InteractiveFly: GeneBrief

Hybrid male rescue & Lethal hybrid rescue: Biological Overview | References

Gene name - Hybrid male rescue & Lethal hybrid rescue

Synonyms -

Cytological map positions - 9D3-9D3 & 54B7-54B7

Functions - chromatin proteins

Keywords - required to repress transposable element and satellite DNA expression - gain-of-function phenotype of causing lethality in F1 male hybrids between D. melanogaster and D. simulans - Hybrid sterility - boundary element-associated proteins - Oogenesis

Symbol - Hmr & Lhr

FlyBase ID: FBgn0001206 &

Genetic map positions - chrX:10,590,920-10,595,855 & chr2R:17,431,032-17,432,219

NCBI classifications - Hmr: Alcohol dehydrogenase transcription factor Myb/SANT-like & Lhr: BESS motif - named after the proteins in which it is found (BEAF, Suvar(3)7 and Stonewall)

Cellular location - nuclear

NCBI link for Hmr: EntrezGene, Nucleotide, Protein
NCBI links for Lhr: EntrezGene, Nucleotide, Protein
Hmr orthologs: Biolitmine
Lhr orthologs: Biolitmine
Recent literature
Anselm, E., Thomae, A. W., Jeyaprakash, A. A. and Heun, P. (2018). Oligomerization of Drosophila Nucleoplasmin-Like Protein is required for its centromere localization. Nucleic Acids Res. PubMed ID: 30357352
The evolutionarily conserved nucleoplasmin family of histone chaperones has two paralogues in Drosophila, named Nucleoplasmin-Like Protein (NLP) and Nucleophosmin (NPH). NLP localizes to the centromere, yet molecular underpinnings of this localization are unknown. Moreover, similar to homologues in other organisms, NLP forms a pentamer in vitro, but the biological significance of its oligomerization has not been explored. This study characterize the oligomers formed by NLP and NPH in vivo and find that oligomerization of NLP is required for its localization at the centromere. It was further shown that oligomerization-deficient NLP is unable to bind the centromeric protein Hybrid Male Rescue (HMR), which in turn is required for targeting the NLP oligomer to the centromere. Finally, using super-resolution microscopy NLP and HMR were found to largely co-localize in domains that are immediately adjacent to, yet distinct from centromere domains defined by the centromeric histone dCENP-A.
Cooper, J. C., Lukacs, A., Reich, S., Schauer, T., Imhof, A. and Phadnis, N. (2019). Altered localization of hybrid incompatibility proteins in Drosophila. Mol Biol Evol. PubMed ID: 31038678
Understanding the molecular basis of hybrid incompatibilities is a fundamental pursuit in evolutionary genetics. In crosses between Drosophila melanogaster females and Drosophila simulans males, an interaction between at least three genes is necessary for hybrid male lethality: Hmr mel, Lhr sim, and gfzf sim. While HMR and LHR physically bind each other and function together in a single complex, the connection between gfzf and either of these proteins remains mysterious. This study shows that GFZF localizes to many regions of the genome in both D. melanogaster and D. simulans, including at telomeric retrotransposon repeats. GFZF localization at telomeres is significantly different between these two species, reflecting the rapid evolution of telomeric retrotransposon copy number composition between the two species. It was next shown that GFZF and HMR normally do not co-localize in D. melanogaster. In inter-species hybrids, however, HMR shows extensive mis-localization to GFZF sites, thus uncovering a new molecular interaction between these hybrid incompatibility factors. Spreading of HMR to GFZF sites requires gfzf sim but not Lhr sim, suggesting distinct roles for these factors in the hybrid incompatibility. Finally, this study found that over-expression of HMR and LHR within species is sufficient to mis-localize HMR to GFZF binding sites, indicating that HMR has a natural low affinity for GFZF sites. Together, these studies provide the first insights into the different properties of gfzf between D. melanogaster and D. simulans, and uncover a molecular interaction between gfzf and Hmr in the form of altered protein localization.
Lukacs, A., Thomae, A. W., Krueger, P., Schauer, T., Venkatasubramani, A. V., Kochanova, N. Y., Aftab, W., Choudhury, R., Forne, I. and Imhof, A. (2021). The Integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila. PLoS Genet 17(8): e1009744. PubMed ID: 34424906
Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. It has been shown that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. This work describes a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. These experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality.

Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr (Hybrid male rescue) and Lhr (Lethal hybrid rescue) genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR's function, this study measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, HMR was found to localize to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. These data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species (Gerland, 2017).

Biodiversity is the result of the emergence and the extinction of species. New species form by pre- and post-zygotic isolation mediated by genetic incompatibility. One of the best characterized examples of hybrid incompatibility is the gene pair Hybrid male rescue (Hmr) and Lethal hybrid rescue (Lhr). Hmr and Lhr cause hybrid incompatibility between the closely related fly species Drosophila melanogaster and D. simulans. Hmr diverged in both Drosophila sibling species under positive selection (Barbash, 2004). HMR and LHR from both species interact physically and localize predominantly to centromeric regions (Thomae, 2013). A reduction of HMR expression results in a misregulation of transposable elements, satellite DNAs and heterochromatic genes (Thomae, 2013; Satyaki, 2014; Wei, 2014). The major difference between HMR and LHR in D. melanogaster and D. simulans is their substantial difference in protein amounts (Thomae, 2013; Maheshwari, 2012), which has been proposed to result in a lethal gain of function in male hybrids. High levels of HMR and LHR in hybrids and overexpression of these proteins in pure species lead to an increased number of binding sites of the complex. Such spreading phenomena based on protein amount have been observed for several chromatin-associated complexes such as the dosage compensation complex, the polycomb complex or components of pericentromeric heterochromatin. In most cases, the precise mechanisms for targeting and spreading are not fully understood. Interestingly, several of the components involved in these processes show signs of adaptive evolution and differ substantially even in very closely related organisms. This observation has spurred a model of a dynamic genome that drives the adaptive evolution of chromatin-associated factors (Sawamura, 2012; Gerland, 2017 and references therein).

Eukaryotic genomes of closely related species differ mostly in the amount and sequence of repetitive DNA. This DNA is often derived from transposable elements, which are highly mutagenic and are therefore under tight transcriptional control by the cellular machinery. During evolution transposons or transposon-derived sequences occasionally adopted structural or novel cis-regulatory functions, thereby contributing to the evolution of new, species-specific, phenotypic traits. Genomic insulators are a particular class of such novel, fast evolving, cis-regulatory elements that show signs of transposon ancestry. A strong expansion of these elements is observed in arthropods, which also experienced a successive gain in the number of insulator binding proteins during evolution. In fact, the Drosophila genome harbours a large variety of insulator proteins such as CTCF, BEAF-32, Su(Hw), Mod(mdg4) and CP190, which all affect nuclear architecture. Different Drosophila species underwent multiple genomic rearrangements and transposon invasions, which presumably resulted in an adaptive response of regulatory DNA binding factors to maintain spatial and temporal gene expression. For example, binding sites for the insulator proteins BEAF-32 and CTCF show a high degree of variability when compared among very closely related species (Stark, 2007; Bosco, 2007). The gain of new insulator sites is associated with chromosome rearrangements, new born genes and species-specific transcription regulation. Similar to insulator proteins, which tend to cluster in specific nuclear regions, the speciation factor HMR clusters at centromeres or pericentromeric regions in diploid cells but is also detected at distinct euchromatic regions along the chromosome arms in polytene chromosomes. A unifying feature for many of these sites is their close proximity to binding sits of the Heterochromatin Protein 1 (HP1a), a HMR interactor and a well-characterized heterochromatic mark (Gerland, 2017).

Various studies describe HMR's localization to heterochromatin, but the molecular details on HMR's binding sites and its recruitment to these sites are not well understood. To get new insights into HMR's association to chromatin, this study measured HMR's genome-wide localization by chromatin immunoprecipitation (ChIP) in the D. melanogaster embryonic S2 cell line. This analysis demonstrated an extensive colocalization of HMR with a subset of insulator sites across the genome. HMR's binding to genomic gypsy insulators, which constitute the major group of its binding sites, is dependent on the residing insulator protein complex. In a second group, HMR borders heterochromatin together with the insulator protein BEAF-32. In agreement with previous low-resolution techniques in cell lines and fly tissue, these binding sites are enriched at pericentromeric regions, the cytological region 31 on the 2nd chromosome and the entire 4th chromosome. At most of these sites, HMR associates to the promoters of actively transcribed genes. Interestingly, these genes code for transcripts that have been reported to be downregulated in Hmr mutant larvae and ovaries. Altogether, these data provide evidence for a functional link between HMR and insulator proteins, which potentially results in hybrid incompatibilities due to the adaptive evolution of these genome-organizing complexes (Gerland, 2017).

HMR localizes to centromeric and pericentromeric regions in D. melanogaster cell lines as well as in mitotically dividing embryonic cells where it has been suggested to act as a repressor of transposable elements. Mutations in Hmr lead to overexpression of satellite DNA and transposable elements in ovaries and larvae (Satyaki, 2014). Such a derepression is also observed in hybrid flies, where HMR and LHR levels are higher than the ones in pure species and result in a widespread distribution of the HMR/LHR complex (Thomae, 2013). To better understand the targeting principles that mediate HMR binding within the D. melanogaster genome, it was asked whether it is possible to identify HMR binding sites by applying ChIP-Seq in the D. melanogaster S2 cell line. Combining this approach with RNAi mediated knockdown experiments this study uncovered a strong colocalization of HMR with gypsy insulator binding sites and demonstrated that HMR binding to these sites depends on the presence of the residing insulator protein complex. Notably, HMR associates only with a subset of all Su(Hw) binding sites, but almost all those sites can be classified as gypsy-like sites bound by CP190 and mod(mdg4) in addition to Su(Hw) (Gerland, 2017).

Besides dispersed binding of HMR at genomic gypsy insulator sites along the chromosome arms, dense clusters of HMR binding sites were observed around the centromere and on the 4th chromosome where it potentially serves to separate HP1a binding domains from highly active genes. This dense clustering of binding sites around the centromere correlates well with the strong colocalization of HMR signals with the centromeric H3 variant CID in immunolocalization experiments. Due to its biochemical interaction and partial colocalization with the heterochromatin protein HP1a in Drosophila embryos, HMR has been suggested to be a bona-fide heterochromatin component. However, in contrast to HP1a, this study detected very distinct HMR binding sites within the genome. When HMR is found close to an HP1a binding domain, it rather borders it than covering the whole domain. The sharp HMR binding signals and the fact that almost all euchromatic HMR binding sites contain putative insulator elements, suggest a role of HMR in separating chromatin domains. A distinct boundary that separates constitutive heterochromatin from the core centromere has also been postulated by Olszak and colleagues who suggest that transition zones between heterochromatin and euchromatin are hotspots for sites of CID misincorporation (Olszak, 2011). Unfortunately, centromeres are notoriously difficult to study by next generation sequencing due to their highly repetitive nature. In addition, the microscopic resolution is not sufficiently high to allow a distinction between a binding to the core centromere chromatin and the chromatin immediately adjacent to it. Therefore, it cannot be ruled that HMR binds large domains at the central region of the Drosophila centromere. However, the fact that the purification of chromatin containing the centromeric H3 variant CID did not identify HMR, suggests that it may very well also form a boundary between pericentromeric heterochromatin and the core centromere. To which extent and by which mechanism HMR fulfils a functional role at these genomic sites remains to be elucidated (Gerland, 2017).

The genomic sites, where HMR was found bound next to an HP1a domain, are highly enriched for recognition sites of the insulator protein BEAF-32. Interestingly, a depletion of BEAF-32 in S2 cells results in an increased rate of mitotic defects, which is very reminiscent of the phenotype detected when HMR is depleted. Similarly to flies carrying a mutation in the Hmr gene, flies in which BEAF-32 is only contributed maternally have defects in female fertility. BEAF-32's role in maintaining associated promoter regions in an environment that facilitates high transcription levels has been suggested to be functionally relevant for this phenotype. Strikingly, this study found most HMR/BEAF-32 binding sites located between HP1a containing heterochromatin and the transcription start site of a highly active gene. HP1a chromatin might fulfil a repressive function at these genomic regions and HMR might block this repressive impact on the neighbouring gene body. However, no extensive spreading of HP1a or H3K9me3 is seen upon HMR knockdown suggesting that the repressive effect is not directly mediated by HP1a binding or the HMR knock down not efficient enough. As there is evidence that HP1a can also promote gene transcription, HMR may also function as a co-activator for HP1a. Currently, HMR binding next to HP1a containing chromatin is considered as a unifying feature of transcriptionally affected genes but the potential mechanism by which HMR exerts its function is as yet unknown (Gerland, 2017).

Although HMR depletion has a substantial effect on the transcription of multiple transposons, HMR was found enriched only at the 5' insulator region of the gypsy or gtwin retrotransposons and to similar sites within the genome that are presumably derived from these elements. These sites are occupied by insulator proteins Su(Hw), CP190 and Mod(mdg4) and often display enhancer blocking activity in transgenic assays. Artificial targeting of HMR to DNA placed between an enhancer and a promoter of a reporter gene can block the transcription activity, suggesting that HMR may indeed play a role in setting up endogenous boundary elements. Similar to what is known for Su(Hw), HMR binding to this class of binding sites is dependent on the presence of the structural protein CP190, which has a key function in the stabilization of insulator protein complexes. However, as no strong physical interaction is observed between CP190 and HMR, the loss of HMR binding upon a reduction of CP190 levels may also be the result of increased nucleosome occupancy. Such increase in Histone H3 binding cannot be observed upon HMR removal suggesting that HMR acts downstream of CP190. Interestingly, CP190 loss impairs HMR binding to gypsy-like insulator sites but has weak effect on HMR binding to sites containing BEAF-32 recognition motifs. Notably, in contrast to BEAF-32, CP190 is not required for oogenesis, suggesting that the lack of HMR binding to the class 1 sites may be responsible for the female sterility phenotype observed in Hmr mutant flies (Gerland, 2017).

How can the current findings be integrated with the lethal phenotype of increased HMR/LHR levels in male hybrids? It is tempting to speculate that multiple additional binding sites that are observed in hybrids and on polytene chromosomes of fly strains over-expressing HMR constitute boundary regions. An increased binding to such boundaries, which have been shown to cluster and form aggregates in vivo, may trigger a massive change in nuclear architecture. In turn, this could indirectly activate multiple transposable elements similar to what is observed when centromere clustering is disturbed. Such a disturbed nuclear architecture may then trigger the activation of a cell cycle checkpoint which has been previously suggested to be a major cause of hybrid lethality (Gerland, 2017).

Altogether, these data provide a novel link between HMR and cis-regulatory elements bound by insulator proteins. It is speculated that divergent evolution of such genomic elements and their corresponding binding factors in sibling species is triggering hybrid incompatibilities (Gerland, 2017).

The hybrid incompatibility genes Lhr and Hmr are required for sister chromatid detachment during anaphase but not for centromere function

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. This study examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster In none of the mutants were defects observed in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin-heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. It is concluded that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, Hmr and Lhr were found to be required in D. melanogaster for detachment of sister chromatids during anaphase (Blum, 2017).

Limited gene misregulation is exacerbated by allele-specific upregulation in lethal hybrids between Drosophila melanogaster and Drosophila simulans

Misregulation of gene expression is often observed in interspecific hybrids and is generally attributed to regulatory incompatibilities caused by divergence between the two genomes. However, it has been challenging to distinguish effects of regulatory divergence from secondary effects including developmental and physiological defects common to hybrids. This study use RNA-Seq to profile gene expression in F1 hybrid male larvae from crosses of Drosophila melanogaster to its sibling species D. simulans. Lethal and viable hybrid males were compared, the latter produced using a mutation in the X-linked D. melanogaster Hybrid male rescue (Hmr) gene, and they were compared with their parental species and to public data sets of gene expression across development. Hmr was found to have drastically different effects on the parental and hybrid genomes, demonstrating that hybrid incompatibility genes can exhibit novel properties in the hybrid genetic background. Additionally, it was found that D. melanogaster alleles are preferentially affected between lethal and viable hybrids. It was further determined that many of the differences between the hybrids result from developmental delay in the Hmr+ hybrids. Finally, surprisingly modest expression differences were found in hybrids when compared with the parents, with only 9% and 4% of genes deviating from additivity or expressed outside of the parental range, respectively. Most of these differences can be attributed to developmental delay and differences in tissue types. Overall, this study suggests that hybrid gene misexpression is prone to overestimation and that even between species separated by approximately 2.5 Ma, regulatory incompatibilities are not widespread in hybrids (Wei, 2014).

Interspecific hybrids have been long studied because they often manifest hybrid incompatibilities that cause reproductive isolation between species. More recently, hybrids have been widely used as a genetic background for investigating gene expression divergence. However, interpreting and analyzing gene expression in hybrids presents challenges. First, because hybrid gene expression is the combination of the two parental-species alleles, determination of expression level is prone to ascertainment biases because the assembly qualities of the two species are typically not equivalent, and/or hybridization probes are designed based on only one of the parental genomes. Second, comparisons of hybrids to parental species are plagued by developmental and physiological defects that are common in hybrids. The net effect is that genes with true regulatory differences are difficult to distinguish from an amalgam of tissue- and developmental stage-specific expression differences. Third, the expected hybrid expression level can be hard to predict for genes that have diverged in expression between species. These analysis challenges may contribute to previous estimations that the proportion of the genome misregulated in hybrids can be as high as 89% (Wei, 2014).

This study attempted to account for these issues when examining gene expression in D. melanogaster/D. simulans hybrids. To accurately quantitate expression RNA-Seq reads were analyzed for species-specific SNPs to determine the allele-specific expression for all orthologous sites. Using data sets describing developmental stage differences among wild-type larvae and tissue-specific gene expression, the extent that developmental delay and gonadal degeneration in hybrids contribute to differential expression was evaluated. For comparison to parental expression, several metrics were evaluated including transgressive expression and deviation from additivity (Wei, 2014).

Wild-type hybrids (hyb-Hmr+) were found to have substantial differences from the parental species. Some of these differences are due to hybrid incompatibility rather than regulatory divergence, because viable hyb-Hmr- hybrids are closer in expression to the parental species. Furthermore, hyb-Hmr+ is more similar to earlier larvae than hyb-Hmr-, demonstrating that developmental delay is also contributing to gene expression differences. After accounting for these factors, it was found that most genes in hybrids conform to additivity and only a limited number are misregulated. These results are surprising considering that D. melanogaster and D. simulans are relatively old species with synonymous divergence of approximately 10%. It is concluded that regulatory incompatibilities may not be as wide spread as previously thought (Wei, 2014).

Using a similar framework with RNA-Seq, McManus (2010) examined the transcriptome of adult female hybrids between D. melanogaster and D. sechellia and found extensive nonadditivity. Although the discrepancy could result from the different species pairs used (and/or different sexes), this is unlikely as D. sechellia and D. simulans are closely related sister species. Instead, two other more likely causes are suggested. First, the difference may reflect the life stages investigated. Regulation of gene expression is under stronger purifying selection during development than in adulthood. As a consequence, regulatory incompatibilities may be less likely to accumulate in larvae compared with adults. Second, the hybrid adult females likely have significant physiological differences compared with their parent species because they lack ovaries, which may increase nonadditive gene expression when whole adults are sampled (Wei, 2014).

The X-chromosome has distinct properties from the autosomes. Its smaller effective population size and hemizygosity in males result in a faster rate of evolution than the autosomes, the so-called fast-X effect. Additionally, the X accumulates more hybrid sterility loci than the autosomes, known as the large-X effect. One might therefore expect that hybrids have an excess of X-linked misregulation, but the results are mixed. Sterile hybrid male mice show a disproportionate amount of X-linked upregulation, but sterile Drosophila hybrid males show the opposite, with X-linked misregulation underrepresented. These differences between species might reflect different processes of sex chromosome silencing in the male germline. For example, although there is some dispute on the issue, the X-chromosome does not appear to be strongly silenced in the Drosophila male germline. The current results show that hybrid male larvae have neither a higher nor lower proportion of X-linked genes that differ from additivity. Additionally, the lethality induced by the X-linked Hmr is also not associated with more differences among X-linked genes. Together, these findings suggest that increased X-linked misregulation is not a rule in hybrid males (Wei, 2014).

A previous microarray study revealed few genes differentially expressed between lethal and viable hybrids. In this study, RNA-Seq offered several advantages, including unbiased determination of allele-specific expression. This allowed identification of many more genes, and allele-specific effects on gene regulation in hybrids were observed. However, overlap between the two studies is small both in terms of GO terms enriched and specific genes. One likely explanation is that the current samples were from older larvae than in the previous study, such that different sets of developmental genes may be affected. Additionally, developmental differences between lethal and viable hybrids will likely become exacerbated over time, resulting in a larger set of differentially regulated genes in the current study. Nonetheless, nearly all genes shared between the two experiments show the same direction of change, potentially revealing genes implicated in causing hybrid lethality. Overall, the small number of genes identified suggests that hybrid lethality is neither caused by nor causes significant changes in gene regulation (Wei, 2014).

A small set of genes was upregulated in mel-Hmr-, indicating that Hmr functions as a negative regulator in D. melanogaster. In contrast, more genes were downregulated in hyb-Hmr-, reflecting an activating role for Hmr in hybrids. This result is unlikely to be due to developmental differences between the hybrids, because one would expect to see a similar directional bias in the modENCODE data when comparing different developmental stages. However, no such bias exists. Additionally, candidate targets of Hmr identified in D. melanogaster are not differentially expressed between the hybrids. Therefore, the current results indicate that the repressive effect of Hmr is not maintained in hybrids (Wei, 2014).

This difference between Hmr function in D. melanogaster versus hybrids was also apparent in an analysis of TEs (Satyaki, 2014). Hmr is required for TE repression in wild-type D. melanogaster, yet much higher TE expression occurs in hyb-Hmr+ compared with hyb-Hmr-. Together with the current findings, these results strongly argue that Hmr has neomorphic function in the hybrid and that the associated hybrid lethality is a gain-of-function phenotype, as suggested by earlier genetic analyses (Barbash, 2000; Orr, 2000). One scenario for this gain of function is that Hmr protein acquires new binding partners in the hybrid background, allowing it to localize to new targets and reverse its repressive activity. This is supported by the observation of mislocalization of Hmr protein in hybrids (Thomae, 2013). Given that Hmr is required to repress a wide range of heterochromatic repeats (Satyaki, 2014), hybrid lethality and the observed misregulation may result from alterations in heterochromatin that affect chromosome function (Wei, 2014).

Hmr's activating effects in hybrids is particularly intriguing, in light of the observation that hybrids have significantly more upregulation when compared with the additive expectation. It is speculated that the overexpression may be detrimental to hybrids, either broadly affecting the stoichiometry of many complexes and pathways or through misexpression of a small set of genes with large effects. The partial mitigation of this effect through downregulation of some genes in Hmr-hybrids may therefore be requisite for hybrid viability (Wei, 2014).

In the absence of Hmr in hybrids (hyb-Hmr-), the D. melanogaster alleles of many genes are downregulated, whereas the D. simulans alleles are unchanged. Because this excess is exclusive to D. melanogaster alleles, it seems unlikely to reflect general misregulation associated with hybrid death. One possible cause of this pattern is intraspecific regulatory differences in developmental genes. Because hyb-Hmr+ has just reached stage L3 while hyb-Hmr- is approaching puff stage 1 of L3, the observed mel-specific regulation may be revealing a set of genes that are differentially expressed between the two developmental time points only in D. melanogaster but not in D. simulans. This differential pattern likely results from cis-regulatory differences between the species, because both alleles are exposed to the same set of trans-factors in hybrids (Wei, 2014).

An alternative possibility is that Hmr causes allele-specific activation in hybrids. If true, it again points to a neomorphic hybrid function because the genes affected are not regulated by Hmr in pure species. Additionally, this allele-specific regulation may indicate that the interacting partner is of D. melanogaster origin. One possibility is that it is X-linked as genetic studies have suggested that, in addition to Hmr, incompatibility genes on the D. melanogaster X are required for fully penetrant hybrid lethality. It is suggested that such HI genes may be contributing to the allele-specific patterns that were observed in this study (Wei, 2014).

2.5-fold higher expression of Hmr was observed in D. simulans male larvae compared with D. melanogaster. This result is consistent with Northern blot analysis of mixed-sex larvae and also is apparent, albeit to a lesser extent (1.78-fold higher), in RNA-Seq analysis of white prepupae. Interestingly, the opposite result is seen at the protein level, with D. melanogaster being higher than D. simulans (Thomae, 2013). The discrepancy between protein and RNA levels strongly argues that Hmr levels are controlled by a complex combination of transcriptional and posttranscriptional effects, and that this regulation has changed drastically between the species. These differences are likely to be due at least in part to cis-regulatory divergence because the flanking noncoding regions of Hmr show evidence of adaptive evolution (Barbash, 2004; Wei, 2014. and references therein).

At face value, lower protein level in D. simulans predicts weaker suppression of Hmr targets. However, to the contrary, this study found that most of the genes repressed by Hmr in D. melanogaster experience a significantly stronger silencing in D. simulans. One possibility is that the stronger repression of the targets is the result of protein coding differences between the Hmr orthologs, either through stronger binding affinity to the targets or stronger recruitment of associated factors. Genetic assays have revealed that Hmr has diverged with respect to its hybrid lethal activity, as D. simulans Hmr does not cause lethality to hybrid males. The current results provide further evidence of the functional consequences of the rapid divergence of Hmr between the two species, which is likely the product of both protein-coding and expression-level differences (Wei, 2014).

Given Hmr's role in regulation of repetitive sequences, its stronger repression of targets in D. simulans has intriguing implications for the evolution of heterochromatin in the two species. Because stronger repression reduces TE and satellite DNA activity, the difference in repressive capabilities of the Hmr orthologs may contribute to a stronger defense system against selfish elements in D. simulans. This proposal is consistent with the significantly higher TE and satellite DNA content of D. melanogaster compared with D. simulans. Furthermore, it underscores that defense against selfish DNA plays a pivotal role in the evolution of genome size and architecture (Wei, 2014).

The Hmr and Lhr hybrid incompatibility genes suppress a broad range of heterochromatic repeats

Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. This study reports that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses it was discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans several satellite DNAs and TEs were identified that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. It was further found that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. These results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background (Satyaki, 2014).

Previous work has shown that Lhr (also known as HP3) interacts with HP1a. This study report that Hmr also interacts with Lhr, and both are present in a complex together with HP1a. Consistent with this interaction, many of the roles reported in this study for Lhr and Hmr have been described for HP1a, including localizing to heterochromatin, regulating TE and pericentric gene expression, and controlling telomere length. However, unlike mutations in Su(var)205 which enodes HP1a, mutations in Hmr and Lhr are viable. Furthermore, Hmr and Lhr do not localize to the 359 bp satellite which forms a substantial fraction of X-linked pericentric heterochromatin. These findings suggest that Hmr and Lhr are not ubiquitous heterochromatin proteins, leaving open the intriguing question of what guides their localization specificity (Satyaki, 2014).

The interaction of Hmr and Lhr with HP1a has recently been independently reported. Thomae (2013) also report other findings similar to the current observations including repressive effects of Hmr and Lhr on TEs in somatic tissues and their localization to telomeres. Several conclusions are similar between the two studies and with previously published conclusions. Thomae observe upregulation of TEs in hybrids but conclude that they are unlikely to be the direct cause of hybrid lethality, a conclusion that was reach in this study using different methods. Their conclusion that hybrids are highly sensitive to Hmr dosage is in concordance with previous studies, such as the previous observation that a ~9.7 kb Hmr+ transgene causes dosage-dependent lethality to hybrid females (Barbash, 2003). This conclusion also fits well with the discovery that hybrids are highly sensitive to Lhr dosage (Satyaki, 2014).

One area of possible discrepancy is the viability effects and cellular phenotypes associated with Hmr and Lhr mutants versus RNAi knockdown. Thomae (2013) reports a high rate of mitotic defects in Lhr RNAi knockdown tissue culture cells, yet this study found that LhrKO flies are almost fully viable, as are Lhr RNAi knockdown animals. This study also has not observed the lethality or morphological defects in Hmr mutants that are reported for Hmr RNAi cells and animals. For example, Aruna (2009) found reduced longevity but no effect on viability up to eclosion of flies carrying the Df(1)Hmr- allele, a deletion of the 5' end of Hmr. Further work is necessary to determine if these discrepancies reflect phenotypes associated with the use of RNA interference or differences between assaying whole animals versus tissue-culture cells, such as the aneuploid state of cultured cell lines (Satyaki, 2014).

Several HIs involve heterochromatin proteins or heterochromatic sequences, leading to the suggestion that genetic conflicts between selfish DNAs and host fitness are an important force that is driving the evolution of HI (Satyaki, 2014 and references therein).

TE and satellite abundance varies widely among species and is a major contributor to genome-size variation. The evolutionary causes of this variation have been widely debated for many years. When considering genetic conflict theories, it is important to first exclude alternative evolutionary causes of repetitive DNA variation. One explanation is neutrality, with repeat variation governed by mutational processes, in particular the balance between insertions and deletions. Insertion/deletion models are particularly appropriate for inactive and degenerate TEs, and perhaps also for certain classes of satellites that are no longer homogenized by concerted evolution (Satyaki, 2014).

Selectionist models fit better for active repeats, and must be invoked if the adaptive evolution of heterochromatin proteins is proposed to reflect co-evolution with repetitive DNA. One model is that some repeats are co-opted for host functions. Drosophila's telomeric retrotransposons are a relevant example that is discussed below. Three, non-mutually exclusive selective costs associated with repetitive DNA are considered when discussing the evolution of Hmr and Lhr (Satyaki, 2014).

One potential cost arises from the overall load of repetitive DNAs, including increased genome size and instability. A second is direct genetic conflict. Genetic conflict is defined here to refer to fitness costs imposed by selfish DNAs that have evolved specific mechanisms to increase their transmission. Such conflicts could be caused by highly active individual repeats, for example during hybrid dysgenesis caused by introduction of a TE family into naive strains. Finally, genetic conflicts can have indirect costs, such as pleiotropic fertility defects caused by repeat expansions involved in meiotic drive. They infect most genomes, can self-mobilize and increase their copy number, and destabilize genomes via spontaneous mutations, ectopic recombination, and deleterious increases in genome size. Adaptive evolution of TE-defense genes can therefore be readily interpreted as the host species responding to the fitness cost of TEs (Satyaki, 2014).

Like Hmr and Lhr, many piRNA pathway genes are also evolving under positive selection. This raises the possibility that Lhr and Hmr are co-evolving with the piRNA pathway proteins. However, the lack of major perturbations in the piRNA pool in LhrKO suggests that Lhr and Hmr function downstream or independently of piRNA biogenesis. Piwi, guided by piRNA, has been proposed to recruit repressive heterochromatin components including HP1a and histone methyl transferases to transposable elements. One possibility is that Lhr and Hmr function downstream of HP1a to repress TEs via RNA degradation machinery such as the nuclear exosome (Satyaki, 2014).

It is noted that Ago3 is moderately down-regulated in both LhrKO (3.4 fold) and Hmr- (~2 fold), likely because the gene is peri-centromeric. Two results demonstrate that this modest reduction in Ago3 cannot explain the broad effects on TEs in Hmr and Lhr mutants. First, Ago3 expression is unaffected in D. simulans Lhr1, which also shows widespread TE derepression. Second, Ago3 mutants have major disturbances to their piRNA pool, which was not observed in LhrKO (Satyaki, 2014).

While TE repression is typically viewed in terms of genetic conflicts, the relationship between Lhr, Hmr and the telomeric TEs resembles symbiosis. These TEs have been domesticated by Drosophila species for tens of millions of years to serve a vital host function, and thus are not considered selfish DNA. The telomeric TEs were among the most strongly derepressed in Hmr and Lhr mutants, in some cases more than 100 fold. Increases were also observed in HeT-A DNA copy number in Hmr and Lhr stocks. Increased telomeric TE expression does not necessarily increase HeT-A DNA copy number and cause longer telomeres, suggesting that multiple factors control telomere length. If so, then Lhr and Hmr must control multiple processes at the telomere. This is supported by the localization of both proteins to the telomere cap, a protective structure that prevents telomere fusions. The strong reduction in LhrKO of piRNAs from three TAS-repeat containing sub-telomeric piRNA clusters is particularly intriguing. piRNA production from clusters is dependent on them maintaining a heterochromatic state, which could explain why Lhr is required for TAS piRNA expression while it acts as a repressor in most other circumstances (Satyaki, 2014).

This study discovered several striking examples that suggest species-specific co-evolution of Hmr and Lhr with satellite DNAs. D. melanogaster Hmr and Lhr proteins were found to localize to and repress transcripts from GA-rich satellites. GA-rich satellites are ~8 fold less abundant in D. simulans but are cytologically detectable; nevertheless it was found that sim-Lhr does not localize to them. GA-rich satellites also have low abundance in the outgroup species D. erecta, implying that the differential abundance with D. simulans reflects an increase in D. melanogaster. Similarly it was discovered that mel-Lhr-HA localizes to AACAC in D. melanogaster, a repeat that is absent in D. simulans. Furthermore, moderate up-regulation of several other satellite transcripts was detected only in D. melanogaster. These results suggest that Lhr and Hmr may have evolved in D. melanogaster to mitigate the deleterious consequences of satellite expansion, which can include ectopic recombination, increased genome size, and destabilized chromosome segregation (Satyaki, 2014).

Satellite transcripts have been reported from various tissues in wild type D. melanogaster but little is known about their production. They could be products of either non-specific transcription or read-through from adjacent TEs. Increased levels of satellite transcripts are observed in D. melanogaster spn-E mutants, suggesting that RNA interference or piRNA pathways control satellite transcript levels (Satyaki, 2014).

At a broad scale, Lhr and Hmr from both D. melanogaster and D. simulans regulate heterochromatic repetitive DNAs but very few genes. This finding is consistent with previous analyses demonstrating that some functions of these genes are conserved between species. But many of the repeats regulated by Lhr and Hmr are rapidly evolving, raising the question of whether specific repetitive DNAs are directly driving the adaptive evolution of the Lhr and Hmr coding sequences between species. A simple prediction is that D. simulans orthologs should fail to fully repress such repeats when placed into D. melanogaster, a prediction that was tested for Hmr (Satyaki, 2014).

The BS non-LTR retrotransposon is significantly derepressed in D. melanogaster Hmr- and LhrKO, and in D. simulans Lhr1 mutants. Interestingly, BS appears to be transpositionally active in D. melanogaster but inactive in D. simulans. One interpretation is that BS was active in the common ancestor and regulated by Hmr and Lhr. The genes would continue to co-evolve with BS in D. melanogaster, making the sim-Hmr ortholog less effective at repressing BS elements in D. melanogaster. In this scenario Hmr and Lhr are engaged in a recurrent genetic conflict with BS elements that leads to their sequence divergence. Consistent with this prediction, significantly higher expression was found in Hmr-; ø{sim-Hmr-FLAG}/+ compared to Hmr-; ø{mel-Hmr-FLAG}/+ (Satyaki, 2014).

Copia shows a different pattern, with ~20-fold up-regulation in LhrKO but only ~2-fold in Lhr1 (and only when mapping to the consensus-sequence database), as well as significant derepression in Hmr-. Copia expression level can be high in D. melanogaster but is variable among populations. In contrast, copia elements in D. simulans typically contain deletions in regulatory elements required for expression, and transcripts are undetectable by Northern blot analysis. These results suggest that Hmr and Lhr could be D. melanogaster host factors that defend against a TE that is currently active within the species. However, copia was fully repressed in Hmr-; ø{sim-Hmr-FLAG}/+, demonstrating that adaptive divergence of Hmr by itself does not affect copia regulation (Satyaki, 2014).

Overall, this study found surprisingly few cases of overexpression associated with Hmr divergence, including no effects on satellite DNAs. It is also noted that most of the TEs identified other than BS and Doc6 are likely transpositionally inactive in D. melanogaster, which makes it more challenging to fit a scenario of direct and recurrent evolution between Hmr and specific TEs (Satyaki, 2014).

Several possible interpretations of these results are suggested. One is that Hmr and Lhr adaptive divergence is in fact driven largely or solely by BS and/or Doc6, a hypothesis that will require understanding the mechanism by which Hmr and Lhr affect expression of these TEs. Second is that Hmr and Lhr may be co-evolving with other genes, and that multiple diverged genes need to be replaced simultaneously in order to detect their effects on other TEs and satellite DNAs. Third is that more sensitive assays are needed, for example monitoring TE transposition rates over multiple generations. A fourth possibility is an alternative to genetic conflict scenarios that arises from population-genetic models. These models suggest that the fitness costs of individual TE families are likely extremely weak under most circumstances. The adaptive evolution of repressor proteins may therefore reflect the cumulative load of repeats within a genome. This alternative view could be applicable to Hmr and Lhr since they repress a large number of TEs and satellites. Finally, Hmr and Lhr may have additional unidentified phenotypes that are also the targets of adaptive evolution (Satyaki, 2014).

D. simulans has a smaller genome with ~4-fold less satellite DNA and significantly fewer TEs compared to D. melanogaster. This large difference in repeat content between D. melanogaster and D. simulans may have wider consequences. Reduced expression from pericentric heterochromatin genes was found in Hmr and Lhr mutants in D. melanogaster. This reduction may reflect the fact that pericentric genes have evolved to use heterochromatin proteins such as Lhr and Hmr to maintain gene expression in a repeat-rich environment. Pericentric genes in species with fewer repeats would presumably not require these proteins. Consistent with this model, this study found that Lhr loss in D. simulans has a negligible impact on pericentric gene expression. This finding suggests that Lhr and Hmr have an adaptive role in blocking effects on gene expression arising from increasing repetitive DNA copy number (Satyaki, 2014).

If each genome is uniquely adapted to its repetitive DNA content, then the shock of hybridization may lead to misregulation of TEs and satellites. TEs are activated in various animal and plant hybrids but the consequences, if any, for hybrid fitness are largely unclear. This study found substantial TE misregulation in hybrid male larvae. Since these hybrids are agametic, this TE expression comes from somatic tissues. The fitness cost of this upregulation is unclear as somatic TE overexpression is not necessarily lethal within D. melanogaster. Comparison of lethal Hmr+ and viable Hmr- hybrid males demonstrates that lethal hybrids have more TE expression than the viable hybrids, which in turn have more TE expression than either of its parents. However, this TE misregulation seems unconnected with Hmr as the TEs differentially expressed between Hmr+ and Hmr- hybrid male larvae are largely distinct from those between Hmr+ and Hmr- D. melanogaster male larvae. Further, while Hmr- causes rampant TE over-expression within D. melanogaster, it is associated with reduced TE levels in hybrids. These observations argue that the TE derepression in hybrids is unrelated to the pure species function of Hmr. This finding is consistent with previous genetic studies that demonstrate that the wild type Hmr+ allele causes hybrid lethality and thus behaves as a gain-of-function allele in hybrids. More generally it underscores the unique nature of the hybrid genetic background. Somatic TE overexpression may result from breakdown in the siRNA or piRNA pathways due to incompatibilities among multiple rapidly evolving TE regulators (Satyaki, 2014).

One clear example is known where a species-specific difference in a satellite DNA causes incompatibility between Drosophila species. But the toll caused by heterochromatic differences may more commonly be indirect, as heterochromatin proteins diverge in response to changes in heterochromatic DNA repeats. Recent work suggests that hybrid female sterility may be caused by incompatibilities among rapidly evolving piRNA proteins rather than by species-specific differences in TEs. It is suggested that the role of Hmr and Lhr in regulating the activity of three highly dynamic classes of heterochromatin has led to their recurrent adaptive evolution, and secondarily, to their involvement in interspecific hybrid lethality (Satyaki, 2014).

A screen for F1 hybrid male rescue reveals no major-effect hybrid lethality loci in the Drosophila melanogaster autosomal genome

Hybrid sons between Drosophila melanogaster females and D. simulans males die as 3rd instar larvae. Two genes, D. melanogaster Hybrid male rescue (Hmr) on the X chromosome, and D. simulans Lethal hybrid rescue (Lhr) on chromosome II, interact to cause this lethality. Loss-of-function mutations in either gene suppress lethality, but several pieces of evidence suggest that additional factors are required for hybrid lethality. This study screened the D. melanogaster autosomal genome by using the Bloomington Stock Center Deficiency kit to search for additional regions that can rescue hybrid male lethality. This screen is designed to identify putative hybrid incompatibility (HI) genes similar to Hmr and Lhr which, when removed, are dominant suppressors of lethality. After screening 89% of the autosomal genome, no regions were found that rescue males to the adult stage. Several regions, however, were identified that rescue up to 13% of males to the pharate adult stage. This weak rescue suggests the presence of multiple minor-effect HI loci, but it was not possible to map these loci to high resolution, presumably because weak rescue can be masked by genetic background effects. Attempts were made to test one candidate, the dosage compensation gene male specific lethal-3 (msl-3), by using RNA interference with short hairpin microRNA constructs targeted specifically against D. simulans msl-3, but knockdown was not achieved, in part due to off-target effects. It is concluded that the D. melanogaster autosomal genome likely does not contain additional major-effect HI loci. It was also shown that Hmr is insufficient to fully account for the lethality associated with the D. melanogaster X chromosome, suggesting that additional X-linked genes contribute to hybrid lethality (Cuykendall, 2014).

A pair of centromeric proteins mediates reproductive isolation in Drosophila species

Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. However, the molecular basis of hybrid lethality and the evolutionary driving forces that provoke it remain largely elusive. The Hybrid male rescue (Hmr) and the Lethal hybrid rescue (Lhr) genes serve as a model to study speciation in Drosophilids because their interaction causes lethality in male hybrid offspring. This study shows that HMR and LHR form a centromeric complex necessary for proper chromosome segregation. The Hmr expression level is substantially higher in Drosophila melanogaster, whereas Lhr expression levels are increased in Drosophila simulans. The resulting elevated amount of HMR/LHR complex in hybrids results in an extensive mislocalization of the complex, an interference with the regulation of transposable elements, and an impairment of cell proliferation. These findings provide evidence for a major role of centromere divergence in the generation of biodiversity (Thomae, 2013).

Hmrmel and Lhrsim constitute members of a few identified examples of genes that form a classical Dobzhansky-Muller gene pair and mediate postzygotic isolation of the two closely related species D. mel and D. sim. This study shows that the gene products of Hmr and Lhr form a complex in D. mel with an important centromeric function. This function is exquisitely dose dependent as an increase as well as a decrease of complex levels result in an increased number of mitotic defects. At the same time, an increase was observed in the number of transcripts derived from TEs upon alteration of the complex levels, suggesting that HMR/LHR has a function in setting up a repressive chromatin structure at these genomic regions. Although it has not been proven that the increased transcription from the transposable elements is the main cause for the mitotic defects, the centromeric binding pattern of the complex in mitotically cycling cells, its function in interphase, and the fact that a heterochromatic structure is beneficial for the generation of a functional centromere suggest that the HMR/LHR complex may contribute to a functional chromatin structure at the centromere. On first glance, the strong effects of an HMR depletion seen in cell culture as well as in fly strains expressing an Hmr RNAi construct would have predicted a stronger phenotype of D. mel flies carrying Hmr mutations than the one reported by Barbash (Aruna, 2009). At least for the result in cell lines, off-target effects were excluded because two independently derived RNAi constructs were used with a similar outcome. In flies, it may well be that compensatory mechanisms can at least partially substitute HMRs function at centromeres, leading to a less pronounced effect. Compared to classical mutations, such compensatory effects are less frequent in knockdown experiments, which may also be the cause of the difference in viability (Thomae, 2013).

Based on the fact that HMR and LHR show strong signatures of positive selection and an Hmrsim transgene does not cause hybrid male lethality, Barbash and colleagues proposed that Hmrmel causes hybrid incompatibilities as a consequence of primary amino acid sequence divergence (Barbash, 2003, Maheshwari, 2008). Evidence from the current study that the HMR/LHR complex is not crucial for proper centromere function in D. sim cells might partially lend support for such a functional divergence. On the other hand, the orthologs from both species behave virtually identical in all other tested assays. Considering that residues in HMR that are conserved between species are critical for hybrid lethality and that Hmrsim can partially rescue the fertility defect of D.mel Hmr mutant females (Aruna, 2009), an alternative model is proposed. The current data strongly support a scenario in which the asymmetric lethal effects of Hmrmel and Lhrsim, respectively, are due to the divergence in regulatory pathways that modulate their levels, which is the most apparent difference between the orthologs that was identified (Thomae, 2013).

The driving force that led to the increased expression of Hmr in D. mel is subject to speculation. Considering the finding that HMR/LHR levels are critical for setting up a repressive chromatin structure at centromeric regions, it is striking that D. sim and D. mel strongly differ in the number of TEs, and these elements are highly enriched at centromeres. Interestingly, whereas most of the copies are degraded to small fragments in D. sim, D. mel contains substantially more intact copies. This might be reflected in higher levels of HMR/LHR complex in D. mel and its crucial role in centromere functionality in tissues with mitotic cell cycles (Thomae, 2013).

The species inverted higher expression of Hmr and Lhr results in increased complex amounts relative to its target sites, which are diluted in the hybrid genetic background. It is proposed that this misbalance results in the lethal gain of function in hybrids. This model also implicates that factors that influence the abundance of the complex are modifiers of hybrid lethality. In fact, early genetic experiments hinted toward such modifiers. For instance, hybrid males from D. mel mothers and D. sim fathers are not lethal if they carry two third chromosomes from D. mel (3mel). This implies that either a sensitizer locus on 3sim or a haploinsufficient suppressor locus on 3mel exists. The suppressor model, in which a negative regulator of complex abundance is diluted in hybrids, is favored. This hypothesis is based on the observation that Lhrsim does not require Hmrsim for its high levels in D. sim, but its abundance depends on the presence of Hmrmel in hybrids. This becomes apparent by the decreased level of Lhrsim in Hmr3 mutant hybrids (Thomae, 2013).

Complete lethality further requires the presence of the D.mel X chromosome as the sole presence of an autosomal copy of Hmrmel does not kill male hybrids carrying an Xsim. In this respect, it was already postulated that disturbed dosage compensation may cause hybrid male lethality due to species-specific divergence of the involved components. It has been demonstrated that the D. mel dosage compensation system shows particularly strong signatures of positive selection, which may render the D. sim DCC components incompetent to properly compensate the D. mel X chromosome. In contrast, another study postulated that a key component of dosage compensation is not expressed in the lethal hybrid males. This conclusion was based on the failure to detect MSL2 on lethal hybrid male X chromosomes with an Xmel, but not with an Xsim. The latter findings are in contrast to the current results, as this study detected X-chromosome-specific binding of MSL2 on Xmel/Ysim hybrid male polytene chromosomes. This discrepancy might be due to a different fixation procedure or the use of a more sensitive antibody. It is important to note, however, that based on the current data, the existence of subtle differences in DCC function in lethal male hybrids cannot be fully excluded. The possibility has been examined that impaired dosage compensation causes hybrid male inviability making use of different D. mel dosage compensation complex (DCC) mutants. These mutations rather increase than decrease hybrid male viability. Furthermore, considering that female hybrid lethality is higher at elevated temperatures in an Hmr-dependent manner, another plausible scenario has been put forward in which hybrid lethality is caused by a disturbed chromatin state of Xmel. In fact, chromatin structure of the male X is known to be extremely sensitive toward the amount of heterochromatin proteins. Strikingly, two of the factors that strongly affect X chromosome morphology (HP1a and Su(var)3-7) copurify with HMR and LHR. Alternatively, global HMR/LHR-induced changes in chromatin structure, increases in mitotic defects, or deregulation of TEs might trigger a cell-cycle checkpoint leading to the observed cell-cycle arrest (Thomae, 2013).

In summary, the current experiments underscore the importance of tight regulation of protein levels to sustain their functional capacity. this study shows that altered expression levels of the DM pair Hmr and Lhr in hybrids result in detrimental problems concerning centromere function and silencing of transposable elements. The combination of these defects finally results in the observed lethality of hybrids from D. mel und D. sim, whereby HMR and LHR contribute to the reproductive isolation of the two species (Thomae, 2013).

An indel polymorphism in the hybrid incompatibility gene lethal hybrid rescue of Drosophila is functionally relevant

Hybrid incompatibility (HI) genes are frequently observed to be rapidly evolving under selection. This observation has led to the attractive conjecture that selection-derived protein-sequence divergence is culpable for incompatibilities in hybrids. The Drosophila simulans HI gene Lethal hybrid rescue (Lhr) is an intriguing case, because despite having experienced rapid sequence evolution, its HI properties are a shared function inherited from the ancestral state. Using an unusual D. simulans Lhr hybrid rescue allele, Lhr2, this study identified a conserved stretch of 10 amino acids in the C terminus of LHR that is critical for causing hybrid incompatibility. Altering these 10 amino acids weakens or abolishes the ability of Lhr to suppress the hybrid rescue alleles Lhr1 or Mhr1, respectively. Besides single-amino-acid substitutions, Lhr orthologs differ by a 16-aa indel polymorphism, with the ancestral deletion state fixed in D. melanogaster and the derived insertion state at very high frequency in D. simulans. Lhr2 is a rare D. simulans allele that has the ancestral deletion state of the 16-aa polymorphism. Through a series of transgenic constructs this study demonstrates that the ancestral deletion state contributes to the rescue activity of Lhr2. This indel is thus a polymorphism that can affect the HI function of Lhr (Maheshwari, 2012).

Functional conservation of the Drosophila hybrid incompatibility gene Lhr

Hybrid incompatibilities such as sterility and lethality are commonly modeled as being caused by interactions between two genes, each of which has diverged separately in one of the hybridizing lineages. The gene Lethal hybrid rescue (Lhr) encodes a rapidly evolving heterochromatin protein that causes lethality of hybrid males in crosses between Drosophila melanogaster females and D. simulans males. Previous genetic analyses showed that hybrid lethality is caused by D. simulans Lhr but not by D. melanogaster Lhr, confirming a critical prediction of asymmetry in the evolution of a hybrid incompatibility gene. This study examined the functional properties of Lhr orthologs from multiple Drosophila species, including interactions with other heterochromatin proteins, localization to heterochromatin, and ability to complement hybrid rescue in D. melanogaster/D. simulans hybrids. These properties are conserved among most Lhr orthologs, including Lhr from D. melanogaster, D. simulans and the outgroup species D. yakuba. It is concluded that evolution of the hybrid lethality properties of Lhr between D. melanogaster and D. simulans did not involve extensive loss or gain of functions associated with protein interactions or localization to heterochromatin (Brideau, 2011).

Reduced fertility of Drosophila melanogaster hybrid male rescue (Hmr) mutant females is partially complemented by Hmr orthologs from sibling species

The gene Hybrid male rescue (Hmr) causes lethality in interspecific hybrids between Drosophila melanogaster and its sibling species. Hmr has functionally diverged for this interspecific phenotype because lethality is caused specifically by D. melanogaster Hmr but not by D. simulans or D. mauritiana Hmr. Hmr was identified by the D. melanogaster partial loss-of-function allele Hmr1, which suppresses hybrid lethality but has no apparent phenotype within pure-species D. melanogaster. This study has investigated the possible function of Hmr in D. melanogaster females using stronger mutant alleles. Females homozygous for Hmr mutants have reduced viability posteclosion and significantly reduced fertility. Reduced fertility of Hmr mutants is caused by a reduction in the number of eggs laid as well as reduced zygotic viability. Cytological analysis reveals that ovarioles from Hmr mutant females express markers that distinguish various stages of wild-type oogenesis, but that developing egg chambers fail to migrate posteriorly. D. simulans and D. mauritiana Hmr+ partially complement the reduced fertility of a D. melanogaster Hmr mutation. This partial complementation contrasts with the complete functional divergence previously observed for the interspecific hybrid lethality phenotype. This study also investigated the molecular basis of hybrid rescue associated with a second D. melanogaster hybrid rescue allele, In(1)AB. In(1)AB is mutant for Hmr function, likely due to a missense mutation in an evolutionarily conserved amino acid. Two independently discovered hybrid rescue mutations are therefore allelic (Aruna, 2009).

Recurrent positive selection of the Drosophila hybrid incompatibility gene Hmr

Lethality in hybrids between Drosophila melanogaster and its sibling species Drosophila simulans is caused in part by the interaction of the genes Hybrid male rescue (Hmr) and Lethal hybrid rescue (Lhr). Hmr and Lhr have diverged under positive selection in the hybridizing species. This study tested whether positive selection of Hmr is confined only to D. melanogaster and D. simulans. Hmr was found to continue to diverge under recurrent positive selection between the sibling species D. simulans and Drosophila mauritiana and along the lineage leading to the melanogaster subgroup species pair Drosophila yakuba and Drosophila santomea. Hmr encodes a member of the Myb/SANT-like domain in ADF1 (MADF) family of transcriptional regulators. This study show that although MADF domains from other Drosophila proteins have predicted ionic properties consistent with DNA binding, the MADF domains encoded by different Hmr orthologs have divergent properties consistent with binding to either the DNA or the protein components of chromatin. These results suggest that Hmr may be functionally diverged in multiple species (Maheshwari, 2008).

Two Dobzhansky-Muller genes interact to cause hybrid lethality in Drosophila

The Dobzhansky-Muller model proposes that hybrid incompatibilities are caused by the interaction between genes that have functionally diverged in the respective hybridizing species. This study shows that Lethal hybrid rescue (Lhr) has functionally diverged in Drosophila simulans and interacts with Hybrid male rescue (Hmr), which has functionally diverged in D. melanogaster, to cause lethality in F1 hybrid males. LHR localizes to heterochromatic regions of the genome and has diverged extensively in sequence between these species in a manner consistent with positive selection. Rapidly evolving heterochromatic DNA sequences may be driving the evolution of this incompatibility gene (Brideau, 2006).

Functional divergence caused by ancient positive selection of a Drosophila hybrid incompatibility locus

Interspecific hybrid lethality and sterility are a consequence of divergent evolution between species and serve to maintain the discrete identities of species. The evolution of hybrid incompatibilities has been described in widely accepted models by Dobzhansky and Muller where lineage-specific functional divergence is the essential characteristic of hybrid incompatibility genes. Experimentally tractable models are required to identify and test candidate hybrid incompatibility genes. Several Drosophila melanogaster genes involved in hybrid incompatibility have been identified but none has yet been shown to have functionally diverged in accordance with the Dobzhansky-Muller model. By introducing transgenic copies of the X-linked Hybrid male rescue (Hmr) gene into D. melanogaster from its sibling species D. simulans and D. mauritiana, this study demonstrates that Hmr has functionally diverged to cause F1 hybrid incompatibility between these species. Consistent with the Dobzhansky-Muller model, Hmr has diverged extensively in the D. melanogaster lineage, but extensive divergence was found in the sibling-species lineage. Together, these findings implicate over 13% of the amino acids encoded by Hmr as candidates for causing hybrid incompatibility. The exceptional level of divergence at Hmr cannot be explained by neutral processes because this study used phylogenetic methods and population genetic analyses to show that the elevated amino-acid divergence in both lineages is due to positive selection in the distant past-at least one million generations ago. These findings suggest that multiple substitutions driven by natural selection may be a general phenomenon required to generate hybrid incompatibility alleles (Barbash, 2004).

A rapidly evolving MYB-related protein causes species isolation in Drosophila

Matings among different species of animals or plants often result in sterile or lethal hybrids. Identifying the evolutionary forces that create hybrid incompatibility alleles is fundamental to understanding the process of speciation, but very few such alleles have been identified, particularly in model organisms that are amenable to experimental manipulation. This study reports the cloning of the first Drosophila melanogaster gene involved in hybrid incompatibilities, Hybrid male rescue (Hmr). Hmr causes lethality and female sterility in hybrids among D. melanogaster and its sibling species. Hmr encodes a protein with homology to a family of MYB-related DNA-binding transcriptional regulators. The HMR protein has evolved both amino acid substitutions and insertions and deletions at an extraordinarily high rate between D. melanogaster and its sibling species, including in its predicted DNA-binding domain. These results suggest that hybrid lethality may result from disruptions in gene regulation, and it is also proposed that rapid evolution may be a hallmark of speciation genes in general (Barbashi, 2003).

Genetic analysis of the hybrid male rescue locus of Drosophila

Several hybrid rescue mutations-alleles that restore the viability of normally lethal hybrids-have been discovered in Drosophila melanogaster and its relatives. This study analyzed one of these genes, Hybrid male rescue (Hmr), asking two questions about its role in hybrid inviability. (1) Does the wild-type allele from D. melanogaster (Hmrmel) cause hybrid embryonic inviability? (2) Does Hmrmel cause hybrid larval inviability? The results show that the wild-type product of Hmr is neither necessary nor sufficient for hybrid embryonic inviability. Hmrmel does, however, appear to lower the viability of hybrid larvae. The data further suggest (though do not prove) that Hmrmel acts as a gain-of-function poison in hybrids. These findings support previous claims that hybrid embryonic and larval lethalities are genetically distinct and suggest that Hmrmel is at least one of the proximate causes of hybrid larval inviability (Orr, 2000).

The Drosophila melanogaster hybrid male rescue gene causes inviability in male and female species hybrids

The Drosophila melanogaster mutation Hmr rescues inviable hybrid sons from the cross of D. melanogaster females to males of its sibling species D. mauritiana, D. simulans, and D. sechellia. This study extended previous observations that hybrid daughters from this cross are poorly viable at high temperatures and have shown that this female lethality is suppressed by Hmr and the rescue mutations In(1)AB and D. simulans Lhr. Deficiencies defined in this study as Hmr(-) also suppressed lethality, demonstrating that reducing Hmr(+) activity can rescue otherwise inviable hybrids. An Hmr(+) duplication had the opposite effect of reducing the viability of female and sibling X-male hybrid progeny. Similar dose-dependent viability effects of Hmr were observed in the reciprocal cross of D. simulans females to D. melanogaster males. Finally, Lhr and Hmr(+) were shown to have mutually antagonistic effects on hybrid viability. These data suggest a model where the interaction of sibling species Lhr(+) and D. melanogaster Hmr(+) causes lethality in both sexes of species hybrids and in both directions of crossing. These results further suggest that a twofold difference in Hmr(+) dosage accounts in part for the differential viability of male and female hybrid progeny, but also that additional, unidentified genes must be invoked to account for the invariant lethality of hybrid sons of D. melanogaster mothers. Implications of these findings for understanding Haldane's rule-the observation that hybrid breakdown is often specific to the heterogametic sex-are also discussed (Barbash, 2000).


Search PubMed for articles about Drosophila Hybrid male rescue & Lethal hybrid rescue

Aruna, S., Flores, H. A. and Barbash, D. A. (2009). Reduced fertility of Drosophila melanogaster Hybrid male rescue (Hmr) mutant females is partially complemented by Hmr orthologs from sibling species. Genetics 181(4): 1437-1450. PubMed ID: 19153254

Barbash, D. A., Roote, J. and Ashburner, M. (2000). The Drosophila melanogaster hybrid male rescue gene causes inviability in male and female species hybrids. Genetics 154(4): 1747-1771. PubMed ID: 10747067

Barbash, D. A., Siino, D. F., Tarone, A. M. and Roote, J. (2003). A rapidly evolving MYB-related protein causes species isolation in Drosophila. Proc Natl Acad Sci U S A 100(9): 5302-5307. PubMed ID: 12695567

Barbash, D. A., Awadalla, P. and Tarone, A. M. (2004). Functional divergence caused by ancient positive selection of a Drosophila hybrid incompatibility locus. PLoS Biol 2(6): e142. PubMed ID: 15208709

Blum, J. A., Bonaccorsi, S., Marzullo, M., Palumbo, V., Yamashita, Y. M., Barbash, D. A. and Gatti, M. (2017). The Hybrid Incompatibility genes Lhr and Hmr are required for sister chromatid detachment during anaphase but not for centromere function. Genetics 207(4): 1457-1472. PubMed ID: 29046402

Bosco, G., Campbell, P., Leiva-Neto, J. T. and Markow, T. A. (2007). Analysis of Drosophila species genome size and satellite DNA content reveals significant differences among strains as well as between species. Genetics 177(3): 1277-1290. PubMed ID: 18039867

Brideau, N. J., Flores, H. A., Wang, J., Maheshwari, S., Wang, X. and Barbash, D. A. (2006). Two Dobzhansky-Muller genes interact to cause hybrid lethality in Drosophila. Science 314(5803): 1292-1295. PubMed ID: 17124320

Brideau, N. J. and Barbash, D. A. (2011). Functional conservation of the Drosophila hybrid incompatibility gene Lhr. BMC Evol Biol 11: 57. PubMed ID: 21366928

Cuykendall, T. N., Satyaki, P., Ji, S., Clay, D. M., Edelman, N. B., Kimchy, A., Li, L. H., Nuzzo, E. A., Parekh, N., Park, S. and Barbash, D. A. (2014). A screen for F1 hybrid male rescue reveals no major-effect hybrid lethality loci in the Drosophila melanogaster autosomal genome. G3 (Bethesda) 4(12): 2451-2460. PubMed ID: 25352540

Gerland, T.A., Sun, B., Smialowski, P., Lukacs, A., Thomae, A.W. and Imhof, A. (2017). The Drosophila speciation factor HMR localizes to genomic insulator sites. PLoS One 12: e0171798. PubMed ID: 28207793

Maheshwari, S., Wang, J. and Barbash, D. A. (2008). Recurrent positive selection of the Drosophila hybrid incompatibility gene Hmr. Mol Biol Evol 25(11): 2421-2430. PubMed ID: 18755760

Maheshwari, S. and Barbash, D. A. (2012). An indel polymorphism in the hybrid incompatibility gene lethal hybrid rescue of Drosophila is functionally relevant. Genetics 192(2): 683-691. PubMed ID: 22865735

McManus, C. J., Coolon, J. D., Duff, M. O., Eipper-Mains, J., Graveley, B. R. and Wittkopp, P. J. (2010). Regulatory divergence in Drosophila revealed by mRNA-seq. Genome Res 20(6): 816-825. PubMed ID: 20354124

Olszak, A. M., van Essen, D., Pereira, A. J., Diehl, S., Manke, T., Maiato, H., Saccani, S. and Heun, P. (2011). Heterochromatin boundaries are hotspots for de novo kinetochore formation. Nat Cell Biol 13(7): 799-808. PubMed ID: 21685892

Orr, H. A. and Irving, S. (2000). Genetic analysis of the hybrid male rescue locus of Drosophila. Genetics 155(1): 225-231. PubMed ID: 10790397

Satyaki, P. R., Cuykendall, T. N., Wei, K. H., Brideau, N. J., Kwak, H., Aruna, S., Ferree, P. M., Ji, S. and Barbash, D. A. (2014). The Hmr and Lhr hybrid incompatibility genes suppress a broad range of heterochromatic repeats. PLoS Genet 10(3): e1004240. PubMed ID: 24651406

Sawamura, K. (2012). Chromatin evolution and molecular drive in speciation. Int J Evol Biol 2012: 301894. PubMed ID: 22191063

Stark, A., et al. (2007). Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures. Nature 450(7167): 219-232. PubMed ID: 17994088

Thomae, A. W., Schade, G. O., Padeken, J., Borath, M., Vetter, I., Kremmer, E., Heun, P. and Imhof, A. (2013). A pair of centromeric proteins mediates reproductive isolation in Drosophila species. Dev Cell 27(4): 412-424. PubMed ID: 24239514

Wei, K. H., Clark, A. G. and Barbash, D. A. (2014). Limited gene misregulation is exacerbated by allele-specific upregulation in lethal hybrids between Drosophila melanogaster and Drosophila simulans. Mol Biol Evol 31(7): 1767-1778. PubMed ID: 24723419

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date revised: 20 February, 2018

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