Figure 3.—

A complex interaction between loss of dSETDB1 and of SU(VAR)3-9 impacts heterochromatin formation, with the double mutant showing a less severe phenotype. (A) dSETDB1 and dG9a act with SU(VAR)3-9 to produce methyl-H3K9 in vivo. Western blot showing quantities of H3K9 methylation in double mutants (adults). Values at the bottom of each blot express the fraction of mono-, di-, or trimethylation in double mutants by comparison with homozygous Su(var)3-9⁰⁶. Knockdown of dG9a and dSETDB1 was achieved by combining the Act5CGAL4 driver with hairpins dG9ahp²²⁰¹ or dSETDB1hp⁰⁴⁰⁸. (B) Loss of silencing in dSETDB1^KD lines is partially restored by depletion of SU(VAR)3-9, except at pericentric fourth chromosome heterochromatin. Pigment values for dominant suppression by Su(var)3-9⁰⁶/+, dSETDB1^KD and dSETDB1^KD;Su(var)3-9⁰⁶/+ are compared with parental (no HMT mutant) values. Knockdown of dSETDB1 was achieved by combining the daGAL4 driver with hairpin dSETDB1hp^2101B. Error bars represent standard deviations (n = 3). (C) Comparison of fold suppression changes from the parental pigment values are expressed for single and double mutants on the basis of the data from B. Enhancement of PEV (reduced pigmentation) is expressed as a negative fold change.