The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower—approximately 3-fold or more—for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution.

The evolution of different sex chromosomes (e.g., X and Y) has occurred many times in animals and plants. One consequence of having different chromosome copy numbers between the sexes (XY males and XX females) is the evolution of sex chromosome-specific regulation, both in the soma (i.e., X chromosome dosage compensation) and in the male germline (i.e., meiotic sex chromosome inactivation). Understanding how the X is regulated in the male germline has implications for gene expression, the evolution of sex chromosome-specific gene content, and speciation. Surprisingly, how the X chromosome is regulated in the Drosophila melanogaster male germline remains unclear. We have characterized X suppression, a novel form of X chromosome transcriptional regulation specific to the Drosophila male germline. Our results reveal that transcription of the X is suppressed 2- to 4-fold for endogenous genes. We show that the X chromosome has evolved strong testis-specific promoters via the gene-by-gene recruitment of sequence elements that counteract transcriptional suppression of the X chromosome. These findings reveal a novel form of X chromosome regulation and lead to a new model for the control of gene expression in the Drosophila male germline.

Funding: DCP was supported by grant no. 000596 from the David and Lucile Packard Foundation ( www.packard.org ), grant no. BR-5006 from the Alfred P. Sloan Foundation ( www.sloan.org ), and funds from the University of Rochester. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here we further characterize the regulation of X chromosome gene expression in the Drosophila male germline. First, we test if X suppression is restricted to genes with testis-specific promoters or is more general. Second, we test if X suppression is limited to transgene constructs having transposable elements as vectors—i.e., does X suppression correspond to a form of transposon silencing that differs between the X and autosomes? Third, we test if X suppression is specific to the male germline. Fourth, we test if X-autosome translocations show evidence of X suppression (or MSCI) in Drosophila testes. Finally, we present evidence that X-linked genes have adapted to X suppression via the recruitment of strong testis-specific promoters. We computationally identify and then functionally validate a promoter element that drives strong expression in the testis, is especially enriched in the promoters of testis-specific genes on the X chromosome, and is evolutionarily conserved. Our results reveal that the X chromosome has evolved strong testis-specific promoters via the gene-by-gene recruitment of sequence elements that counteract sex chromosome-wide transcriptional suppression in the Drosophila male germline. The strong promoters on the X chromosome effectively compensate the effects of transcriptional suppression, rendering X suppression undetectable except via genetic manipulations that move genes between the X and autosomes. These findings lead to a new model for the control of gene expression in the male germline and have clear implications for the evolution of gene expression, gene duplication, and gene location in the genome.

Two genetic findings have been suggested as evidence for MSCI in Drosophila. First, ~75% of X-autosome reciprocal translocations cause dominant male sterility (autosome-autosome translocations do not), as might be expected if putative allocyclic condensation of the sex chromosomes, and hence MSCI, is disrupted ([ 50 ]; but see Results below). Second, and more direct, the expression levels of transgene reporters in the Drosophila male germline are consistently lower for X-linked insertions than autosomal ones ([ 51 – 53 ]; see also [ 54 ]). In particular, promoters from five genes (two autosomal, three X-linked) with normally strong testis expression have been found to drive 3- to 8-fold lower expression of the lacZ reporter when the transgenes reside on the X chromosome ([ 51 – 53 ]; see also [ 54 ]). If the X chromosome undergoes MSCI, then X-linked transgenes may be prematurely silenced in primary spermatocytes, yielding lower average expression than autosomal transgenes [ 51 , 54 ]. The transgene findings are compelling, but some aspects of the data are difficult to reconcile with MSCI. For one, endogenous X-linked genes are not expressed ≥3-fold lower than autosomal genes in testes [ 40 , 41 , 48 ]. For another, RNA in situ analyses show that some X-linked transgene reporters initiate transcription relatively late in primary spermatocytes—i.e., at precisely the stage that MSCI is expected to silence the X [ 53 ]. Finally, the transcriptional suppression of some X-linked transgene reporters is detectable early in the male germline, in cells enriched for mitotic gonialblasts, prior to any putative MSCI [ 40 ]. Thus, while the transcriptional suppression of X-linked transgenes—which, for convenience, we hereafter term X suppression—is a real and robust phenomenon, it probably does not correspond to canonical MSCI (as in mammals or worms).

How sex chromosome gene expression is regulated in the Drosophila male germline has proved surprisingly difficult to resolve. Despite early claims that the X chromosome and autosomes are expressed at similar levels (e.g., [ 35 , 36 ]), sex chromosome-specific dosage compensation appears absent in the Drosophila male germline. First, key components of the MSL complex are not expressed in testes, and those that are do not localize to the X chromosome, indicating a lack of MSL-mediated sex chromosome dosage compensation [ 37 – 39 ]. Second, median expression of the X chromosome is ~1.5-fold lower relative to autosomes, consistent with basal but not sex chromosome dosage compensation [ 40 – 42 ]. Similarly, MSCI may also be absent from the Drosophila male germline, as previous data from cytology, microarray analyses, and indirect genetic evidence have failed to settle the question. Direct cytological evidence is inconclusive or lacking [ 34 , 43 – 46 ], and microarray analyses do not demonstrate the expected strong global down-regulation of X-linked gene expression as cells progress from premeiotic to meiotic stages of spermatogenesis ([ 40 , 47 ]; but see [ 48 , 49 ]).

MSCI has also evolved independently in taxa with XY (e.g., mammal) and XO systems (e.g., nematode, grasshoppers [ 25 – 27 ]); it is unclear if MSCI acts in ZW systems [ 28 , 29 ]. During MSCI in XY and XO systems, sex chromosomes are sequestered into a subcompartment of the nucleus and decorated with epigenetic modifications characteristic of heterochromatin formation and/or transcriptional silencing [ 26 – 29 ]. In mice, MSCI strongly impacts gene expression, resulting in the ~10-fold down-regulation of ~80% of X-linked genes in spermatocytes [ 30 , 31 ]. Like sex chromosome dosage compensation, the molecular basis of MSCI differs among taxa [ 12 , 13 ], but, unlike dosage compensation, the function of MSCI is still unclear [ 13 ]. It has been suggested that MSCI is an epigenetic form of host genome defense against selfish genetic elements [ 13 , 32 , 33 ] or that it functions to prevent recombination events between non-homologous X and Y chromosomes [ 34 ].

Sex chromosome dosage compensation has evolved in taxa with XY (Drosophila, mammal), XO (nematode), and, to varying degrees, ZW systems [ 14 – 17 ]. While the mode and molecular basis of dosage compensation differs among taxa, the function is the same [ 10 , 18 ]. In the somatic cells of Drosophila melanogaster males, the single X chromosome is dosage compensated by two mechanisms. First, generic basal dosage compensation mechanisms—including buffering and gene-specific regulation—result in an average ~1.5-fold increase in expression from the X [ 19 ]. Second, sex chromosome-specific dosage compensation up-regulates X-linked genes a further ~1.35-fold via the recruitment of the Male-Specific Lethal (MSL) protein-RNA complex to chromatin entry sites enriched for a GA-rich ~21-bp MSL recognition element (MRE) [ 20 , 21 ]. In several Drosophila lineages, neo-X chromosomes—i.e., ancestral autosomes that now segregate as sex chromosomes—have independently co-opted MSL-mediated dosage compensation via the de novo evolution of MREs [ 22 – 24 ].

Heteromorphic sex chromosomes—e.g., XY males in Drosophila and mammals and ZW females in birds and butterflies—have evolved independently numerous times in animals and in plants [ 1 , 2 ]. The different chromosome copy numbers between the sexes and the general lack of recombination between X and Y (Z and W) chromosomes have resulted in the evolution of sex chromosome-specific gene contents, rates of mutation, rates of evolution, and chromosome-wide forms of regulation [ 3 – 8 ]. Two types of sex chromosome regulation have evolved independently in disparate taxa: sex chromosome dosage compensation, a process that results in roughly equal X:autosome expression levels between the sexes [ 9 , 10 ], and meiotic sex chromosome inactivation (MSCI), the precocious heterochromatinization and transcriptional silencing of the sex chromosomes during meiosis I in the heterogametic sex [ 11 – 13 ].

Results

X Suppression Is Not Limited to Testis-Specific Promoters All previous evidence for transcriptional suppression on the X chromosome in the Drosophila male germline (hereafter, “X suppression”) has come from the study of P-element transgenes in which testis-specific promoters drive the expression of reporter genes [51,53,54]. It is therefore unclear if X suppression is restricted to testis-specific promoters or affects all promoters. We therefore tested if promoters that drive less tissue-specific expression profiles are subject to X suppression. We first confirmed X suppression for lacZ transgene reporters driven by the promoter of the autosomal testis-specific gene ocnus with a subset of X-linked and autosomal inserts used in previous work [51]: ocnus transgenes inserted into X chromosome locations (n = 5) are expressed 14.5-fold lower than those inserted into autosomal ones (n = 5; Table 1). To test if X suppression occurs for less tissue-specific promoters, we assayed testis expression of mini-white for the same transgenes (white is expressed in the male germline [40]): mini-white is expressed 1.7-fold lower from X-linked transgenes than autosomal transgenes (Table 1). Next, to test if X suppression affects promoters that mediate broad expression profiles, we assayed testis expression of transgene reporters driven by Actin 5c (Act5c) and Ubiquitin (Ubi). As Table 1 shows, Act5c and Ubi transgenes are expressed 23.2-fold and 9.6- to 13.8-fold lower on the X compared to autosomes. These results show, for a small sample of promoters (but see below), that X suppression is not limited to genes with testis-specific expression. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Testis expression of transgene reporters driven by testis-specific and non-testis–specific promoters. https://doi.org/10.1371/journal.pbio.1002499.t001