Figure 1. UpSET is a SET-containing nuclear protein required for Drosophila oogenesis.

(A) Schematic of the upSET locus on chromosome 3L. Location of the SET and PHD domains are highlighted. Arrowhead indicates P-element insertion in upSET^e00365. Graph shows the locus conservation in the Drosophilidae family.

(B) UpSET shares only ~15% identity with mouse MLL5 in the SET domain. The mouse MLL5 SET domain shares 60% identity with that of mouse SETD5.

(C) Sequence alignment of the SET and PHD domains between Drosophila UpSET and mouse MLL5. % identity is indicated.

(D) UpSET is mainly a nuclear protein. Western blot analysis of nuclear (NE) and cytoplasmic (Cyt) extracts from Kc cells probed with anti-UpSET N-end monoclonal antibodies. Full-length UpSET protein is denoted by the arrow. *:Non-specific band.

(E–E’) UpSET forms foci in the nucleus. Confocal projection of 10 serial 0.2μm optical sections of a Kc cell stained with a mix anti-UpSET NH3-terminus monoclonal antibodies (green; F–F’) and DAPI to visualize DNA (F’).

(F–H) Third larval instar polytene chromosomes stained with a mix of antibodies to UpSET NH3- and COOH-terminus (green) and DNA counterstained with DAPI (blue). Higher magnification view of X chromosome (H). UpSET recruitment is enriched in euchromatic regions (I).

(I) UpSET does not exhibit histone methyltransferase activity (HMT). Radioactive HMT assays performed with bacterially purified GST or GST-UpSET SET domain and purified histones as substrate (J). Recombinant proteins were incubated with Super Sex Combs (SXC) or nuclear extracts (NE). SET7 was used as positive control. Loading controls for the histone methyltransferase assay are shown.

(K) Polytene chromosome from upSET^e00365 mutant third instar larva stained with a mix of the 5 UpSET monoclonal antibodies (green) and DNA counterstained with DAPI (blue).

(J) Western blot analysis of larval nuclear extracts from wildtype and upSET^e00365 mutants blotted with a mix of the 5 antibodies against UpSET NH3- and COOH-termini. Lamin Dm0 serves as loading control.

(L–M) Lack of UpSET disrupts oogenesis. Confocal projections of 5 serial 1μm optical sections of ovaries from wildtype (N) and upSET^e00365 mutants (O) actin stained with phalloidin. Scale bars represent 100μm. See also Figure S1.