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Stepwise Evolution of Essential Centromere Function in a Drosophila Neogene

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Science  07 Jun 2013:
Vol. 340, Issue 6137, pp. 1211-1214
DOI: 10.1126/science.1234393

Essential Novelty

The evolution of essential function for newly originated genes presents a conundrum, in that prior to the gene's origin either the essential function was absent or else performed by another gene or set of genes. In order to better understand how new genes acquire essential function, Ross et al. (p. 1211) investigated the origin of the Drosophila gene Umbrea. Umbrea became an essential protein in certain Drosophila species through the gain of localization at the centromere and a role in chromosome segregation.

Abstract

Evolutionarily young genes that serve essential functions represent a paradox; they must perform a function that either was not required until after their birth or was redundant with another gene. How young genes rapidly acquire essential function is largely unknown. We traced the evolutionary steps by which the Drosophila gene Umbrea acquired an essential role in chromosome segregation in D. melanogaster since the gene's origin less than 15 million years ago. Umbrea neofunctionalization occurred via loss of an ancestral heterochromatin-localizing domain, followed by alterations that rewired its protein interaction network and led to species-specific centromere localization. Our evolutionary cell biology approach provides temporal and mechanistic detail about how young genes gain essential function. Such innovations may constantly alter the repertoire of centromeric proteins in eukaryotes.

Young essential genes (1) challenge long-standing dogmas about the relationship between essentiality and conservation (2). Partitioning of essential, ancestral functions (subfunctionalization) between (old) parental and (young) daughter genes (3, 4) explains one route by which young genes become essential. More difficult to understand is how new genes become essential via the emergence of novel function (neofunctionalization) (5). This could result from partial duplication of ancestral genes, novel gene fusions, or rapid amino acid changes (6). The contribution of each of these processes to the acquisition of essential function is unknown, as are the underlying molecular changes.

To gain insight into the birth and evolution of essential function, we focused on one newly evolved gene in Drosophila. Umbrea (also known as HP6 and CG15636) arose via duplication of the intronless Heterochromatin Protein 1B (HP1B) gene into an intron of the dumpy gene (Fig. 1A) (7). HP1B is a chromosomal protein that predominantly localizes to heterochromatin in D. melanogaster cells and regulates gene expression (8). HP1B is dispensable for viability (8), yet RNA interference (RNAi) knockdown phenotypes show Umbrea to be essential in D. melanogaster (1, 9). The 100% late larval-pupal lethality upon Umbrea knockdown could be rescued by an Umbrea–green fluorescent protein (GFP) fusion (fig. S1). Genetic knockout experiments (fig. S1) further confirmed that Umbrea is essential in D. melanogaster.

Fig. 1 Neofunctionalization of Umbrea.

(A) Umbrea originated via gene duplication of the intronless HP1B gene into an intron of the dumpy locus. (B) GFP-tagged HP1B localizes to heterochromatin in D. melanogaster Kc cells [magenta, anti-Cid; green, GFP; blue, 4′,6-diamidino-2-phenylindole (DAPI); colocalization appears white]. (C) In contrast, Umbrea-GFP localizes to centromeres. (D and E) Endogenous Umbrea colocalizes with centromeres in testes and in larval imaginal discs (magenta, anti-Cid; green, anti-Umbrea; blue, DAPI; scale bar, 5 μm). (F and G) S2 cells depleted of Umbrea by RNAi revealed increased mitotic errors (green, anti-Cid; blue, phospho-H3-staining mitotic chromosomes; red, anti-tubulin) relative to double-stranded RNA control (**P < 0.05).

We traced Umbrea's evolutionary path after duplication from HP1B to understand when and how essential function was gained by comparing the localization of HP1B and Umbrea proteins in D. melanogaster Kc cells. GFP-tagged HP1B proteins from both D. melanogaster and D. ananassae [whose divergence predates the birth of Umbrea (7)] localized to pericentric heterochromatin and euchromatin (Fig. 1B and fig. S2). In contrast, Umbrea-GFP predominantly localized to interphase centromeres, but not telomeres (Fig. 1C and fig. S3, A and B). Specific antibodies raised against Umbrea (fig. S4A) confirmed its centromere localization in developing spermatocytes and larval imaginal discs (Fig. 1, D and E, and fig. S4, B and C).

On the basis of its essentiality and centromere localization, we hypothesized that Umbrea was required for chromosome segregation. Upon depletion of Umbrea by RNAi knockdown (fig. S5A), relative to control cells, D. melanogaster S2 cells displayed increased mitotic errors, including delayed chromosome alignment, early anaphase onset, lagging anaphase chromosomes, and multipolar configurations (P < 0.05) (Fig. 1, F and G, fig. S5B, and movies S1 to S3). These data suggest that Umbrea promotes proper chromosome segregation, but is not required for the localization of the centromeric histone Cid (Fig. 1F).

To date the origin of Umbrea and subsequent changes, we sequenced the Umbrea locus from 32 Drosophila species (fig. S6A). Whereas HP1B was preserved (7), we found Umbrea in only 20 of 32 species, dating its monophyletic origin to 12 to 15 million years ago (Fig. 2A and fig. S6B). Using maximum likelihood methods, we observed evidence of both episodic and recurrent positive selection acting on Umbrea (fig. S7, A to D). These findings, together with the altered localization, lead us to conclude that neofunctionalization, not subfunctionalization, drove the divergence of Umbrea (10). Although Umbrea is essential in D. melanogaster, it was lost at least three independent times—in D. fuyamai, D. eugracilis, and in the suzukii clade (Fig. 2A)—which suggests that Umbrea was not essential at or immediately after its birth.

Fig. 2 Dynamic evolution of Umbrea after its birth.

(A) Polymerase chain reaction to shared syntenic sites followed by sequencing (fig. S6) revealed the presence and structure of Umbrea genes. Asterisks indicate strong support for key branch points in the phylogeny (25), suggesting that Umbrea was lost at least three times. Umbrea is presented with HP1 canonical domains: chromodomain (CD, green) and chromoshadow domain (CSD, blue). (B) Localization of GFP-tagged HP1B lacking its CD is diffuse in D. melanogaster Kc cell nuclei (magenta, anti-Cid; green, GFP; blue, DAPI staining of DNA; scale bar, 5 μm). (C) In contrast, HP1BmelCD+hinge fused to Umbreamel delocalizes it from centromeres.

Four lineages retained full-length Umbrea genes, two of which encode an intact chromodomain (CD) and ancestral residues essential for binding histone H3 trimethyl Lys9 (H3K9me) (fig. S8) (11). However, most extant Umbrea genes have lost their CDs, and encode only the chromoshadow domain (CSD), which mediates protein-protein interactions (12) (Fig. 2A). We first tested how CD loss affected HP1B function. We found that an HP1B-GFP fusion lacking the CD lost heterochromatin localization (Fig. 2B), consistent with the requirement of HP1 CD for H3K9me binding (13). Furthermore, fusion of the HP1B CD and hinge to Umbrea-GFP reverted localization from centromeres to heterochromatin (Fig. 2C), which suggests that loss of the ancestral CD was necessary for Umbrea to gain new function. Our findings support a model of neofunctionalization that is facilitated via intermediate loss of function (14). Although CD loss was necessary, it was not sufficient for Umbrea neofunctionalization; both full-length (D. fuyamai) and CSD-only (D. eugracilis and the suzukii clade) Umbrea genes have been lost in evolution.

We next investigated the consequences of evolution in the Umbrea-CSD. CSDs are only found in HP1-family proteins and mediate interactions with other HP1s or proteins possessing degenerate PxVxL motifs (P, Pro; V, Val; L, Leu; x, any amino acid) (15). An amino acid alignment of HP1B and Umbrea revealed conservation of residues defining the CSD structural fold (Fig. 3A). In contrast, three of the nine residues that mediate specificity for PxVxL recognition (16) changed along the branch leading to the melanogaster species subgroup (Fig. 3A and fig. S9). We found that D. melanogaster Umbrea CSD localized to centromeres (Fig. 3B). This property was not shared with HP1B CSD or even other Umbrea CSDs, because neither "parental" HP1Bmel CSD (from D. melanogaster) nor Umbreaptak CSD (from D. pseudotakahashii) could localize to centromeric regions in D. melanogaster cells (Fig. 3B and fig. S10B). We conclude that a discrete transition for centromere localization occurred in Umbrea CSD after divergence of the melanogaster and takahashii subgroups, coincident with changes in the PxVxL recognition residues. Indeed, reversion of these three residues (Cys15, Ile57, and Phe59; Fig. 3A and fig. S9) to the ancestral state delocalized Umbreamel CSD from centromeres (Fig. 3D). Moreover, replacement of the same residues in Umbreaptak CSD to corresponding residues in Umbreamel resulted in a gain of centromere localization (Fig. 3E). These results suggest that centromere localization by Umbrea CSD originated in the common ancestor of the melanogaster species subgroup 5 to 7 million years ago. Consistent with this, we found that GFP-Umbreatei localized to centromeres in D. teissieri cells (Fig. 3F). Centromeric localization may have also coincided with gain of essentiality, as Umbrea was lost three times prior to, but not after, CSD modification (Fig. 2A).

Fig. 3 Chromoshadow changes led to Umbrea centromere localization via altered protein-protein interactions.

(A) An amino acid alignment of HP1B and Umbrea CSDs reveals conservation of fold-defining residues but divergence in PxVxL recognition residues. In particular, three changes (bold) are predicted to affect the binding specificity of Umbrea CSD. Abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. (B) GFP-tagged Umbreamel CSD (green) colocalizes with Cid (magenta) at centromeres in D. melanogaster Kc cells (scale bar, 5 μm, colocalization appears white). (C) However, GFP-tagged Umbreaptak CSD does not localize to centromeres. (D) Reversion of Umbreamel PxVxL recognition residues (Cys-Ile-Phe) to ancestral states (Thr-Leu-Trp) causes delocalization from centromeres. (E) By contrast, introduction of PxVxL recognition residues (Cys-Ile-Phe) is sufficient to localize Umbreaptak CSD to centromeres (compare to Fig. 3C). (F) Umbreatei colocalizes with centromeric protein CENP-C in D. teissieri cells. (G) Immunoprecipitation of Flag (F)– and hemagglutinin (HA)–tagged Umbrea pulls down protein complexes in S2 cells. (H) Analysis of these complexes reveals that Umbrea and HP1B have mutually exclusive protein-protein interactions. Umbrea interacts with centromere and heterochromatin proteins [table S2; bold lines indicate confirmation of previously reported interactions (9, 17)], but not with the primary targets of HP1B (18).

To test the prediction that mutation of PxVxL recognition resulted in CSD centromere localization by alteration of protein interactions, we performed proteomic analyses to identify proteins that coimmunoprecipitate with Umbrea in S2 cells (Fig. 3G). Many chromatin factors were found in this set (table S1), including heterochromatin proteins HP4/Hip and HP5 [previously shown to be direct interactors of Umbrea (9, 17)], as well as novel interactions with the H3K9 methyltransferase Su(var)3-9 and the centromeric protein Cenp-C. We found no overlap with protein partners of HP1B, which include the euchromatic proteins HP1C, Woc, and Row (18) (Fig. 3H); this suggests a rewiring of the protein interaction network of Umbrea.

Our evolutionary analyses (fig. S7, A to D) indicated that the most recent innovations in Umbrea occurred in the short tail sequences that flank the CSD. We tested how these changes contributed to Umbrea neofunctionalization. HP1Bmel CSD alone showed no discrete localization (Fig. 2B), whereas the addition of Umbreamel tails was sufficient to confer centromere localization (Fig. 4A). These data indicate that Umbrea may target centromeres using both the CSD and the tails. Whereas the CSD likely mediates its localization via protein-protein interactions, Umbrea tails may bind centromeric nucleic acids, analogous to the hinge region of mammalian HP1α, which binds DNA in vitro (19). Because centromeric DNA sequence diverges rapidly (20), we tested whether rapid evolution of the Umbrea tails resulted in species specificity. We found that Umbreasim localized (Fig. 4B) to centromeres in D. melanogaster. However, Umbreatei and Umbreayak did not (Fig. 4, C and D), localizing instead to distinct foci. Although positive selection of Umbrea preceded its centromere localization (fig. S7), these data suggest that positive selection in the melanogaster species subgroup resulted in species-specific centromere targeting, reminiscent of CenH3/Cid in Drosophila (21). For example, despite mislocalizing in D. melanogaster cells, Umbreatei appropriately localized to D. teissieri centromeres (Fig. 3F).

Fig. 4 Species-specific centromere targeting of Umbrea.

(A) GFP fusion of Umbreamel tails with HP1B-CD (green) localizes to centromeres (magenta, anti-Cid; colocalization appears white; scale bar, 5 μm). (B to D) D. melanogaster Kc cell centromere localization (magenta, Cid) of Umbrea orthologs (green, GFP) from D. simulans, D. teissieri, and D. yakuba worsens with increased divergence. (E) Steps to essential neofunctionalization by Umbrea after gene duplication (Ma, millions of years ago).

Our analyses suggest that gain of essential function evolved in discrete steps (Fig. 4E) (5) that involved the loss of an ancestral domain (CD), rewiring of protein interaction networks (CSD), and species-specific changes (tails). Umbrea was likely not essential for much of its evolutionary history; intermediate forms were lost multiple times.

Our finding that Umbrea rapidly became essential for the conserved process of chromosome segregation is unexpected. Drosophila species that never possessed or lost Umbrea still carry out chromosome segregation. This suggests that the essential function of Umbrea might be a result of a lineage-specific requirement. Just as genetic conflicts arising during meiosis may drive rapid evolution of existing centromeric proteins (22), we propose that recurrent changes at centromeric DNA satellites could drive the retention of duplicate genes such as Umbrea to alleviate selective pressure on essential centromeric proteins. This is analogous to pathogen-driven genetic conflict, which promotes the diversification of existing and new antiviral immune genes (23). This process would result in idiosyncratic retention of centromeric proteins that become essential as they integrate into existing networks. Intriguingly, other HP1B-derived CSD-only genes are found in other Drosophila species that diverged before the birth of Umbrea (7), raising the possibility of convergent evolution of Umbrea-like centromere factors. This process may explain the broad diversity and divergence among centromeric proteins across taxa (24). Although a large fraction of the many young, essential genes identified in Drosophila (1) may result from subfunctionalization, others (like Umbrea) may illuminate other essential processes that could require recurrent genetic innovation to mitigate previously unappreciated adaptive challenges within the cell.

Supplementary Materials

www.sciencemag.org/cgi/content/full/340/6137/1211/DC1

Materials and Methods

Figs. S1 to S10

Table S1

References (2638)

Movies S1 to S3

References and Notes

  1. Acknowledgments: We thank J. Bloom, M. Daugherty, M. Emerman, D. Gottschling, M. Levine, M. Patel, N. Phadnis, K. Peichel, and W. Shou for helpful comments, and Drosophila colleagues for generous sharing of reagents. Supported by NIH Training grant T32HG000035 and an NSF predoctoral fellowship (B.D.R.), an EU network grant (EpiGeneSys 257082, A.I.), NSF award 1024973 (B.G.M.), and a grant from the Mathers Foundation and NIH grant R01GM074108 (H.S.M.). H.S.M. is an HHMI Early Career Scientist. Umbrea DNA sequences have been submitted to GenBank under accession numbers KC660086 to KC660100. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (PRIDE partner repository dataset identifier PXD000163).
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