BACKGROUND
the biological species concept is a widely accepted definition of species in which species are defined as groups of interbreeding populations that are reproductively isolated from other groups  <cit> .
hybrids between closely related species are often inviable or sterile, and this inviability and sterility is collectively called hybrid incompatibility .
crosses between drosophila melanogaster and its sibling species d. simulans produce incompatible hybrids.
in the cross of d. melanogaster females to d. simulans males, the f <dig> daughters are semi-viable but sterile, and the sons are inviable  <cit> .
two genes, hybrid male rescue  in d. melanogaster, and lethal hybrid rescue  in d. simulans, are major-effect contributors to hybrid incompatibility on the basis that loss-of-function mutations in either gene can suppress hybrid male lethality  <cit> .
lhr encodes a small protein  and appears to be drosophila specific, as we are unable to identify homologs in vertebrates or in other invertebrates, including in other insect genera  <cit> .
furthermore, among drosophila species the coding sequence of lhr is also highly divergent, with limited regions of identity as well as extensive indel variation.
for example, d. melanogaster lhr shares  <dig>   <dig>  and  <dig> percent protein identity with d. simulans, d. yakuba and d. virilis lhr orthologs, respectively.
between d. melanogaster and d. simulans, lhr has a high ka/ks value of  <dig> , and population genetic analyses demonstrated that this high divergence is consistent with positive selection  <cit> .
lhr localizes to heterochromatic sites on salivary gland polytene chromosomes in a pattern similar to, but not completely overlapping with, heterochromatin protein  <dig>   <cit> .
hp <dig> is a well-studied chromosomal protein that is found in eukaryotes ranging from fission yeast to humans  <cit> , and is encoded by the su2- <dig> gene in drosophila  <cit> .
hp <dig> localizes to pericentric and telomeric heterochromatin, and to approximately  <dig> euchromatic sites throughout the genome  <cit> .
the localization of hp <dig> is consistent with its multiple functions in establishment and spreading of heterochromatin, telomere capping, and both silencing and activation of gene expression  <cit> .
greil et al.
<cit>  identified four new heterochromatin proteins  based on their association with hp <dig>  which they named hp <dig>  hp <dig>  hp <dig> and hp <dig>  hp <dig> was independently identified as lhr  <cit> ; hp <dig> was independently identified as the hp1-interacting protein   <cit> ; and hp <dig> was independently identified as the protein umbrea  <cit> .
like mutations in hp <dig>  mutations in hp <dig> and hp <dig> dominantly suppress position-effect variegation, and analysis of hp <dig> suggests that it has a role in telomere protection  <cit> .
hp <dig> , hp <dig>  and hp <dig> all have similar expression patterns as hp <dig>  being enriched in ovaries and early embryos  <cit> .
based on their common localization to heterochromatin, their similar expression patterns, and their proposed physical interactions, hp <dig>  lhr, hp <dig>  and hp <dig> may form a multi-protein heterochromatic complex in vivo.
of particular interest is whether and how the interactions and functions of lhr may be evolving.
this interest stems from a fundamental prediction of the dobzhansky-muller model that his are asymmetric  <cit> .
here this means that hybrid male lethality is caused specifically by d. simulans lhr , and not by d. melanogaster lhr .
we previously confirmed this prediction of asymmetry by demonstrating that a loss-of-function mutation in sim-lhr suppresses hybrid male lethality, but loss-of-function deletions of mel-lhr do not  <cit> .
asymmetry of hybrid lethality properties has also been experimentally demonstrated for the gene hmr  <cit> .
what is the nature of this functional divergence for an hi protein like lhr, especially in light of the evidence that lhr is rapidly evolving and has diverged under adaptive evolution?
one possibility is that sim-lhr or mel-lhr has evolved a new function  distinct from their common ancestor.
another possibility is that sim-lhr or mel-lhr has lost ancestral functions.
finally, one could envision that divergence has led to complex or subtle changes in function.
we showed previously that sim-lhr and mel-lhr share the ability to interact with mel-hp <dig> in a yeast two-hybrid assay  <cit> , suggesting that lhr has not undergone a complete change in functional properties between d. melanogaster and d. simulans.
here we have examined several properties of mel-lhr, including interactions with other hps and localization to the chromocenter.
to potentially discover differences among lhr orthologs, we have specifically addressed whether the localization and interaction properties of mel-lhr are conserved for other lhr orthologs, and have tested for the ability to induce hybrid male lethality.
surprisingly, we find that all lhr orthologs tested appear to have the same molecular interaction and localization properties, and most strikingly, three have the same ability to induce d. melanogaster-d. simulans hybrid male lethality.
RESULTS
variable rates of hp gene evolution
four heterochromatin proteins have been characterized and named  based on their association with hp <dig>  <cit> .
interestingly, these hp <dig> interacting proteins have two distinct patterns of molecular evolution .
both hp <dig> and hp <dig> are well conserved between d. melanogaster and d. simulans with low ka/ks values that are typical of genes evolving under stabilizing selection, while lhr , hp <dig>  and hp <dig> have high ka/ks values that suggest either adaptive evolution or relaxed constraint.
the different patterns of molecular evolution are intriguing given that several of these hps interact with one another, and all interact with the well-conserved hp <dig> .
we were therefore interested to explore further the relationships between lhr and these four other hps.
*- values are from ref.
<cit> .
lhr binds directly to hp1
lhr has been shown to interact in yeast two-hybrid assays with hp <dig> and hp <dig>  <cit> .
because hp <dig> is required to localize lhr , we wanted to determine whether these proteins interact directly.
we therefore performed immunoprecipitation assays with extracts from two sources.
first, we used nuclear extracts from drosophila kc cells that were transiently transfected with a lhr-myc fusion construct.
we found that endogenously expressed hp <dig> immunoprecipitates with myc-tagged lhr .
it is possible, however, that lhr and hp <dig> co-associate by each binding to another protein rather than directly binding to each other.
therefore, to test if lhr can bind directly to hp <dig>  we performed an in vitro binding assay.
we found that in vitro synthesized lhr-ha and hp <dig> also co-immunoprecipitate .
in combination with the published yeast two-hybrid and immunofluorescence data, our in vitro results strongly suggest that lhr and hp <dig> co-localize in heterochromatin due to direct physical binding to each other.
because it has been suggested that lhr is part of a network of hp1-associated proteins  <cit>  we used yeast two-hybrid to test if lhr interacts with hp <dig>  hp <dig> and hp <dig>  of these three other hps we examined, only hp <dig> interacts with lhr , consistent with previously reported results  <cit> .
mapping the interacting regions of hp <dig> and lhr
hp <dig> has multiple functions that are mediated by different domains  <cit> .
to define which region of hp <dig> is responsible for interacting with lhr, we created a series of derivatives of hp <dig> and tested them in a yeast two-hybrid assay.
we found that the c-terminal chromo-shadow domain  is both necessary and sufficient to interact with full length lhr .
this result is consistent with other findings showing that the csd of hp <dig> mediates protein-protein interactions  <cit> .
since hp <dig> also interacts with lhr and has a chromo-shadow domain similar to hp <dig>  it is possible that lhr simply interacts with any protein containing a csd.
this explanation is unlikely, however, because we observed no interaction between lhr and the d. melanogaster hp <dig> paralog rhino, which also contains a csd .
many proteins that interact with the chromo-shadow domain of hp <dig> contain either the consensus peptide pentamer pxvxl, or variations of this motif  <cit> .
we were unable to detect any canonical hp1-binding motifs in d. melanogaster lhr, but did find five potential pxvxl variants .
however, mutagenesis of each motif demonstrated that none of the  <dig> motifs are individually essential for the hp <dig> interaction .
we therefore mapped the hp <dig> interaction domain in lhr by testing a series of lhr fragments for interaction in yeast two-hybrid assays.
by analyzing the interacting and non-interacting two-hybrid fragments, we inferred that the smallest hp1-interacting fragment of lhr contains  <dig> amino acids and is located in the c-terminal half of the protein .
heterochromatic localization of lhr depends on hp <dig> in drosophila
lhr was previously shown to mislocalize in drosophila embryonic tissue-culture cells when hp <dig> is knocked down by rnai  <cit> .
we created lhr transgenes in order to confirm this result in whole animals and to develop an assay system for examining lhr ortholog evolution.
we made a fusion of mel-lhr to yfp  under control of the gal4/uas expression system, and generated transgenic flies that carry this uas-mel\lhr::yfp transgene.
we used a salivary-gland specific gal <dig> driver and examined the localization of lhr by live yfp analysis or anti-gfp staining in fixed tissues.
we found that lhr-yfp loses heterochromatic targeting and is redistributed throughout the nucleus in hp <dig> mutant larvae .
it is possible that the heterochromatic localization of lhr-yfp is lost in the hp <dig> mutant because the protein is unstable.
western blots indicate, however, that the protein is present at a similar level in the mutant hp <dig> background as in the wild type background .
in contrast to these results with hp <dig> mutants, we did not observe any localization differences for lhr-yfp in salivary glands from hp <dig> mutant animals .
lhr orthologs interact with hp <dig> and hp6
to test if the property of hp <dig> interaction is conserved among lhr orthologs we cloned the coding sequences from seven species and found that all of them interact with d. melanogaster hp <dig> in a yeast two-hybrid assay .
we then tested whether these lhr orthologs can also interact with d. melanogaster and d. simulans hp <dig>  because hp <dig> has a much higher rate of divergence between d. melanogaster and d. simulans than hp <dig> .
we found that all seven lhr orthologs tested interact with both mel-hp <dig> and sim-hp <dig> .
these data demonstrate that lhr interactions with heterochromatin proteins discovered in d. melanogaster are likely to be conserved among drosophila despite highly variable rates of evolution of these proteins, especially the c-terminal region of lhr .
lhr orthologs localize to heterochromatin when expressed in d. melanogaster
we have demonstrated that lhr from all species tested can interact with d. melanogaster hp <dig>  and that hp <dig> is required in d. melanogaster for lhr to localize to heterochromatin.
other studies in d. melanogaster, however, suggest that lhr localizes to some sites by an unknown mechanism, independently of hp <dig>  <cit> .
therefore, we wished to test if other drosophila lhr orthologs would also localize to heterochromatin.
because of the difficulty in directly determining lhr localization in the absence of an antibody recognizing lhr orthologs, we developed an assay system for localization of lhr orthologs expressed in d. melanogaster.
we tested lhr orthologs from d. simulans, d. yakuba, and d. virilis, which together represent a range of divergence from d. melanogaster lhr.
we transformed uas-driven lhr-yfp fusion constructs from these species into a common attp site on chromosome  <dig> of d. melanogaster using the Φ c <dig> site-specific integration system  <cit> .
using this system, transgenes can be inserted into the same position in the genome, allowing a direct comparison between lhr orthologs without complications from variation due to position effects on transgene expression.
we were unable to obtain a transformant of d. melanogaster lhr-yfp into the same attp site, so as an alternative, we used p element transformation and obtained three independent transgene insertion lines.
live yfp analysis in whole mount salivary glands showed similar bright foci for all three lhr orthologs when compared to d. melanogaster lhr-yfp .
anti-gfp staining in polytene squashes demonstrated that the foci observed in the whole mount tissues for all four lhr orthologs overlap with the pericentric heterochromatin at the chromocenter.
we also co-stained for hp <dig> and found that all four lhr orthologs co-localize with d. melanogaster hp <dig> in heterochromatin when expressed in d. melanogaster .
these results demonstrate that the property of heterochromatin localization is likely conserved among lhr orthologs.
three lhr orthologs have hybrid lethal activity
the above results suggest that lhr has not likely evolved different interaction or localization properties between d. melanogaster and d. simulans, or among outgroup species.
these findings raise the question of whether the hybrid lethality activity of sim-lhr is unique.
we tested this question using the genetic assay previously developed to identify the sim-lhr gene  <cit> .
we crossed d. melanogaster females heterozygous for the different uas driven lhr-yfp transgenes and the actin5c-gal <dig> driver to d. simulans lhr <dig> males, and scored the hybrid male progeny .
in these crosses, we are testing whether the ubiquitous lhr expression from the actin5c-gal <dig> driver can suppress the hybrid male rescue from the loss-of-function d. simulans lhr <dig> mutation.
we first performed control crosses to d. melanogaster.
p{uas-mel\lhr::yfp} <dig> and p{uas-mel\lhr::yfp} <dig> will segregate independently from the gal <dig> transgene .
in the absence of viability effects one expects a 2:1: <dig> ratio of eye colors, which we observed.
p{uas-mel\lhr::yfp} <dig> is on chromosome  <dig> and was thus trans-heterozygous to the gal <dig> transgene in the mothers of the crosses.
progeny ratios in the control cross again did not deviate significantly from the expected ratios, demonstrating that the p{uas-mel\lhr::yfp} <dig> transgene is unlinked to the gal <dig> transgene and that its expression has no affect in d. melanogaster.
similar results were obtained in control crosses with the lhr-yfp transgenes from d. simulans, d. yakuba and d. virilis .
we conclude that expression of these four lhr orthologs does not affect viability of d. melanogaster.
in experimental crosses where uas-sim\lhr::yfp was crossed to the d. simulans lhr <dig> strain to generate hybrids, the progeny ratios differed significantly from those in the control cross.
this difference reflects suppression of hybrid male rescue by lhr <dig> as judged by the presence of approximately half of the gal4-containing sons  compared to the control cross .
this result is similar to what we previously observed with untagged sim-lhr transgenes  <cit>  and thus demonstrates that the yfp tag does not alter lhr function.
we also observed here a deficit in uas/+ progeny compared to their +/+ siblings, which we also saw previously and attributed to maternally inherited gal <dig> protein activating expression of the zygotically inherited uas transgene.
a - females heterozygous for the indicated uas transgene and for p{act5c-gal4}17bfo <dig> were crossed to either d. melanogaster w1118/y  or to d. simulans lhr <dig> males.
for the first two sets of crosses the females were of genotype w/w; uas/+; gal4/+; for the remaining crosses the females were of genotype w/w; uas +/gal <dig> +.
b - for the control crosses the ratios of red eye: orange eye: white eye progeny were tested for deviation from a 2:1: <dig> ratio using a chi-square test.
n.s.
= p >  <dig>
c - the ratios of red eye: orange eye: white eye for hybrid crosses were compared to control crosses using a  <dig> ×  <dig> contingency table and chi-square test.
*** = p <  <dig> × 10-3; n.s.
= p >  <dig> ,  <dig> degrees of freedom.
surprisingly, progeny in experimental crosses with lhr-yfp transgenes from d. melanogaster and d. yakuba also differed significantly from their respective control crosses.
we confirmed by pcr that the rescued red-eyed males only carried the gal <dig> transgene , thus concluding that the reduction in red-eyed males is caused by lethality of sons inheriting both the gal <dig> and uas-lhr-yfp transgenes.
in two of the three crosses with uas-mel\lhr::yfp and in the cross with uas-yak\lhr::yfp we also again observed a strong reduction in the viability of uas/+  sons.
these results demonstrate that the hybrid lethality activity of lhr is not unique to d. simulans lhr, at least when expressed under control of actin-gal <dig>  experimental crosses with the d. virilis transgene had a slight reduction in the number of red-eyed males, but this cross did not differ significantly from the controls.
this result suggests that vir-lhr does not induce lethality in d. melanogaster-d. simulans hybrids.
discussion
lhr interaction and localization properties
giot et al.
<cit>  reported that lhr interacts with hp <dig> and hp <dig> in a large-scale yeast two-hybrid screen.
we previously confirmed this result for hp <dig>  and have done so here for hp <dig>  we have further shown here that hp <dig> immunoprecipitates with lhr in cell culture extracts, and that lhr binds to hp <dig> in an in vitro assay.
these data indicate that the interaction between the lhr and hp <dig> proteins is likely to be direct.
we also discovered that lhr interacts with the chromo-shadow domain of hp <dig>  unlike many other csd-interacting proteins  <cit> , we did not find a pxvxl-like motif in lhr that is individually responsible for mediating interaction with hp <dig>
joppich et al.
<cit>  have reported that similar to lhr, hp <dig>  binds to hp <dig> and hp <dig>  in vitro, and that these proteins colocalize at pericentric heterochromatin and form a complex in vivo.
these authors further proposed that the association between hp <dig> and hp <dig> is mediated by heterodimerization of the chromo-shadow domain  in each protein, similar to the known homodimerization of the csd in hp <dig>  structural investigation suggests that csd dimerization results in formation of a protein-protein interaction pit that is predicted to bind one peptide per csd dimer  <cit> .
lhr, hp <dig>  and hp <dig> each interact with both hp <dig> and hp <dig>  but do not interact with each other.
these interaction results combined with the limitation of csd dimers to only bind one peptide, suggest that lhr, hp <dig> and hp <dig> are unlikely to simultaneously bind to hp <dig> or hp <dig> csd dimers or to hp1/hp <dig> heterodimers.
instead, lhr, hp <dig>  and hp <dig> likely interact separately with hp <dig> in distinct complexes.
greil et al.
<cit>  demonstrated that lhr  is dependent on hp <dig> for proper localization to heterochromatin in tissue culture cells.
here we developed an in vivo localization assay using yfp-tagged lhr transgenes expressed in salivary glands, in order to take advantage of the resolution available in their large polytenized nuclei.
we have confirmed with this assay that hp <dig> is essential for proper heterochromatic localization of lhr.
one potential complication of our assay is that it relies on the gal4-uas expression system, and may overexpress lhr compared to the wild type.
the fact that lhr colocalizes with hp <dig> in our assay, consistent with previous reports  <cit>  suggests that lhr is unlikely to be localizing outside of its normal pattern.
we also found that lhr does not depend on hp <dig> for heterochromatic localization in salivary glands.
one possibility is that hp <dig> depends on lhr for localization, as suggested by greil et al.
<cit>  and predicted by a recent bayesian network inference analysis based on genomic mapping studies  <cit> .
a second possible explanation is that lhr interacts with hp <dig> in tissues other than third-instar larval salivary glands, and our conclusion is limited to this tissue and stage of development.
a third possibility is that lhr and hp <dig> are differentially expressed and thus do not interact in vivo.
we have found that available d. melanogaster mutations of some of these hp genes do not suppress hybrid male lethality .
rescue of male lethality by lhr, however, requires a mutant of d. simulans, not d. melanogaster.
therefore a more comprehensive test of whether these other hp genes have any role in hybrid male lethality will require obtaining mutants in d. simulans.
conserved interactions, localization and functional properties of lhr orthologs
the high rate of evolution of lhr raised our interest in examining whether lhr functional properties are conserved among drosophila orthologs.
more specifically, the previous discoveries that lhr diverged between d. melanogaster and d. simulans under adaptive evolution, and that only sim-lhr but not mel-lhr causes hybrid lethality, strongly suggested that lhr has undergone significant functional changes between these two species and possibly among other species.
we found, however, that two heterochromatin-related properties of the lhr protein are conserved across species.
first, lhr from seven drosophila species including d. melanogaster interacts in yeast two-hybrid assays with two d. melanogaster heterochromatin proteins, hp <dig> and hp <dig>  as well as with d. simulans hp <dig>  second, we found that the four lhr orthologs we tested co-localize with hp <dig> at heterochromatic regions when expressed in d. melanogaster.
we infer from these results that lhr localization to heterochromatin via binding to hp <dig> is both an ancestral and conserved property among drosophila.
from our data, we cannot exclude an alternative possibility that some lhr orthologs do not localize to heterochromatin in their native species despite doing so when expressed in d. melanogaster.
this alternative, however, seems unlikely.
first, we have shown that lhr heterochromatic localization likely depends on direct binding to hp <dig>  and hp <dig> is highly conserved among drosophila.
second, such a scenario would require an lhr ortholog to have evolved so as to have lost or changed protein interactions that likely occur in the ancestral state, while retaining the ability to interact with the same proteins in d. melanogaster.
our data strongly suggest that lhr has not undergone a complete change of function between d. melanogaster and d. simulans, or among other drosophila species.
we therefore tested lhr from four species for its ability to induce hybrid male lethality.
because we previously observed an asymmetry in the effects of d. melanogaster and d. simulans lhr mutants on hybrid rescue, we predicted that the addition of a d. melanogaster lhr transgene  would not complement the d. simulans lhr <dig> mutation, and thus not be lethal.
contrary to our expectation, we found that lhr from both d. melanogaster and d. simulans as well as from the outgroup species d. yakuba suppresses lhr <dig> rescue and thus prevents rescue of lethal hybrid males .
how do we reconcile this discovery with our previous report?
we suggest that both mel-lhr and sim-lhr have at least some degree of hybrid lethal activity, but that sim-lhr is stronger.
therefore the functional changes between d. melanogaster and d. simulans lhr orthologs are likely quantitative and thus easily masked by gene dosage effects.
the transgenic complementation experiments reported here expressed lhr orthologs in hybrids in addition to that expressed from the endogenous mel-lhr gene.
in contrast, in our previous deletion experiments hybrids expressed only a single endogenous copy of either mel-lhr or sim-lhr.
in addition, the gal4-uas system used here likely expresses lhr to a level higher than in wild type animals, and it is possible that hybrid viability is more sensitive to lhr dosage than suggested by the gal4-driven localization studies above.
what then is the functional difference between sim-lhr and mel-lhr that causes the asymmetry in their hybrid lethality properties?
one possibility is that there is a subtle difference in their localization properties that is not detectable in our assays.
mel-lhr has been reported to bind to several hundred sites along the chromosome arms, predominantly in pericentric regions  <cit> .
a second possibility is that sim-lhr and mel-lhr have different protein interaction properties, perhaps with as-yet undiscovered partners.
finally, they may differ in effector functions such as modifying chromatin states or affecting gene regulation.
our results with expression of lhr orthologs in foreign-species backgrounds contrast with other studies of rapidly and adaptively evolving heterochromatin proteins.
one example is the centromeric protein cid.
the distantly related d. bipectinata cid fails to localize to the centromere when expressed in d. melanogaster tissue-culture cells  <cit> .
this study also found that conserved amino acids were essential for proper targeting of cid to the centromere, and suggested that the rapid evolution of cid may be a response to rapid turnover of dna at satellites and centromeres.
a second example is the odsh protein.
the hybrid sterility proteins odsh from d. simulans  and d. mauritiana  have different localization patterns when expressed in d. simulans  <cit> .
sim-odsh associates with the x pericentric region and the 4th chromosome, both repeat-rich regions of the d. simulans genome.
similarly, mau-odsh localizes to the d. simulans x and 4th chromosomes, but also to the d. simulans y chromosome, and it has been proposed that this unusual localization causes decondensation of the y leading to hybrid sterility.
this change in localization is likely associated with the large number of amino-acid changes in the odsh homeodomain between these species  <cit>  and has been proposed to be driven by co-evolution with satellite dnas  <cit> .
we proposed previously that lhr also has co-evolved with rapidly evolving heterochromatic dnas  <cit> .
if so, this co-evolution may be indirect, since lhr does not appear to have a dna binding domain, and its localization to heterochromatin depends on other proteins.
rapid and adaptive evolution of lhr may therefore more directly be a consequence of its interactions with other rapidly evolving heterochromatin proteins.
CONCLUSIONS
one of the major goals in the study of speciation is to unravel the molecular bases of the genetic phenomena.
here, we have shown that despite the high levels of divergence among lhr orthologs, lhr properties associated with localizing to heterochromatin and interacting with other hps are conserved across species.
in addition, we also show that the property of inducing d. melanogaster-d. simulans hybrid male lethality is conserved for multiple lhr orthologs.
this conservation of lethal activity includes d. melanogaster lhr, which was previously thought to be functionally distinct from d. simulans lhr in this respect.
our results suggest that the hybrid background is highly sensitive to small functional differences and that hybrid incompatibilities may result from an accumulation of subtle molecular changes.

 the biological species concept is a widely accepted definition of species in which species are defined as groups of interbreeding populations that are reproductively isolated from other groups. hybrids between closely related species are often inviable or sterile, and this inviability and sterility is collectively called hybrid incompatibility. crosses between drosophila melanogaster and its sibling species d. simulans produce incompatible hybrids. in the cross of d. melanogaster females to d. simulans males, the f daughters are semi-viable but sterile, and the sons are inviable. two genes, hybrid male rescue in d. melanogaster, and lethal hybrid rescue in d. simulans, are major-effect contributors to hybrid incompatibility on the basis that loss-of-function mutations in either gene can suppress hybrid male lethality. lhr encodes a small protein and appears to be drosophila specific, as we are unable to identify homologs in vertebrates or in other invertebrates, including in other insect genera. furthermore, among drosophila species the coding sequence of lhr is also highly divergent, with limited regions of identity as well as extensive indel variation. for example, d. melanogaster lhr shares and percent protein identity with d. simulans, d. yakuba and d. virilis lhr orthologs, respectively. between d. melanogaster and d. simulans, lhr has a high ka/ks value of, and population genetic analyses demonstrated that this high divergence is consistent with positive selection. lhr localizes to heterochromatic sites on salivary gland polytene chromosomes in a pattern similar to, but not completely overlapping with, heterochromatin protein. hp is a well-studied chromosomal protein that is found in eukaryotes ranging from fission yeast to humans, and is encoded by the su2- gene in drosophila. hp localizes to pericentric and telomeric heterochromatin, and to approximately euchromatic sites throughout the genome. the localization of hp is consistent with its multiple functions in establishment and spreading of heterochromatin, telomere capping, and both silencing and activation of gene expression. greil et al. identified four new heterochromatin proteins based on their association with hp which they named hp hp hp and hp hp was independently identified as lhr; hp was independently identified as the hp1-interacting protein; and hp was independently identified as the protein umbrea. like mutations in hp mutations in hp and hp dominantly suppress position-effect variegation, and analysis of hp suggests that it has a role in telomere protection. hp, hp and hp all have similar expression patterns as hp being enriched in ovaries and early embryos. based on their common localization to heterochromatin, their similar expression patterns, and their proposed physical interactions, hp lhr, hp and hp may form a multi-protein heterochromatic complex in vivo. of particular interest is whether and how the interactions and functions of lhr may be evolving. this interest stems from a fundamental prediction of the dobzhansky-muller model that his are asymmetric. here this means that hybrid male lethality is caused specifically by d. simulans lhr, and not by d. melanogaster lhr. we previously confirmed this prediction of asymmetry by demonstrating that a loss-of-function mutation in sim-lhr suppresses hybrid male lethality, but loss-of-function deletions of mel-lhr do not. asymmetry of hybrid lethality properties has also been experimentally demonstrated for the gene hmr. what is the nature of this functional divergence for an hi protein like lhr, especially in light of the evidence that lhr is rapidly evolving and has diverged under adaptive evolution? one possibility is that sim-lhr or mel-lhr has evolved a new function distinct from their common ancestor. another possibility is that sim-lhr or mel-lhr has lost ancestral functions. finally, one could envision that divergence has led to complex or subtle changes in function. we showed previously that sim-lhr and mel-lhr share the ability to interact with mel-hp in a yeast two-hybrid assay, suggesting that lhr has not undergone a complete change in functional properties between d. melanogaster and d. simulans. here we have examined several properties of mel-lhr, including interactions with other hps and localization to the chromocenter. to potentially discover differences among lhr orthologs, we have specifically addressed whether the localization and interaction properties of mel-lhr are conserved for other lhr orthologs, and have tested for the ability to induce hybrid male lethality. surprisingly, we find that all lhr orthologs tested appear to have the same molecular interaction and localization properties, and most strikingly, three have the same ability to induce d. melanogaster-d. simulans hybrid male lethality.  variable rates of hp gene evolution four heterochromatin proteins have been characterized and named based on their association with hp. interestingly, these hp interacting proteins have two distinct patterns of molecular evolution. both hp and hp are well conserved between d. melanogaster and d. simulans with low ka/ks values that are typical of genes evolving under stabilizing selection, while lhr, hp and hp have high ka/ks values that suggest either adaptive evolution or relaxed constraint. the different patterns of molecular evolution are intriguing given that several of these hps interact with one another, and all interact with the well-conserved hp. we were therefore interested to explore further the relationships between lhr and these four other hps. *- values are from ref.. lhr binds directly to hp1 lhr has been shown to interact in yeast two-hybrid assays with hp and hp. because hp is required to localize lhr, we wanted to determine whether these proteins interact directly. we therefore performed immunoprecipitation assays with extracts from two sources. first, we used nuclear extracts from drosophila kc cells that were transiently transfected with a lhr-myc fusion construct. we found that endogenously expressed hp immunoprecipitates with myc-tagged lhr. it is possible, however, that lhr and hp co-associate by each binding to another protein rather than directly binding to each other. therefore, to test if lhr can bind directly to hp we performed an in vitro binding assay. we found that in vitro synthesized lhr-ha and hp also co-immunoprecipitate. in combination with the published yeast two-hybrid and immunofluorescence data, our in vitro results strongly suggest that lhr and hp co-localize in heterochromatin due to direct physical binding to each other. because it has been suggested that lhr is part of a network of hp1-associated proteins we used yeast two-hybrid to test if lhr interacts with hp hp and hp of these three other hps we examined, only hp interacts with lhr, consistent with previously reported results. mapping the interacting regions of hp and lhr hp has multiple functions that are mediated by different domains. to define which region of hp is responsible for interacting with lhr, we created a series of derivatives of hp and tested them in a yeast two-hybrid assay. we found that the c-terminal chromo-shadow domain is both necessary and sufficient to interact with full length lhr. this result is consistent with other findings showing that the csd of hp mediates protein-protein interactions. since hp also interacts with lhr and has a chromo-shadow domain similar to hp it is possible that lhr simply interacts with any protein containing a csd. this explanation is unlikely, however, because we observed no interaction between lhr and the d. melanogaster hp paralog rhino, which also contains a csd. many proteins that interact with the chromo-shadow domain of hp contain either the consensus peptide pentamer pxvxl, or variations of this motif. we were unable to detect any canonical hp1-binding motifs in d. melanogaster lhr, but did find five potential pxvxl variants. however, mutagenesis of each motif demonstrated that none of the motifs are individually essential for the hp interaction. we therefore mapped the hp interaction domain in lhr by testing a series of lhr fragments for interaction in yeast two-hybrid assays. by analyzing the interacting and non-interacting two-hybrid fragments, we inferred that the smallest hp1-interacting fragment of lhr contains amino acids and is located in the c-terminal half of the protein. heterochromatic localization of lhr depends on hp in drosophila lhr was previously shown to mislocalize in drosophila embryonic tissue-culture cells when hp is knocked down by rnai. we created lhr transgenes in order to confirm this result in whole animals and to develop an assay system for examining lhr ortholog evolution. we made a fusion of mel-lhr to yfp under control of the gal4/uas expression system, and generated transgenic flies that carry this uas-mel\lhr::yfp transgene. we used a salivary-gland specific gal driver and examined the localization of lhr by live yfp analysis or anti-gfp staining in fixed tissues. we found that lhr-yfp loses heterochromatic targeting and is redistributed throughout the nucleus in hp mutant larvae. it is possible that the heterochromatic localization of lhr-yfp is lost in the hp mutant because the protein is unstable. western blots indicate, however, that the protein is present at a similar level in the mutant hp background as in the wild type background. in contrast to these results with hp mutants, we did not observe any localization differences for lhr-yfp in salivary glands from hp mutant animals. lhr orthologs interact with hp and hp6 to test if the property of hp interaction is conserved among lhr orthologs we cloned the coding sequences from seven species and found that all of them interact with d. melanogaster hp in a yeast two-hybrid assay. we then tested whether these lhr orthologs can also interact with d. melanogaster and d. simulans hp because hp has a much higher rate of divergence between d. melanogaster and d. simulans than hp. we found that all seven lhr orthologs tested interact with both mel-hp and sim-hp. these data demonstrate that lhr interactions with heterochromatin proteins discovered in d. melanogaster are likely to be conserved among drosophila despite highly variable rates of evolution of these proteins, especially the c-terminal region of lhr. lhr orthologs localize to heterochromatin when expressed in d. melanogaster we have demonstrated that lhr from all species tested can interact with d. melanogaster hp and that hp is required in d. melanogaster for lhr to localize to heterochromatin. other studies in d. melanogaster, however, suggest that lhr localizes to some sites by an unknown mechanism, independently of hp. therefore, we wished to test if other drosophila lhr orthologs would also localize to heterochromatin. because of the difficulty in directly determining lhr localization in the absence of an antibody recognizing lhr orthologs, we developed an assay system for localization of lhr orthologs expressed in d. melanogaster. we tested lhr orthologs from d. simulans, d. yakuba, and d. virilis, which together represent a range of divergence from d. melanogaster lhr. we transformed uas-driven lhr-yfp fusion constructs from these species into a common attp site on chromosome of d. melanogaster using the Φ c site-specific integration system. using this system, transgenes can be inserted into the same position in the genome, allowing a direct comparison between lhr orthologs without complications from variation due to position effects on transgene expression. we were unable to obtain a transformant of d. melanogaster lhr-yfp into the same attp site, so as an alternative, we used p element transformation and obtained three independent transgene insertion lines. live yfp analysis in whole mount salivary glands showed similar bright foci for all three lhr orthologs when compared to d. melanogaster lhr-yfp. anti-gfp staining in polytene squashes demonstrated that the foci observed in the whole mount tissues for all four lhr orthologs overlap with the pericentric heterochromatin at the chromocenter. we also co-stained for hp and found that all four lhr orthologs co-localize with d. melanogaster hp in heterochromatin when expressed in d. melanogaster. these results demonstrate that the property of heterochromatin localization is likely conserved among lhr orthologs. three lhr orthologs have hybrid lethal activity the above results suggest that lhr has not likely evolved different interaction or localization properties between d. melanogaster and d. simulans, or among outgroup species. these findings raise the question of whether the hybrid lethality activity of sim-lhr is unique. we tested this question using the genetic assay previously developed to identify the sim-lhr gene. we crossed d. melanogaster females heterozygous for the different uas driven lhr-yfp transgenes and the actin5c-gal driver to d. simulans lhr males, and scored the hybrid male progeny. in these crosses, we are testing whether the ubiquitous lhr expression from the actin5c-gal driver can suppress the hybrid male rescue from the loss-of-function d. simulans lhr mutation. we first performed control crosses to d. melanogaster. p{uas-mel\lhr::yfp} and p{uas-mel\lhr::yfp} will segregate independently from the gal transgene. in the absence of viability effects one expects a 2:1: ratio of eye colors, which we observed. p{uas-mel\lhr::yfp} is on chromosome and was thus trans-heterozygous to the gal transgene in the mothers of the crosses. progeny ratios in the control cross again did not deviate significantly from the expected ratios, demonstrating that the p{uas-mel\lhr::yfp} transgene is unlinked to the gal transgene and that its expression has no affect in d. melanogaster. similar results were obtained in control crosses with the lhr-yfp transgenes from d. simulans, d. yakuba and d. virilis. we conclude that expression of these four lhr orthologs does not affect viability of d. melanogaster. in experimental crosses where uas-sim\lhr::yfp was crossed to the d. simulans lhr strain to generate hybrids, the progeny ratios differed significantly from those in the control cross. this difference reflects suppression of hybrid male rescue by lhr as judged by the presence of approximately half of the gal4-containing sons compared to the control cross. this result is similar to what we previously observed with untagged sim-lhr transgenes and thus demonstrates that the yfp tag does not alter lhr function. we also observed here a deficit in uas/+ progeny compared to their +/+ siblings, which we also saw previously and attributed to maternally inherited gal protein activating expression of the zygotically inherited uas transgene. a - females heterozygous for the indicated uas transgene and for p{act5c-gal4}17bfo were crossed to either d. melanogaster w1118/y or to d. simulans lhr males. for the first two sets of crosses the females were of genotype w/w; uas/+; gal4/+; for the remaining crosses the females were of genotype w/w; uas +/gal +. b - for the control crosses the ratios of red eye: orange eye: white eye progeny were tested for deviation from a 2:1: ratio using a chi-square test. n.s. = p > c - the ratios of red eye: orange eye: white eye for hybrid crosses were compared to control crosses using a × contingency table and chi-square test. *** = p < × 10-3; n.s. = p >, degrees of freedom. surprisingly, progeny in experimental crosses with lhr-yfp transgenes from d. melanogaster and d. yakuba also differed significantly from their respective control crosses. we confirmed by pcr that the rescued red-eyed males only carried the gal transgene, thus concluding that the reduction in red-eyed males is caused by lethality of sons inheriting both the gal and uas-lhr-yfp transgenes. in two of the three crosses with uas-mel\lhr::yfp and in the cross with uas-yak\lhr::yfp we also again observed a strong reduction in the viability of uas/+ sons. these results demonstrate that the hybrid lethality activity of lhr is not unique to d. simulans lhr, at least when expressed under control of actin-gal experimental crosses with the d. virilis transgene had a slight reduction in the number of red-eyed males, but this cross did not differ significantly from the controls. this result suggests that vir-lhr does not induce lethality in d. melanogaster-d. simulans hybrids. discussion lhr interaction and localization properties giot et al. reported that lhr interacts with hp and hp in a large-scale yeast two-hybrid screen. we previously confirmed this result for hp and have done so here for hp we have further shown here that hp immunoprecipitates with lhr in cell culture extracts, and that lhr binds to hp in an in vitro assay. these data indicate that the interaction between the lhr and hp proteins is likely to be direct. we also discovered that lhr interacts with the chromo-shadow domain of hp unlike many other csd-interacting proteins, we did not find a pxvxl-like motif in lhr that is individually responsible for mediating interaction with hp joppich et al. have reported that similar to lhr, hp binds to hp and hp in vitro, and that these proteins colocalize at pericentric heterochromatin and form a complex in vivo. these authors further proposed that the association between hp and hp is mediated by heterodimerization of the chromo-shadow domain in each protein, similar to the known homodimerization of the csd in hp structural investigation suggests that csd dimerization results in formation of a protein-protein interaction pit that is predicted to bind one peptide per csd dimer. lhr, hp and hp each interact with both hp and hp but do not interact with each other. these interaction results combined with the limitation of csd dimers to only bind one peptide, suggest that lhr, hp and hp are unlikely to simultaneously bind to hp or hp csd dimers or to hp1/hp heterodimers. instead, lhr, hp and hp likely interact separately with hp in distinct complexes. greil et al. demonstrated that lhr is dependent on hp for proper localization to heterochromatin in tissue culture cells. here we developed an in vivo localization assay using yfp-tagged lhr transgenes expressed in salivary glands, in order to take advantage of the resolution available in their large polytenized nuclei. we have confirmed with this assay that hp is essential for proper heterochromatic localization of lhr. one potential complication of our assay is that it relies on the gal4-uas expression system, and may overexpress lhr compared to the wild type. the fact that lhr colocalizes with hp in our assay, consistent with previous reports suggests that lhr is unlikely to be localizing outside of its normal pattern. we also found that lhr does not depend on hp for heterochromatic localization in salivary glands. one possibility is that hp depends on lhr for localization, as suggested by greil et al. and predicted by a recent bayesian network inference analysis based on genomic mapping studies. a second possible explanation is that lhr interacts with hp in tissues other than third-instar larval salivary glands, and our conclusion is limited to this tissue and stage of development. a third possibility is that lhr and hp are differentially expressed and thus do not interact in vivo. we have found that available d. melanogaster mutations of some of these hp genes do not suppress hybrid male lethality. rescue of male lethality by lhr, however, requires a mutant of d. simulans, not d. melanogaster. therefore a more comprehensive test of whether these other hp genes have any role in hybrid male lethality will require obtaining mutants in d. simulans. conserved interactions, localization and functional properties of lhr orthologs the high rate of evolution of lhr raised our interest in examining whether lhr functional properties are conserved among drosophila orthologs. more specifically, the previous discoveries that lhr diverged between d. melanogaster and d. simulans under adaptive evolution, and that only sim-lhr but not mel-lhr causes hybrid lethality, strongly suggested that lhr has undergone significant functional changes between these two species and possibly among other species. we found, however, that two heterochromatin-related properties of the lhr protein are conserved across species. first, lhr from seven drosophila species including d. melanogaster interacts in yeast two-hybrid assays with two d. melanogaster heterochromatin proteins, hp and hp as well as with d. simulans hp second, we found that the four lhr orthologs we tested co-localize with hp at heterochromatic regions when expressed in d. melanogaster. we infer from these results that lhr localization to heterochromatin via binding to hp is both an ancestral and conserved property among drosophila. from our data, we cannot exclude an alternative possibility that some lhr orthologs do not localize to heterochromatin in their native species despite doing so when expressed in d. melanogaster. this alternative, however, seems unlikely. first, we have shown that lhr heterochromatic localization likely depends on direct binding to hp and hp is highly conserved among drosophila. second, such a scenario would require an lhr ortholog to have evolved so as to have lost or changed protein interactions that likely occur in the ancestral state, while retaining the ability to interact with the same proteins in d. melanogaster. our data strongly suggest that lhr has not undergone a complete change of function between d. melanogaster and d. simulans, or among other drosophila species. we therefore tested lhr from four species for its ability to induce hybrid male lethality. because we previously observed an asymmetry in the effects of d. melanogaster and d. simulans lhr mutants on hybrid rescue, we predicted that the addition of a d. melanogaster lhr transgene would not complement the d. simulans lhr mutation, and thus not be lethal. contrary to our expectation, we found that lhr from both d. melanogaster and d. simulans as well as from the outgroup species d. yakuba suppresses lhr rescue and thus prevents rescue of lethal hybrid males. how do we reconcile this discovery with our previous report? we suggest that both mel-lhr and sim-lhr have at least some degree of hybrid lethal activity, but that sim-lhr is stronger. therefore the functional changes between d. melanogaster and d. simulans lhr orthologs are likely quantitative and thus easily masked by gene dosage effects. the transgenic complementation experiments reported here expressed lhr orthologs in hybrids in addition to that expressed from the endogenous mel-lhr gene. in contrast, in our previous deletion experiments hybrids expressed only a single endogenous copy of either mel-lhr or sim-lhr. in addition, the gal4-uas system used here likely expresses lhr to a level higher than in wild type animals, and it is possible that hybrid viability is more sensitive to lhr dosage than suggested by the gal4-driven localization studies above. what then is the functional difference between sim-lhr and mel-lhr that causes the asymmetry in their hybrid lethality properties? one possibility is that there is a subtle difference in their localization properties that is not detectable in our assays. mel-lhr has been reported to bind to several hundred sites along the chromosome arms, predominantly in pericentric regions. a second possibility is that sim-lhr and mel-lhr have different protein interaction properties, perhaps with as-yet undiscovered partners. finally, they may differ in effector functions such as modifying chromatin states or affecting gene regulation. our results with expression of lhr orthologs in foreign-species backgrounds contrast with other studies of rapidly and adaptively evolving heterochromatin proteins. one example is the centromeric protein cid. the distantly related d. bipectinata cid fails to localize to the centromere when expressed in d. melanogaster tissue-culture cells. this study also found that conserved amino acids were essential for proper targeting of cid to the centromere, and suggested that the rapid evolution of cid may be a response to rapid turnover of dna at satellites and centromeres. a second example is the odsh protein. the hybrid sterility proteins odsh from d. simulans and d. mauritiana have different localization patterns when expressed in d. simulans. sim-odsh associates with the x pericentric region and the 4th chromosome, both repeat-rich regions of the d. simulans genome. similarly, mau-odsh localizes to the d. simulans x and 4th chromosomes, but also to the d. simulans y chromosome, and it has been proposed that this unusual localization causes decondensation of the y leading to hybrid sterility. this change in localization is likely associated with the large number of amino-acid changes in the odsh homeodomain between these species and has been proposed to be driven by co-evolution with satellite dnas. we proposed previously that lhr also has co-evolved with rapidly evolving heterochromatic dnas. if so, this co-evolution may be indirect, since lhr does not appear to have a dna binding domain, and its localization to heterochromatin depends on other proteins. rapid and adaptive evolution of lhr may therefore more directly be a consequence of its interactions with other rapidly evolving heterochromatin proteins.  one of the major goals in the study of speciation is to unravel the molecular bases of the genetic phenomena. here, we have shown that despite the high levels of divergence among lhr orthologs, lhr properties associated with localizing to heterochromatin and interacting with other hps are conserved across species. in addition, we also show that the property of inducing d. melanogaster-d. simulans hybrid male lethality is conserved for multiple lhr orthologs. this conservation of lethal activity includes d. melanogaster lhr, which was previously thought to be functionally distinct from d. simulans lhr in this respect. our results suggest that the hybrid background is highly sensitive to small functional differences and that hybrid incompatibilities may result from an accumulation of subtle molecular changes. 
we conclude 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. the gene lethal hybrid rescue 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. here we have 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. we find that these properties are conserved among most lhr orthologs, including lhr from d. melanogaster, d. simulans and the outgroup species d. yakuba. 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.
 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 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.  here we have 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. we find that these properties are conserved among most lhr orthologs, including lhr from d. melanogaster, d. simulans and the outgroup species d. yakuba.  we conclude 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. 