Figure 1. Deletion of the proline-rich domain of MML-1 does not affect its DNA binding and O-GlcNAc modification: (A) Venn diagram showing an overlap between ZFP-1 and DOT-1.1 chromatin localization peaks (ChIP-chip data, Cecere et al. 2013) and O-GlcNAc chromatin localization peaks (ChIP-chip data, Love et al. 2010). (B) Schematic of the C. elegans MML-1 protein: Mondo-specific domains are indicated in green, proline-rich domain is depicted as a grey oval, and the DNA binding/dimerization domain is shown in yellow. The mutant alleles used are indicated above the protein schematic; a loss-of-function
mml-1(
ok849) allele is an in-frame internal deletion, shown by grey box. The region recognized by the anti-MML-1 antibody (Pickett et al. 2007) is underlined. (C) Western immunoblotting showing anti-MML-1 antibody specificity using
mml-1 mutant alleles designated on the schematic. Asterisks indicate non-specific bands. (D) Detection of O-GlcNAc modification of MML-1: O-GlcNAc IP followed by MML-1 western with the N-terminus-specific antibody is shown; the truncated
mml-1(
ok849) product is modified. (E) MML-1 ChIP-qPCR experiment showing MML-1 enrichment at indicated promoters (% Input) in wild type and mutant L3 larvae. Dashed lines denote ChIP background, based on ChIP with unrelated IgG and/or MML-1 ChIP with
mml-1(
gk402844) null mutant extracts. Error bars denote SD of triplicate qPCR runs. Asterisks *, ** and *** denote P values (Student's t-test) less than 0.05, 0.01 and 0.001, respectively, mutant vs wild type or as indicated. MML-1 ChIP-qPCR was repeated three times. (F) Hermaphrodite-specific neuron (HSN) under-migration in
mml-1(
gk402844) and
mml-1(
ok849) mutant worms. HSNs are visualized by the Tryptophan Hydroxylase (TPH) promoter driven GFP reporter marking serotonergic neurons. Asterisks indicate the vulva and arrows denote the HSN position. Images combine Differential Interference Contrast (DIC) and GFP fluorescence; scale bar is 100 micrometers.