X chromosome inactivation during Drosophila spermatogenesis

Abstract

Genes with male- and testis-enriched expression are under-represented on the Drosophila melanogaster X chromosome. There is also an excess of retrotransposed genes, many of which are expressed in testis, that have "escaped" the X chromosome and moved to the autosomes. It has been proposed that inactivation of the X chromosome during spermatogenesis contributes to these patterns: genes with a beneficial function late in spermatogenesis should be selectively favored to be autosomal in order to avoid inactivation. However, conclusive evidence for X inactivation in the male germline has been lacking. To test for such inactivation, we used a transgenic construct in which expression of a lacZ reporter gene was driven by the promoter sequence of the autosomal, testis-specific ocnus gene. Autosomal insertions of this transgene showed the expected pattern of male- and testis-specific expression. X-linked insertions, in contrast, showed only very low levels of reporter gene expression. Thus, we find that X linkage inhibits the activity of a testis-specific promoter. We obtained the same result using a vector in which the transgene was flanked by chromosomal insulator sequences. These results are consistent with global inactivation of the X chromosome in the male germline and support a selective explanation for X chromosome avoidance of genes with beneficial effects late in spermatogenesis.

Figure 1. Schematic Diagram of the ocn-lacZ Expression Constructs

(A) The pP[wFl-ocn-lacZ] vector. The ocn promoter fused to the lacZ open reading frame was inserted into the pP[wFl] transformation vector, which contains the white gene as a selectable marker. The boundaries of the DNA inserted into the Drosophila genome are indicated by “P”. The portion of the plasmid used for replication in E. coli is labeled “pUC”. (B) The pP[YEStes-lacZ] vector. The ocn promoter and 3′ UTR were fused to respective ends of the lacZ open reading frame and inserted into the YES transformation vector. Binding sites for the Suppressor of Hairy-wing protein, which functions as a chromosomal insulator, are labeled “S”.

Figure 2. Reporter Gene Expression in Testis

Testes were dissected and incubated with S-GAL, which forms a black precipitate in the presence of β-galactosidase. Shown are testes from y w males (negative control) (A), y w males with an autosomal insertion of P[wFl-ocn-lacZ] (B), and y w males with an X-linked insertion of P[wFl-ocn-lacZ] (C). Staining was performed in parallel for the same length of time. The strongest signal is in the proximal region of the autosomal-insert testis. Note that weak staining is visible in the proximal region of the X-insert testis.

Figure 3. Average β-Galactosidase Activity of Adult Male Flies with Autosomal (Solid Bars) or X-Linked (Open Bars) Insertions of P[wFl-ocn-lacZ]

Each bar represents a different transformed line with a unique, independent transgene insertion. Error bars indicate the standard error of the mean, calculated from the variance among all replicate measurements within each independent insertion line.

Figure 4. Expression Levels of Autosomal (Solid Bars) and X-Linked (Open Bars) Insertions of the Two ocn-lacZ Transformation Vectors Shown in Figure 1

(A) Average β-galactosidase activity of adult males. (B) Relative expression measured by qRT-PCR. Transcript abundance was standardized to that of the ribosomal protein gene RpL32 and is given in arbitrary units. Error bars indicate the standard error of the mean, calculated from the variance among the means of the independent insertion lines.

Figure 5. Chromosomal Location of the Transgene Insertions

Arrows indicate the insertion sites of P[wFl-ocn-lacZ] (black) and P[YEStes-lacZ] (gray) transgenes as determined by inverse PCR. Nine additional inserts could be assigned only to the X chromosome or autosomes by genetic crosses and are not shown.