TTK69 binds Drosophila MEP1. (A) A yeast two-hybrid screen identified Drosophila MEP1 (CG1244) as a TTK69-interacting protein. TTK69 interacts with MEP1 aa 257 to 536, encoded by a partial cDNA that was expressed fused to the activation domain (AD) in the original screen. The potential C2H2 zinc fingers are indicated. (B) A series of TTK69 truncation constructs fused to the LexA DNA-binding domain were used to map the MEP1-binding domain. The BTB/POZ domain, the double zinc finger DNA-binding domain, and the PEST domain are diagramed. Dark green indicates the TTK69 sequence; light green, the sequence shared with TTK88. A region spanning aa 317 to 500 retained full MEP1-binding activity. Amino acids 392 to 500 bound somewhat more weakly but still displayed robust binding to MEP1. The same constructs have been used previously to delineate the Mi2 binding domain (27), revealing an overlap between the MEP1 and Mi2 binding regions of TTK69. Representative streaks of yeast expressing the two-hybrid fusions are shown. The relative interaction strengths are indicated as follows: −, no detectable interaction; (+), very weak; +, weak; ++, strong; +++, very strong. (C) TTK69 binds MEP1 directly. [³⁵S]methionine-labeled full-length TTK69 was incubated with either GST-MEP1 aa 257 to 536 or GST alone. Following washes, bound material was resolved by SDS-PAGE and visualized by autoradiography. Duplicate samples and 10% of the input were loaded.

TTK69, MEP1, and Mi2 interact genetically. Concomitant reduction of MEP1 and TTK69 or MEP1 and Mi2 causes an enhancement of the rough-eye phenotype. Representative scanning electron micrographs of adult eyes from flies with the indicated genotypes are shown. The GMR driver was used to direct the expression of specific interfering RNAs targeting TTK69 (B), Mi2 (C), or MEP1 (D), which had only a slight effect on eye development and the arrangement of ommatidia. However, a combined reduction in the levels of TTK69 and MEP1 (E) or Mi2 and MEP1 (F) strongly enhanced the rough-eye phenotype.

Genomewide expression profiling reveals that TTK69, MEP1, and Mi2 control overlapping transcriptomes. (A) Selective depletion of TTK69, MEP1, and Mi2. S2 cells were either mock treated or treated with dsRNA against TTK69, MEP1, or Mi2. Whole-cell extracts were analyzed by Western immunoblotting using the indicated antibodies. ISWI and α-tubulin served as loading controls. (B) The TTK69 transcriptome correlates well with that of NuRD, but not with that of ISWI or (P)BAP. Agglomerative hierarchical cluster analysis of the expression profiles of TTK69, NuRD, ISWI, and the (P)BAP core subunits SNR1, BRM, and MOR was performed. Clustering is based on Spearman R values. TTK69 and NuRD (green), ISWI (blue), and (P)BAP (red) clusters are indicated. (C) Principal-component (PC) analysis of gene expression profiles reveals the close clustering of TTK69 with Mi2 and MEP1, but not with ISWI or the (P)BAP core subunits. (D) Venn diagram depicting the numbers of genes that are coordinately regulated by TTK69, Mi2, and MEP1. Arrows indicate either upregulation or downregulation following depletion. The numbers of genes affected are given. (E) GO analysis and biological pathway clustering of genes coordinately regulated by TTK69, Mi2, and MEP1. morphogen., morphogenesis.

TTK69 recruits NuRD to selective target loci. The models depict the hierarchical relationship between the sequence-specific transcription factor TTK69 and the ATP-dependent chromatin remodeling factor NuRD. (A) NuRD cannot bind TTK69 loci by itself, whereas TTK69 can bind its binding site independently of NuRD. (B) TTK69 can target NuRD through direct protein-protein interactions with its MEP1 and Mi2 (27) subunits. (C) TTK69 tethers the NuRD complex to specific loci, where it attenuates target gene transcription by modulating the local structure of chromatin. We note that the precise molecular nature of NuRD remodeling in vivo is still unclear. Our results support the notion that TTK69 recruits NuRD to selective loci, not the other way around. See the text for details.

MEP1 and CDK2AP1/DOC1 are Drosophila NuRD subunits. (A and B) Immunopurification (IP) of MEP1 (A) and Mi2 (B) from embryo nuclear extracts. Embryo nuclear extracts were incubated with protein A-Sepharose beads coated with either a control (anti-GST) antibody (mock) or an affinity-purified anti-MEP1 or anti-Mi2 antibody. Input, unbound material, and proteins retained on the beads after extensive washing were resolved by SDS-PAGE and visualized by Coomassie staining. Bands were excised, and proteins were identified by nanoflow liquid chromatography-tandem mass spectrometry. The mass spectrometry scores of the indicated proteins are listed in Table 1. (C) Coimmunoprecipitations of NuRD and TTK69 with MEP1. Embryo nuclear extracts were incubated either with preimmune serum (mock) or with an anti-Mi2 or anti-MEP1 antibody. Immunopurified proteins were resolved by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. Ten percent of the input material was loaded for reference. (D) CDK2AP1/DOC1 is a NuRD subunit. Results of coIPs using anti-CDK2AP1/DOC1 antibodies are shown. (E) The majority of NuRD is stably associated with CDK2AP1/DOC1. Nuclear extracts were immunodepleted with beads that were coated either with preimmune serum (mock) or with an antibody directed against CDK2AP1/DOC1. The supernatants were then resolved by SDS-PAGE and analyzed by Western immunoblotting with the indicated antibodies. Whereas NuRD subunits were strongly depleted, ISWI remained unaffected. Because RPD3 is part of multiple complexes, its depletion is expected to be less complete. (F) Cartoon summarizing our proteomic results. Drosophila NuRD comprises the ATPase Mi2, the HDAC RPD3, MTA1-like, CAF1 p55, p66/68-like, MBD-like isoforms A and B, MEP1, and CDK2AP1/DOC1. We note that mammalian CDK2AP1 was identified previously as a mammalian NuRD-associated protein by Le Guezennec et al. (19). We failed to detect additional proteins that have been reported incidentally as binding to NuRD. We do not consider TTK69 a NuRD subunit, although this cannot be formally concluded from the proteomics results. Rather, we view TTK69 as a transcription factor that interacts with NuRD by binding MEP1 and Mi2.

TTK69 colocalizes with MEP1 and Mi2 on many loci. (A to E) The distributions of TTK69 (red) and MEP1 (green) on Drosophila salivary gland chromosomes were determined by immunostaining. (F to J) Likewise, the distributions of TTK69 (red) and Mi2 (green) were determined. DNA stained blue (with DAPI) in the merged panels. The colocalization of TTK69 and NuRD on many sites is demonstrated by the yellow staining in the merged panels and the similar patterns in split polytenes. However, TTK69 and NuRD also occupy unique loci.

NuRD binding to selective promoters depends on TTK69, whereas TTK69 binding is independent of NuRD. (A) Upregulation of ARK, Hairy, and En after TTK69, MEP1, or Mi2 knockdown. RNA was extracted and quantified by RT-qPCR using appropriate primers. mRNA levels were normalized against those of CG11874, a gene whose expression did not change in our microarray experiments. Normalized mRNA levels were expressed relative to those in mock-treated cells. Graphs represent the results of three independent biological replicate experiments. Error bars represent the standard errors of the means. (B to E) ChIP-qPCR analysis of the binding of TTK69 (B), Mi2 (C), and MEP1 (D) to ARK, Hairy, and En. The BAP target SK1 is not bound by TTK69 or NuRD and serves as a negative control. ChIPs were performed on chromatin from mock-treated or RNAi-depleted cells for TTK69, MEP1, or Mi2. (E) The binding of BAP111 to its targets SK1 and KCNQ is not affected by RNAi against TTK69, MEP1, or Mi2. Cross-linked chromatin was prepared from mock-treated or RNAi-treated cells. All ChIP data are the results of at least 3 independent biological replicates. Error bars represent the standard errors of the means.