaster ovaries
(Klattenhoff et al. 2009). HP1D/Rhino is a critical component
of TE silencing in Drosophila females; mutant females are
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Recurrent Gene Duplication of HP1D/Rhino in Drosophila Evolution . doi:10.1093/molbev/msw053
Results
Oxpecker Is a Recent Gene Duplicate of HP1D/Rhino
Like all canonical HP1 proteins, HP1D/Rhino has a tripartite
structure, consisting of three domains (fig. 1A) (Canzio et al.
2014; Eissenberg and Elgin 2014). The N-terminal chromodomain typically recognizes histone modifications associated
with silencing, for example, di- and tri-methyl H3K9 (Jacobs
and Khorasanizadeh 2002; Nielsen et al. 2002). The middle
hinge domain interacts with DNA and RNA (Meehan et al.
2003; Badugu et al. 2005), whereas the C-terminal chromoshadow self-dimerizes and interacts with other chromosomal
proteins (Smothers and Henikoff 2000; Canzio et al. 2014). We
previously carried out a phylogenomic analysis of all
Drosophila HP1 proteins based on 12 published Drosophila
species’ genome sequences (Levine et al. 2012). In this analysis,
we found that the Oxpecker (“Oxp” from now on) gene was
born via gene duplication from HP1D within the last 15 million years (fig. 1A). Like HP1D, Oxp is primarily expressed in
D. melanogaster ovaries. We ruled out the possibility of an
Oxp-HP1D fusion gene in D. melanogaster by RT-PCR analyses
(supplementary fig. S1, Supplementary Material online). Oxp
encodes a protein that contains only a chromodomain in
D. melanogaster and other Drosophila genomes. In D. melanogaster, the Oxp and HP1D/Rhino chromodomains share
62% amino acid identity (supplementary fig. S2,
Supplementary Material online). We hypothesize that the
HP1D gene duplication event likely only spanned the chromodomain. However, it is also possible that a full-length
~10-15 mya
A
Oxp
HP1D/Rhino
chromo
(Oxpecker)
B
chromo
hinge
(HP1D/Rhino)
shadow
# progeny (lifetime)
400
**
300
200
100
0
mother’s
genotype:
Oxp+
Oxp1
precise excision
of P element
imprecise excision
of P element
( )
P-element
C
# progeny
completely sterile (Volpe et al. 2001; Klattenhoff et al. 2009).
De-repressed TEs wreak havoc on the female germline and
the consequent loss of genome integrity results in embryo
inviability (Klattenhoff et al. 2009). We previously showed
that HP1D/Rhino evolves under strong positive selection, consistent with its role in a genetic conflict with TEs (Vermaak
et al. 2005). We also previously discovered a few partial or
complete HP1D/Rhino paralogs in a phylogenomic survey of
the HP1 gene family in Drosophila (Levine et al. 2012; Levine
and Malik 2013). We predict that this recurrent HP1D/Rhino
gene duplication diversifies host defense.
To test this hypothesis, we investigated the function of one
of these HP1D paralogs, Oxpecker (Oxp). Oxp is an evolutionarily young gene (10–15 million-years-old) in Drosophila.
We show that Oxpecker mutant females have impaired fertility
despite being wildtype for HP1D/Rhino. We also find that Oxp
silences many TE families in a wildtype HP1D/Rhino genetic
background. Using the newly available genome sequences in
the melanogaster species group, we further discovered that
the 15 million-year-old Oxpecker gene is one of many HP1D/
Rhino-like paralogs to arise during this short snapshot of
Drosophila evolution. We find evidence for at least ten independent gene duplication/divergence events. All assayed
paralogs are expressed primarily in the germline where TEs
can successfully proliferate. This combination of functional
and phylogenomic analyses of the HP1D/Rhino gene family
in Drosophila implicates recurrent gene duplication as a frequent source of novelty in host genome defense repertoires.
150
100
**
50
0
mother’s Oxp1/CyO
genotype:
Oxp1/CyO Oxp1/Oxp1 Oxp1/Oxp1
+
+
Oxp transgene
Oxp transgene
FIG. 1. Oxpecker plays a role in female fertility. (A) Oxp is a chromodomain-only containing gene that is found immediately upstream of
HP1D in the D. melanogaster genome. It derived from a HP1D/Rhino
gene duplication 10–15 million years ago. (B) Isogenic females that
possess an intact Oxp (Oxpþ) or not (Oxp1) were crossed to wildtype
males in egg-exhaustion experiments (see “Methods” section) that
measured total progeny produced in their lifetime that developed to
adulthood. Oxp deletion leads to a 20–25% drop in total progeny
produced (“**” P < 0.05). (C) The effect of Oxp deletion on female
fertility can be restored by an Oxp transgene. Compared with Oxp1/
Oxp1 female flies that exhibit compromised fertility, isogenic flies that
express an Oxp transgene produce wildtype-like number of progeny.
HP1D/Rhino gene duplication was followed by the rapid degeneration of the hinge and chromoshadow domain in Oxp.
We previously showed that HP1D/Rhino evolves under
positive selection, which implicated its role in defense against
TEs (Vermaak et al. 2005). This hypothesis was subsequently
confirmed by functional studies that demonstrated HP1D/
Rhino’s critical role in TE silencing via the piRNA pathway
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Levine et al. . doi:10.1093/molbev/msw053
A
fold change in expression
Oxp1/Oxp1
+ Oxp transgene
2
3
B
45
Oxp1/Oxp1 0
1
gypsy12
25
gypsy12
1731
23
19
17
15
13
-log10 pval
21
1731
de-repressed in
Oxp1/Oxp1
412
412
11
9
TART
7
5
fdr<0.01
3
TARHE
unaffected in
Oxp1/Oxp1
1
-3
-2
-1
0
1
TART
log2 fold change TARHE
2
3
FIG. 2. Oxpecker deletion results in the de-repression of retrotransposons in the female germline. (A) RNA-seq comparison between transcriptomes of Oxpþ/Oxpþ and Oxp1/Oxp1 ovaries reveal selected TEs (e.g., gypsy12, 1731, and 412 retrotransposons) that are upregulated upon Oxp
deletion. However, the bulk of TEs are not affected by Oxp deletion, including the TART and TAHRE retrotransposons that are highly upregulated
in HP1D homozygous mutant ovaries. (B) qPCR analyses of ovary-derived cDNA from either Oxp1/Oxp1 flies or Oxp1/Oxp1 flies with a “rescue” Oxp
transgene confirms that TE overexpression (gypsy12, 1731, 412) in the imprecise deletion ovaries can be attributed to the loss of Oxp expression
(“**” P < 0.05).
(Klattenhoff et al. 2009). We speculated that Oxp might participate in the same process as its parent gene HP1D/Rhino,
that is, in genome defense. However, unlike its parent gene,
we previously found that Oxp evolves under purifying selection (Levine et al. 2012). This dichotomy suggested an alternate possibility that Oxp could carry out a distinct function
from HP1D, that is, a function unrelated to genome defense.
Oxp Supports Female Fertility and Silences
Retrotransposons
To investigate its function, we characterized the biological
consequences of creating a genetic lesion at the Oxp locus
in a wildtype HP1D background. We mobilized a P-element
near the Oxp start codon (see “Methods” section). This
P-element mobilization gave rise to two types of strains.
The first of these are “precise excision” stocks, in which the
Oxp genomic sequence reverts to its intact form, leading to
wildtype Oxp allele. The P-element mobilization also generated “imprecise deletion” stocks, in which a segment of the
Oxp 50 UTR and promoter sequence was deleted (fig. 1B). For
subsequent analysis, we selected one “precise excision” control line and one “imprecise deletion” experimental line. We
confirmed that the imprecise deletion removed 510 bp spanning the 50 UTR and upstream sequence; using RT-PCR analyses, we confirmed this perturbed Oxp but not the HP1D/
Rhino locus (supplementary fig. S3, Supplementary Material
online). Using the precise deletion as our wildtype control
(“Oxpþ”), we confirmed that Oxp expression was virtually
undetectable in the ovaries of the imprecise deletion
(“Oxp1”) (supplementary fig. S3, Supplementary Material online). In contrast, there was no significant effect on transcription of the downstream parent gene, HP1D/Rhino
(supplementary fig. S3, Supplementary Material online).
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Thus, our P-element mobilization strategy generated two
near isogenic chromosomes that are both wildtype at the
HP1D/Rhino locus but differ in their expression of the Oxp
gene.
We next tested the consequences of this deletion on female fertility. We crossed Oxpþ/Oxpþ or Oxp1/Oxp1 females
to wildtype males (strain w1118) and counted the total progeny generated over their lifetime. We found that Oxp1/Oxp1
females generated 25% less adult progeny than the otherwise isogenic Oxpþ/Oxpþ females (P < 0.02, fig. 1B). We also
tested females homozygous or heterozygous for the imprecise
excision (Oxp1/Oxp1 and Oxp1/CyO, respectively; CyO is a balancer second chromosome in D. melanogaster) crossed to
wildtype males, counting all the adult progeny produced
from eggs oviposited in a 3-day window. Again, we found a
25% reduction in progeny (P < 0.02, fig. 1C) for Oxp1/Oxp1
mothers. In contrast, Oxp1/CyO showed wildtype levels of
progeny. Despite the reduction in fertility, we did not observe
the characteristic “fused appendage” defects that are observed in eggs deposited by HP1D/Rhino homozygous females that led to its name (Volpe et al. 2001). We
introduced an Oxp transgene driven by the native promoter
from an ectopic location. This transgene restored Oxp expression partially in the Oxp1/Oxp1 strain (“rescue genotype”, sup
plementary fig. S4, Supplementary Material online). We found
that this partial restoration of Oxp expression rescued fertility
to wildtype levels (fig. 1C). These data demonstrate that Oxp
function is non-redundant from HP1D/Rhino in the female
germline.
To investigate the basis of Oxp’s fertility function, we first
considered the well-characterized role of HP1D/Rhino in female fertility (Klattenhoff et al. 2009). HP1D/Rhino is a potent
silencer of retrotransposons in the female germline. Indeed,
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Recurrent Gene Duplication of HP1D/Rhino in Drosophila Evolution . doi:10.1093/molbev/msw053
<15 million
year old ancestor
A
B
D. melanogaster
D. simulans
D. sechellia
D. yakuba
D. erecta
D. eugracilis
D. biarmipes
D. takahashii
D. ficusphila
D. elegans
D. rhopaloa
D. kikkawai
D. bipectinata
D. ananassae
HP1D
HP1D2
HP1D2.2
HP1D4
HP1D5
HP1D6
HP1D7
Oxp
Oxp2
Oxp3
HP1D3csd
HP1D8csd
2R:542D
X:7F5
X:7F5
mel,sim,sec,yak,ere,ele,
bia,eug,tak,fic,bip,ana
sim,sec, yak,rho,bia,tak,
eug fic
bia*,tak
(*double chromodomain)
X:16F7
tak
X:12A4
kik
2L:27C1
kik
2R:54E8
kik
2R:542D
mel,sim,sec,yak,ere, ele,
bia,eug,tak, bip,ana
ele
2R:542D
X:18A6
ana,bip
mel, ere
2L:27C1
kik
2R:542D
FIG. 3. Many HP1D paralogs have arisen by gene duplication during Drosophila evolution. (A) A schematic of the melanogaster species group of
Drosophila investigated in detail in this report. Species that were sequenced by the modENCODE project (Chen et al. 2014), which were previously
not analyzed for HP1 duplications (Levine et al. 2012) are highlighted in boldface. (B) Schematic of all HP1D paralogs identified in the melanogaster
group of Drosophila species, indicating the chromodomain (black) and chromoshadow (gray) domains. Putative orthology is assigned via shared
syntenic position in the genome, with genome coordinates corresponding to the syntenic region of the D. melanogaster genome. Species in which a
particular HP1D paralog is found are indicated on the right.
massive TE derepression in HP1D-mutant females is associated with sterility. To test whether Oxp might support female
fertility through a similar mechanism, we performed RNA-seq
on the transcriptomes of ovaries dissected from Oxpþ/
Oxpþ and Oxp1/Oxp1 females, which are isogenic outside of
the Oxp locus. We analyzed RNA-seq reads that mapped
uniquely to coding genes and reads that mapped to repetitive
elements (see “Methods” section). At a false discovery rate
(fdr) of 0.01, we found that only 40 uniquely mapping coding
genes were differentially expressed between the mutant and
wildtype ovaries (supplementary fig. S5 and Table S1,
Supplementary Material online). These differentially expressed euchromatic genes exhibited no significant bias of
up- or down-regulation in the mutant genotype (24 and
16, respectively, binomial probability >0.1, supplementary
fig. S5A, Supplementary Material online); none of the
down-regulated genes encode a well-documented female fertility factor (supplementary table S1, Supplementary Material
online). In wildtype ovaries, these 40 genes are expressed at
low levels (supplementary fig. S5B, Supplementary Material
online). We found that many of the significantly differentially
expressed genes in Oxp1/Oxp1 ovaries were encoded on the
fourth (“dot”) chromosome. Specifically, we observed a significant enrichment of Oxp-sensitive genes on the heterochromatic fourth chromosome (10% observed vs. <1%
expected, P < 0.0001). In contrast, there was a dearth of
such genes on the X chromosome (2.5% observed vs. 16%
expected, P < 0.0001). This dichotomy suggests that the putatively indirect effects of Oxp on gene regulation strongly
depend on the chromatin environment.
In addition to protein-coding genes, we found that 11
repetitive elements were de-repressed in Oxp1/Oxp1 ovaries at an fdr <0.01 (fig. 2A). In contrast to coding genes,
we found that these differentially expressed repetitive elements were all overexpressed in Oxp mutants. These 11
elements represent nine TE families (supplementary table
S2, Supplementary Material online). We confirmed that
de-repression of these elements was specific to the Oxp
locus. In our “rescue genotype” (Oxp transgene in Oxp1/
Oxp1 genetic background), we were able to re-establish
silencing in the ovaries of the three most significantly derepressed elements (fig. 2B). These three, and indeed all
nine oxp-silenced TE families, overlap with those silenced
by HP1D/Rhino (Klattenhoff et al. 2009). The extent of TE
overexpression, however, is comparatively modest in oxp
mutants (Klattenhoff et al. 2009). Our findings suggest
that although HP1D/Rhino plays a dominant role in the
repression of TEs in the female germline, it is nevertheless
insufficient for repressing a subset of D. melanogaster TEs
targeted by the piRNA pathway. A previous study identified piRNAs that were present in the embryo relative to
the ovary as a surrogate for germline versus somatic expression of piRNAs (Malone et al. 2009). We found that
Oxp-silenced TEs are among those silenced by germline
piRNAs, consistent with the model that Oxp-mediated TE
repression occurs in the germline. Our findings support
the hypothesis that gene duplication is an important evolutionary mechanism of genome defense diversification.
Recurrent Innovation by Gene Turnover in the HP1D/
Rhino Subfamily
Our results show that Oxp supports TE silencing and fertility
in the female germline in D. melanogaster. These findings also
raised the possibility that other young duplications of HP1D
could similarly expand the repertoire of TE silencing in
Drosophila species beyond D. melanogaster and its close relatives. We took advantage of newly available genome sequences generated from eight Drosophila species
(Drosophila modENCODE project (Chen et al. 2014)).
These eight species shared a common ancestor around the
time of Oxp’s birth, that is, 15 million years ago (Tamura et al.
2004). These eight genomes, combined with previously published genomes, lead to a total of 13 species of the melanogaster group (fig. 3A). Together, these species offer a
unique opportunity to explore duplication, divergence,
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Levine et al. . doi:10.1093/molbev/msw053
and degeneration of HP1D-like genes on a short evolutionary
time scale.
To define the HP1D-like gene complement in D. melanogaster and its relatives, we used the tBLASTn algorithm
to query these genomes. We used the chromodomain and
chromoshadow domains of HP1D/Rhino (“HP1D” from now
on) and HP1D-like family members spanning 15 million years
of Drosophila evolution (Levine et al. 2012). Our analysis
identified previously undescribed orthologs of HP1D-like
genes that were present in shared syntenic locations. In addition, we found many previously undescribed HP1D paralogs
in non-syntenic locations (fig. 3B, supplementary table S3,
Supplementary Material online). Using the Bayesian MCMC
package in BEAST (see “Methods” section), we built a phylogenetic tree using the chromodomain of all known orthologs
and paralogs across the entire Drosophila HP1 gene family.
The HP1D orthologs and paralogs discovered via tBLASTn
represent a monophyletic clade that is distinct from all other
known HP1 genes in Drosophila (supplementary fig. S6,
Supplementary Material online). This analysis confirms that
these genes unambiguously belong to the HP1D clade.
We initially focused our analyses of the syntenic HP1D and
Oxp orthologs in different Drosophila species. Every
Drosophila species previously analyzed has been found to
encode HP1D (Vermaak et al. 2005; Levine et al. 2012). To
our surprise, we found that HP1D was not universally conserved in all queried genomes. For instance, we found that
HP1D had degenerated along the lineage leading to a
D. kikkawai ancestor (supplementary fig. S7, Supplementary
Material online); this species represents the only currently
sequenced genome from the species-rich montium subgroup.
We were also unable to find HP1D in D. rhopaloa or
D. takahashii due to a large gap (75 kb) in the publically
available genome assemblies. We therefore sequenced the
syntenic region to obtain HP1D and Oxp from both D. takahashii and D. rhopaloa genomic DNA. We discovered that
D. takahashii encodes both Oxp and HP1D. In contrast,
D. rhopaloa only encodes an intact HP1D but not Oxp (sup
plementary fig. S9, Supplementary Material online). Thus, we
conclude that with one exception, HP1D is preserved in the
melanogaster group of Drosophila species sampled. Oxp is
almost equally well conserved; we found it to be absent
only from D. kikkawai, D. rhopaloa, and D. ficusphila, in which
it has recently degenerated (supplementary figs. S7–S9).
Using shared synteny, we found ten additional HP1Drelated genes among the species we analyzed in addition
to HP1D and Oxp (supplementary table S3,
Supplementary Material online, and fig. 3B). We found
that the full-length HP1D paralog, HP1D2, previously reported to be restricted to D. simulans and D. yakuba
(Levine et al. 2012), was also present in D. rhopaloa, D.
biarmipes, D. ficusphila, D. takahashii, and D. eugracilis in a
shared syntenic location. We infer from this finding that
HP1D2 originated over 15 million years ago, followed by
subsequent loss in D. melanogaster and other species.
Other “full-length” HP1D-related genes were only present
in one or two closely-related species (fig. 3B). For instance,
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MBE
D. biarmipes and D. takahashii retain a tandem duplicate
of HP1D2 (which we refer to as HP1D2.2). The HP1D2
chromodomain has internally duplicated in D. biarmipes,
creating a unique HP1 gene that encodes two chromodomains and a single chromoshadow domain. D. takahashii
also encodes an X chromosome-linked full-length HP1Dlike gene (HP1D4) that is not found in any of the other
sequenced species. D. kikkawai encodes a set of three fulllength HP1D-like genes that are not found in other species: HP1D5, HP1D6, HP1D7 (fig. 3 and supplementary figs.
S10–S12). Considering that D. kikkawai lacks the parental
HP1D gene, it is very likely that at least one, and perhaps
all three, of these younger paralogs have taken over the
primary function of TE-silencing in this species.
Five of the HP1D-related genes were not full-length. In
addition to Oxp, we found D. elegans encodes a tandem
Oxpecker-like CD-only gene (Oxp2) while D. ananassae and
D. bipectinata encode a third, independently derived Oxp3
(fig. 3). We also identified two genes that only encode the
chromoshadow domain: the previously described HP1D3csd
in D. melanogaster and D. erecta, and HP1D8csd found upstream of HP1D6 in D. kikkawai (fig. 3). The observed births
and loss events of HP1D-like genes is consistent with recurrent HP1D-like gene family expansion and turnover.
Although the shared syntenic approach can be a powerful
means to interrogate orthology, it can be misleading if multiple independent paralogs were born via gene-duplication in
close proximity to the parental genes. We therefore carried
out detailed phylogenetic analyses to ascertain orthology relationships. Although these analyses support the monophyletic relationship of all identified HP1D-like genes,
unfortunately they failed to resolve many finer scale relationships (supplementary fig. S6, Supplementary Material online).
The 60 aa chromodomain frequently had too little phylogenetic resolution (by a variety of distance and likelihoodbased methods) to allow us to draw firm conclusions about
orthology versus paralogy within the HP1D clade. For instance, we were unable to clearly establish common ancestry
of the putative Oxp orthologs, which instead fall into two
well-supported clades—one that encompasses the melanogaster subgroup and another exclusively the ananassae
subgroup. It is possible that this bifurcation represents a burst
of evolution immediately following the separation of these
two lineages. But it is also likely that these two Oxp clades
instead represent independent, tandem duplications of
HP1D. Similarly, although the putative HP1D2 orthologs
group together with presumed daughter genes HP1D2.2
and HP1D4, this phylogenetic grouping does not have strong
bootstrap support. Overall, we attribute the poor resolution
within HP1D to rapid divergence between these remarkably
young genes. Thus, our assignment of orthology groups
(fig. 3B) based on shared syntenic positions is not significantly
improved by phylogenetic analyses. This limitation introduces
ambiguity in orthology/paralogy only for tandem paralogs,
such as Oxp and HP1D or HP1D2 and HP1D2.2. In all other
cases, their shared syntenic location is a robust indicator of
orthology.
Recurrent Gene Duplication of HP1D/Rhino in Drosophila Evolution . doi:10.1093/molbev/msw053
gene
domains
chromo shadow
HP1D
Oxp
HP1D3csd
HP1D2
HP1D2.2
HP1D4
HP1D5
HP1D6
HP1D7
HP1D8csd
rp49
(control)
MBE
+
D. melanogaster
+
+
+
+
+
D. kikkawai
-------------
---
n.d*
+
D. takahashii
ovary
ovary
---------------
---------
ubiquitous
-
g H T C H O C
Male
Female
-
g H T C H O C
Male
Female
FIG. 4. Expression analyses of HP1D paralogs in adult tissues of two Drosophila species. All HP1D paralogs analyzed in the
RT-PCR analyses of adult tissues from either D. takahashii or D. kikkawai are indicated on the left, along with the previously
measured expression pattern of their D. melanogaster orthologs. The rp49 gene is shown as a loading control on the bottom
and as a quality measure of the cDNA versus genomic DNA (notice size difference). For adults, heads, germline tissue (testes
or ovaries), and carcass were assayed. Note that many paralogs are specific to either species. When paralogs are not present,
we indicate that as “—” [n.d.* refers to not determined because no adult expression was discovered (Levine et al. 2012)].
HP1D-like Genes Are Germline-Expressed
HP1D and Oxp are both expressed in the female germline in
D. melanogaster (Levine et al. 2012). This tissue-restricted expression pattern is consistent with their role in genome defense against TEs, which increase their copy number only if
transposition occurs in the male or female germline. If the
newly identified HP1D-related genes from non-model
Drosophila species encode similar functions in genome defense as HP1D and Oxp, we predicted that they would also be
expressed primarily in adult germline tissues.
To test this prediction, we chose the two species that
encode the largest subset of newly discovered HP1D-like
genes—D. takahashii and D. kikkawai. In addition to HP1D
and Oxp, D. takahashii encodes HP1D2, HP1D2.2, and HP1D4
(fig. 3). In D. takahashii, we found that HP1D, HP1D2, and
HP1D4 were highly expressed in both ovaries and testes
(fig. 4). In contrast, HP1D2.2 was most highly expressed in
ovaries whereas Oxp was highly expressed only in testes.
The additional testis expression of HP1D in D. takahashii
suggests that this gene may encode an analogous TE silencing
function in the male germline in this species even though
such a function has not been elucidated in D. melanogaster.
D. kikkawai encodes HP1D5, HP1D6, HP1D7, and HP1D8csd
but neither HP1D nor Oxp (fig. 3 and supplementary fig. S7,
Supplementary Material online). Intriguingly, we found that
only D. kikkawai HP1D7 was expressed in ovaries where it
might have replaced HP1D function (fig. 4). D. kikkawai
HP1D5, HP1D6, and HP1D8csd are expressed primarily in testes (fig. 4). In contrast with the HP1D family members, the
three founding HP1 genes, that is, HP1A, HP1B, and HP1C had
ubiquitous expression in adult tissues (Vermaak et al. 2005;
Levine et al. 2012). Based on their gonad-restricted expression
patterns and strikingly dynamic evolution, we propose that
the HP1D subfamily has recurrently undergone gene
duplication to increase the repertoire of TE suppressors in
the germline.
Discussion
Gene Duplications as a Genome Defense Strategy
HP1D is a critical component of piRNA defense against TEs in
the female germline (Klattenhoff et al. 2009). We previously
demonstrated that positive selection drives sequence evolution of HP1D orthologs across Drosophila evolution, suggesting that codon evolution might be one means for Drosophila
host genomes to keep pace with TE sequence evolution and
changing TE repertoires (Vermaak et al. 2005). In this report,
we investigated HP1D gene duplication as a mechanism to
diversify host genome defense against TEs. We first focused
on a single HP1D duplication that gave rise to a tandem
paralog encoding a chromodomain-only protein called
Oxpecker. Despite its young age, we discovered that Oxp plays
a significant role in female fertility. We found that Oxp augments HP1D silencing in the female germline of several TE
families. Thus, for a subset of TEs, HP1D alone is insufficient to
mediate complete suppression. We further demonstrate that
at least 11 HP1D paralogs duplicated and diverged during a
short 15-million-year snapshot of Drosophila evolution. The
seven paralogs investigated further are all expressed in either
or both male and female germlines—the putative battleground of host–TE evolutionary conflicts. Together with
our genetic analysis of Oxp function, this pervasive history
of HP1D-related gene duplication supports our hypothesis
that HP1D gene family expansion has diversified genome defense repertoires in Drosophila genomes.
Other piRNA component gene amplifications have been
documented in Drosophila, mosquito, and aphid genomes
(Campbell et al. 2008; Lu et al. 2011; Lewis et al. 2016). Our
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Levine et al. . doi:10.1093/molbev/msw053
Mobile
elements
Rhino
piRNAs
Oxp
Direct trancriptional repression
of “new” mobile elements
Oxp
Generation of primary piRNA transcripts
from “new” mobile elements
FIG. 5. Two possible mechanisms by which Oxp could augment protection against TEs in the female germline. Oxp might mediate binding and
direct transcriptional repression of novel TE sites in the genome in cis (left). Alternatively, Oxp recruitment may generate piRNAs (either
dependent or independent of subsequent HP1D/Rhino recruitment) that serve to silence TE copies in trans (right).
findings especially complement previous studies that identified functional diversification of TUDOR domain proteins
involved in piRNA processing in Drosophila. For instance, a
recent study found that multiple genes encoding TUDOR
domains (e.g., Vreteno) are essential for piRNA biogenesis
(Handler et al. 2011; Zamparini et al. 2011). The TUDORdomain containing genes may serve to make the ping-pong
piRNA amplification cycle more robust, and thereby increase
the efficiency of TE silencing by the piRNA machinery (Sato,
Iwasaki, Shibuya et al. 2015; Sato, Iwasaki, Siomi et al. 2015;
Wang et al. 2015). Unlike the HP1D gene family, however, the
TUDOR-domain gene duplications appear to be quite ancient. Orthologs of these proteins are present in all sequenced
Drosophila genomes, suggesting that the gene duplications
predate the origin of the Drosophila genus, and have been
strictly conserved since. Thus, they do not display the rapid
“birth-and-death” dynamics we have seen in the HP1D gene
family.
Oxpecker Silencing of TEs in the Germline
How does Oxp influence TE silencing in the female germline?
We hypothesize two mechanisms by which Oxp could directly perform its TE-silencing function. First, Oxp could function analogously to HP1D, that is, supporting transcription of
a primary piRNA transcript that is processed into TE-silencing
piRNAs. This piRNA-related function of Oxp may be either
dependent or independent of HP1D. In the latter scenario,
Oxp may recruit HP1D to “new” genomic sites via an Oxp–
HP1D protein–protein interaction, thereby expanding
HP1D’s range of silencing (fig. 5). Alternatively, Oxp may
act independently of the piRNA pathway, instead helping
to recruit heterochromatin-silencing machinery to individual
TEs or euchromatic genes via its chromodomain (fig. 5). Either
of these mechanisms may allow Oxp to augment silencing of
especially active TEs. We favor the latter because Oxpecker did
not emerge as a strong candidate in screens for novel piRNA
pathway members (Czech et al. 2013; Handler et al. 2013).
Future work characterizing the genomic localization of Oxp
(via ChIP-seq) and characterization of the piRNA repertoire
1648
(via piRNA-seq) in Oxp mutant ovaries will help distinguish
between these possibilities.
The evolutionary lability of TEs obscures the evolutionary
force that drove oxp fixation 10 million years ago. However,
the non-random subset of Oxp-silenced TE families offers
insight into the initial selective force that may have driven
oxp fixation nearly 15 million years ago. These nine families
represent a diverse complement of TEs—long terminal repeat
(LTR), non-LTR, and DNA elements. Nevertheless, copies
within each family harbor high-percent identity and occur
at a lower mean frequency in natural populations of D. melanogaster (supplementary table S4, Supplementary Material
online, P < 0.001—Kelleher and Barbash 2013). These two
features are also observed across the complement of TEs
derepressed in piRNA pathway mutants (Kelleher and
Barbash 2013). These two parameter estimates are consistent
with Oxp silencing TEs that recently inserted into the genome
(i.e., active or recently active elements). Oxp’s role in host
defense might be to ensure more robust HP1D silencing of
especially active TEs families. Thus, there is a net gain of TEsuppression activity by Oxp retention in a genome that already encodes HP1D. Similarly, the evolution of KRAB–ZNF
genes in mammals to expand silencing to “new” TE families is
largely the result of a “tit-for-tat” adaptive evolution (Thomas
and Schneider 2011; Jacobs et al. 2014). Alternatively, Oxp
may simply augment the pre-existing HP1D-generated
piRNA pool, which is sufficient to repress all but the most
active TE families in the host Drosophila genomes. This would
be akin to the TUDOR-domain containing paralogs that increase the efficiency of the piRNA pathway response to active
TE families by favoring the ping-pong amplification response
(Sato, Iwasaki, Shibuya et al. 2015; Sato, Iwasaki, Siomi et al.
2015; Wang et al. 2015). This second possibility might help
explain why Oxp evolves under purifying selection (Levine
et al. 2012) unlike HP1D, which evolves under positive selection (Vermaak et al. 2005; Vermaak and Malik 2009).
We propose that the adaptation to combat recently encountered TE families also helps explain the observed pattern
of recurrent HP1D gene duplication and loss. Gene
Recurrent Gene Duplication of HP1D/Rhino in Drosophila Evolution . doi:10.1093/molbev/msw053
duplications could result in functional redundancy that
relaxes selective pressure for gene retention. For instance,
Oxp birth may have led to a reduced repertoire of TE silencing
by HP1D. Although HP1D has been largely conserved through
this recurrent gene turnover, D. kikkawai represents an example of a genome in which HP1D was lost, likely due to its
functional replacement by its female germline-restricted
paralog, HP1D7. This hypothesis helps explain the dramatic
gene turnover we observe in the HP1D gene family. Candidate
replacement genes (e.g., in D. kikkawai) may support an
equally efficient piRNA pathway-based mechanism of TE silencing, but one that leverages a recently refreshed set of
genes. We propose that just like positive selection acting on
codons in HP1D orthologs and other genome defense genes,
the wholesale turnover of HP1D-like genes can be equally
potent at amplifying the arsenal of host silencing machinery
or making it more robust against newly encountered TEs.
Future evolutionary analysis will determine if the dynamic
gene turnover observed for the HP1D gene family here and
previously in the KRAB–ZNF family (Nowick et al. 2010;
Thomas and Schneider 2011) is a general feature of host defense genes across eukaryotes.
Materials and Methods
Oxpecker Mutant Construction and Confirmation
Bloomington stock 21086 encodes a mini-white-marked
P-element (EPgy2) in the 50 UTR of the Oxpecker gene. We
crossed male flies homozygous for the insertion to virgin females heterozygous for D2-3 transposase (D2-3/CyO). Note
that “CyO” refers to a chromosome II balancer. We crossed
D2-3/21086 F1 males to virgin Bdx/CyO females. Next, we
collected 85 F2 males from which the mini-white marked Pelement had excised (white-eyed males). We backcrossed
again to Bdx/CyO virgin females for long-term maintenance
of each excision chromosome balanced over a CyO
chromosome.
This white-eyed, chromosome II stock collection contained
a both precise (clean) excision events and imprecise (deletion) events at the Oxpecker locus. We screened genomic
DNA prepared from these stocks using primers that spanned
the P-element insertion site (supplementary table S5,
Supplementary Material online) and discovered 41 putatively
precise excision and six imprecise excision chromosomes. We
sequenced the PCR products of one putatively precise excision line and confirmed the genomic site was completely
resurrected with no deletions upon P-element mobilization
(supplementary fig. S3, Supplementary Material online). We
also sequenced the PCR products of the five putatively imprecise excisions. One line harbored a large deletion of both
Oxpecker and the upstream HP1D loci and four harbored
smaller deletions 500 bp long. All four imprecise excisions
deleted sequence upstream of the Oxpecker start codon. We
chose the confirmed precise deletion line as the “wildtype”
control going forward for subsequent functional analyses and
an imprecise excision spanning the Oxp 50 UTR and upstream
flanking sequence—a 510-bp deletion upstream of the oxpecker start codon (supplementary fig. S3, Supplementary
MBE
Material online). Based on our crossing scheme, we infer
that these two lines are isogenic except for the 510-bp
deletion.
Oxpecker is expressed primarily in D. melanogaster ovaries
(Levine et al. 2012). We confirmed loss of Oxp expression in
the imprecise excision line by PCR-amplifying the Oxp transcript from ovary-derived cDNA (supplementary fig. S3,
Supplementary Material online). cDNA from the precise excision served as a control. Primers directed at the ubiquitously
expressed rp49 locus confirmed equivalent cDNA concentrations across the genotypes (supplementary fig. S3,
Supplementary Material online). Primer sequences are reported in supplementary table S5, Supplementary Material
online.
Oxp Influence on Expression of Genes and Repetitive
Elements in D. melanogaster
To investigate the genome-wide consequences of depleting
Oxp expression, we conducted RNA-seq on ovaries dissected
from 3 to 5-day-old females encoding either the precise and
imprecise excision. We included three biological replicates per
genotype. The FHCRC Shared Resources Genomics Core prepared six libraries using Illumina TruSeq Sample Prep Kit v2.
We performed image analysis and base calling with Illumina’s
RTA v1.13.sofware and de-multiplexed with Illumina’s
CASAVA v1.8.2. We used TopHat v1.4.0 to align reads to
BDGP5r66 and samtools v0.1.18 to convert files to sam format. To generate counts/gene and to cull genes with zero
counts across all sample (our cut-off was at least 1 count/
million in at least three samples), we used HTseq-count v0.5.3.
This filtering left us with unique 7,547 genes (supplementary
table S1, Supplementary Material online).
To investigate differences in expression across repetitive
element classes, we first downloaded the dm3 repeat mask
track as a GTF file from the UCSC Genome Browser website.
We generated counts for each repeat using HTseq-count
v0.5.3p9 (default “union” overlapped model). We removed
repeats with <1 count/million in at least two samples. This
filter left us with 469 repeat elements. We identified differentially expressed repeats using edgeR v2.6 (supplementary ta
ble S2, Supplementary Material online). For each TE, we extracted “mean pairwise identity” and “mean frequency” from
Kelleher and Barbash (2013) (supplementary table S4,
Supplementary Material online). Using a Mann–Whitney
test, we compared the significantly differentially expressed
set (identity: n ¼ 7 and frequency: n ¼ 9) with the global
set (identity: n ¼ 66, frequency: n ¼ 88).
Oxpecker Transgene Construction and Rescue of TE
Silencing
To determine if the functional consequences of the imprecise
excision could be attributed to loss of Oxp expression alone,
we engineered a native promoter-driven transgene rescue
stock using the PhC31 technology. We cloned into the
pattB vector the Oxp coding sequence, along with 3,150
and 375 bp 50 and 30 of the Oxp coding sequence, respectively.
The Best Gene, Inc. (Chino Hills, CA) injected this plasmid
into the 8622/attP2 line, which encodes a landing platform at
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Levine et al. . doi:10.1093/molbev/msw053
cytolocation 68A4. We crossed a positive transformant line
with the imprecise excision line, generating a genotype homozygous for the deletion (chromosome II) but heterozygous
for the transgene (chromosomes III).
We performed qPCR on ovary-derived, poly-A selected
cDNA prepared from the genotypes that were homozygous
for the imprecise excision plus or minus a single copy of the
transgene rescue construct. We chose the three most differentially expressed TEs in our RNA-seq dataset—Gypsy12 LTR,
1731, and 412—for downstream analysis. We extracted
the bowtie alignments to each TE instance represented
in the repeat mask GTF file (e.g., instance “Gypsy
12_LTR_range ¼ chr3RHet:640872-643144”). We designed
qPCR primers to amplify the locus to which the majority of
the reads mapped. The loci and primers can be found in
supplementary table S5, Supplementary Material online.
Two different primer pairs per TE, along with rp49 primers
at the endogenous control locus, were used to amplify (SYBER
green UPG with ROX, Life Technologies) each target. Each
sample–primer pair combination was run in triplicate. We
ran the reactions on an ABI 7900HT Real Time PCR Systems.
Fertility Assays
We assessed lifetime fertility of females homozygous for either
the precise excision or the imprecise excision. In 15 replicate
vials, we crossed two virgin females to two wildtype (w1118)
males. Every 4 days, we transferred the parental generation
onto new food for a total of 3 weeks. We counted adult
progeny 8 days after first eclosion. We summed the data
over the five time points per replicate and analyzed the
data using a Mann–Whitney U-test (Prism 6).
To determine if the fertility defect detected in the above
experiment could be attributed to the loss of Oxp expression
alone, we repeated the fertility assay using females homozygous for the imprecise deletion with or without an ectopic
Oxp transgene. We crossed three virgin females representing
these two genotypes plus two additional controls (see fig. 1D)
to two wildtype (genotype w1118) males per vial. We removed parents after 3 days and counted adult progeny on
day 8 after the first eclosion. We analyzed the data again using
a Mann–Whitney test (Prism 6). All fertility assays were conducted on standard cornmeal media at 25 C on 12-h day/
night cycles.
Bioinformatic Survey for HP1D Paralogs
Our previous phylogenomic analysis of the Heterochromatin
Protein 1 gene family in Drosophila reported the discovery of
nine genes that clustered phylogenetically within the HP1D/
Rhino clade (Levine et al. 2012). These paralogs span the 60
million years of Drosophila evolution (Drosophila 12
Genomes et al. 2007). The analysis presented here takes advantage of eight recently sequenced Drosophila species related to D. melanogaster that span <15 million years:
D. ficusphila, D eugracilis, D. biarmipes, D. takahashii, D. elegans, D. rhopaloa, D. kikkawai, and D. bipectinata (fig. 3A)
(Chen et al. 2014). To discover new orthologs and paralogs
derived from an HP1D/rhino or HP1D-like gene duplication,
we first concatenated the known chromodomains or the
1650
chromoshadow domains (Levine et al. 2012) Using the
tBLASTn algorithm (www.flybase.org), we searched for chromodomains and chromoshadow domains in the unannotated genomes of the eight Drosophila species.
For each species search, we initially retained hits to the
genome scoring an e-value <0.1. We used the translated hit
in a tBLASTn search of the D. melanogaster genome.
Although we queried with exclusively HP1D/Rhino orthologs
and paralogs, we identified all the previously characterized
HP1 clades, including HP1A, HP1B, and HP1C in each species.
Chromodomain-encoding, non-HP1 gene family members
like Chromator or Polycomb, also came up in our gene lists
for each species, indicating that our search for specifically
HP1D-like genes was exhaustive. Those hits to HP1D-like
genes were retained for synteny analysis. Specifically, we
used the 5–10 kb region flanking each hit as the query in a
BLAT search of the D. melanogaster genome. The location of
the 10–20 kb region in D. melanogaster allowed us to classify
each HP1D-like gene hit as either a potential ortholog of the
previously known (shared syntenic) HP1D locus, of the HP1Dlike loci reported in Levine et al. (2012), or a previously undescribed paralog (non-syntenic, supplementary table S4,
Supplementary Material online).
A chromodomain and chromoshadow domain hit to a
new, previously uncharacterized genomic location indicated
the discovery of a new two-domain, “full HP1,” protein. For a
subset of such hits, we computationally predicted an intronic
sequence separating a chromodomain hit and a nearby chromoshadow domain hit. We confirmed presence or absence of
a single coding gene in these locations by sequencing a PCR
product amplified from cDNA that spanned the putative
intron(s). Alignments of these transcripts to genomic DNA
sequence are reported in supplementary figs. S10–S12,
Supplementary Material online.
Phylogenetic Analyses
To uncover the phylogenetic relationships among our newly
discovered orthologs and paralogs, we built phylogenetic
trees restricted to the chromodomains. We perform treebuilding using the Bayesian MCMC package BEAST v1.6.2
(Drummond and Rambaut 2007) using an uncorrelated
log-normal relaxed clock (Drummond et al. 2006) and the
SRD06 substitution model (Shapiro et al. 2006), which separates the evolutionary model for the third codon position
from the first two. MCMC Chains ran until inspection of
the traces and effective sample size of each parameter using
the Tracer program v1.6 (http://tree.bio.ed.ac.uk/software/
tracer) indicated acceptable mixing (ESS >200 for every parameter) and stationarity (as evaluated by the independent
runs). Acceptable mixing occurred after running 20 million
iterations. We present a maximum clade credibility tree
(http://beast.bio.ed.ac.uk/summarizing-posterior-trees)
in
cladogram format for ease of presentation.
Expression Profiling of HP1 Paralogs in Adult Tissues
To determine if the newly discovered HP1D-like genes are
germline-restricted, we conducted RT-PCR on a panel of
adult male and female tissues dissected from D. takahashii
Recurrent Gene Duplication of HP1D/Rhino in Drosophila Evolution . doi:10.1093/molbev/msw053
and D. kikkawai. These two species share a common ancestor
about 15 million years ago and encode a non-overlapping set
of HP1D-like genes (also non-overlapping relative to D. melanogaster). We dissected 30 ovaries, 60 heads, and five “remaining carcasses” from adult females and stored the tissues
in RNAlater (Qiagen) until total RNA extraction using the
MirVana kit (Ambion). We performed the same procedure
for males by dissecting 60 testis pairs, 60 heads, and five
carcasses. We generated poly-A selected cDNA using SSIII
reverse transcriptase (Invitrogen). Primers used for amplification of the seven loci can be found in supplementary table S5,
Supplementary Material online.
Supplementary Material
Supplementary figures S1–S12 and Supplementary tables
S1–S5 are available at Molecular Biology and Evolution online
(http://www.mbe.oxfordjournals.org/).
Acknowledgments
The authors thank Danielle Vermaak for sharing results from
her earlier analysis on Oxp and HP1D, and Ryan Basom for
essential bioinformatics support of the RNA-seq analysis. We
also thank Q. Helleu, T. Levin, L. Kursel, Grace Lee, A. Molaro,
R. McLaughlin, J. Young, and S. Zanders for their comments
and all members of the Malik and Peichel labs for valuable
discussions. We especially thank the editor and four anonymous reviewers for their many helpful suggestions. This study
was supported by an NIH K99/R00 Pathway to Independence
Fellowship GM107351 to M.T.L. and grants from the Mathers
Foundation and NIH R01 GM74108 to H.S.M. H.S.M. is an
investigator of the Howard Hughes Medical Institute.
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