Imprinting of the Y chromosome influences dosage compensation in roX1 roX2 Drosophila melanogaster

Abstract

Drosophila melanogaster males have a well-characterized regulatory system that increases X-linked gene expression. This essential process restores the balance between X-linked and autosomal gene products in males. A complex composed of the male-specific lethal (MSL) proteins and RNA is recruited to the body of transcribed X-linked genes where it modifies chromatin to increase expression. The RNA components of this complex, roX1 and roX2 (RNA on the X1, RNA on the X2), are functionally redundant. Males mutated for both roX genes have dramatically reduced survival. We show that reversal of sex chromosome inheritance suppresses lethality in roX1 roX2 males. Genetic tests indicate that the effect on male survival depends upon the presence and source of the Y chromosome, revealing a germ line imprint that influences dosage compensation. Conventional paternal transmission of the Y chromosome enhances roX1 roX2 lethality, while maternal transmission of the Y chromosome suppresses lethality. roX1 roX2 males with both maternal and paternal Y chromosomes have very low survival, indicating dominance of the paternal imprint. In an otherwise wild-type male, the Y chromosome does not appreciably affect dosage compensation. The influence of the Y chromosome, clearly apparent in roX1 roX2 mutants, thus requires a sensitized genetic background. We believe that the Y chromosome is likely to act through modulation of a process that is defective in roX1 roX2 mutants: X chromosome recognition or chromatin modification by the MSL complex.

Figure 1.—

Schemes for producing roX1 roX2 males with reversed sex chromosome inheritance. (A) A mating that produces males with maternal or paternal roX1^ex6 roX2 chromosomes. Females carrying a roX1^ex6 roX2 X chromosome and the Df(1)nod FM7a balancer produce O, X and XX gametes (Table 2). Fertilization of an O gamete with an X-bearing sperm produces XO males. As Df(1)nod FM7a is lethal, no sons carrying this chromosome will be recovered. Sons carrying maternal (X_M) or paternal (X_P) X chromosomes are distinguished by the y⁺ marker. (B) Scheme for producing roX1^mb710 roX2 males bearing maternal X and Y chromosomes (Table 3, matings 1 and 2). Females homozygous for y roX1^mb710 roX2 chromosomes and carrying a y⁺Y chromosome are mated to compound X^Y males. All sons inherit a maternal roX1^mb710 roX2 X chromosome and lack a Y chromosome or carry the maternal Y chromosome. (C) Scheme for producing roX1^mb710 roX2 males bearing maternal X and Y chromosomes and a paternal Y chromosome (Table 3, matings 3 and 4). Females homozygous for y roX1^mb710 roX2 chromosomes and carrying a y⁺Y chromosome are mated to yw males with an unmarked Y chromosome. All sons have a maternal roX1^mb710 roX2 X chromosome and an unmarked paternal Y chromosome. Sons that inherit the maternal Y chromosome are distinguished by the y⁺ marker.

Figure 2.—

MSL1 recruitment to the X chromosome and chromocenter of roX1^mb710 roX2 males is not influenced by Y chromosome origin. (A and B) Polytene preparation from a wild type male. DNA is detected with DAPI (A) and MSL1 is detected by Texas Red (B). The chromocenter, marked by the arrowhead, has no MSL1 staining. (C and D) Polytene preparation from a roX1^mb710 roX2 /O male. The X chromosome (X) is scored as having minor MSL1 staining. The chromocenter (arrowhead) has strong MSL1 staining. (E) The intensity of MSL1 signal on the X chromosome was scored in roX1^mb710 roX2 males carrying a maternal Y chromosome, no Y, a paternal Y chromosome, or both maternal and paternal Y chromosomes. The percentage of nuclei falling into each category is on the Y axis. Over 100 nuclei of each karyotype were scored. (F) The intensity of MSL1 signal at the chromocenter was scored for the same nuclei. All nuclei were scored with labels obscured to prevent bias in scoring.

Figure 3.—

Beadex responds to MSL complex activity and Y chromosome origin. (A) Wing from Dp(1;1) Bx^r49k/+ female. (B) Dp(1;1) Bx^r49k/+ ; [w⁺Hs83-M2]6I female. (C) Dp(1;1) Bx^r49k/+ ; msl1¹/+;[ w⁺Hs83-M2]6I female. (D) Wing from roX1^mb710 roX2 Dp(1;1) Bx^r49k male with paternal Y chromosome. (E) Wing from roX1^mb710 roX2 Dp(1;1) Bx^r49k male with maternal Y chromosome. (F) The amount of wing margin lost is represented as the percentage of L3 vein length (arrowheads in D). Sixteen wings from X_MY_P males and 13 wings from X_PY_M males were measured. P values were determined by a two sample unpaired t-test.

Figure 4.—

X-linked gene expression is increased by a maternal Y chromosome. The expression of the X-linked genes Dlmo, SkpA and Ck-IIβ was measured by qRT PCR in wild type male larvae (open bars) and roX1^mb710 roX2 males with maternal (solid) or paternal (shaded) Y chromosomes. Four groups of 50 larvae contributed to each measurement. Expression was normalized using the autosomal gene Dmn. The significance of differences in expression was determined by a two sample unpaired t-test.