The mof transgene driven by a tubulin promoter is a target for MSL1 and overcomes the requirement for MSL3. (A) Tubulin-driven mof gene. (B,C) Tubulin-driven mof gene (tub > y⁺ > mof) containing a yellow gene cassette between the promoter and the gene such that mof expression becomes inducible upon excision of the yellow gene cassette by expression of flippase. Recruitment of MSL1 before (B) and after (C) excision is shown. (D) Same as in A, however, in this case the transgenic line carrying tub-mof was crossed into the msl-3⁰⁸³ mutant background by using females mutant for msl-3⁰⁸³ expressing low levels of MSL2 from an Hsp83 promoter. Recruitment of MSL1 to the tub-mof transgene is indicated by an arrow. The position of the endogenous mof gene is shown by an asterisk. Red cross (×) represents no transcription. DNA was stained with Hoechst 322 (blue).

Targeting by MSL1 to the mof gene involves DNA sequences embedded in the coding region. MSL1 immunostaining (green) in combination with DNA-FISH (red) for the location of the transgene for insertions containing mof with an antisense promoter (A), tubulin-driven antisense mof gene (B), endogenous promoter-driven mof derivative lacking 670 bp of the 3′ end (C), or endogenous promoter-driven 3′ end of mof (1863–2533 bp) (D). Recruitment of MSL1 to the transgenes is indicated by an arrow. DNA was stained with Hoechst 322 (blue).

Transcriptional-dependent recruitment of MSL1 to the mof gene. (A) ChIP in SL-2 cells on the CG3016 and mof genes with antibodies directed against MSL1, MSL3, and MOF. Levels of enrichment are determined by Q-PCR and depicted as the percentage recovery of input DNA. The position of the primer pairs is schematically shown by green arrows. (B–D) MSL-1 immunostaining (green) in combination with DNA-FISH (red) directed against the mini-white gene of a polytene chromosome carrying an autosomal 6.7-kb P-element insertion encompassing mof and the coding region of CG3016 (B), or an autosomal P-element insertion of mof with its endogenous promoter (C) or without its endogenous promoter (D). Recruitment of MSL1 to the transgenes is indicated by an arrow. The position of the endogenous mof gene is shown by an asterisk. DNA was stained with Hoechst 322 (blue).

Recruitment of MSL1 and MOF on X-chromosomal genes is severely affected upon blocking transcription by α-amanitin. (A) Q-RT–PCR analysis of expression levels normalized to mock (100%) after a 20-h α-amanitin treatment. (B) Western blot analysis from whole-cell extracts prepared from mock- or α-amanitin-treated cells. Western blots were probed with MSL1, MOF, and Tubulin antibodies as indicated. (C) ChIP in SL-2 cells with antibodies directed against MSL1, and MOF after 20-h mock or α-amanitin treatment. Levels of enrichment are determined by Q-PCR and depicted as the percentage recovery of input DNA. Enrichment on each gene was systematically tested using primer pairs located at the beginning, middle, and end of genes. Position of the PCR probes with respect to the transcription start site (+1) is indicated on the X-axis; for example, for UCP4A, the first primer pair is located 367 bp upstream of the transcription start site, and the second and third primer pairs are located 612 bp and 2860 bp downstream, respectively. (D) Immunofluorescence with MSL1 (red) and Lamin (green) in Schneider (SL-2) cells of mock- or α-amanitin-treated cells. DNA was stained with Hoechst 322 (blue).

Transcription-dependent targeting of MSL1 to CG3016 and the identification of a targeting sequence toward the 3′ end of the gene. MSL1 immunostaining (green) in combination with DNA-FISH (red) on an autosomal P-element insertion carrying CG3016 with (A) or without (B) its endogenous promoter. (C) A 339-bp fragment (represented by a white box) corresponding to nucleotide positions 1581–1920 bp from the transcription start site of CG3016. Multimerization of the 339-bp sequence (five tandem repeats) in a wild-type (D) and an msl-3⁰⁸³ mutant (E) background. Recruitment of MSL1 to the transgenes is indicated by an arrow. DNA was stained with Hoechst 322 (blue).