A Comprehensive Drosophila melanogaster Transcription Factor Interactome

Abstract

Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.

Keywords: BiFC; ChIP-seq analysis; Drosophila melanogaster; MITOMI; in vivo bimolecular fluorescence complementation assay; protein-protein interactions; transcription factor-DNA interactions; transcription factors; yeast two-hybrid.

Figure 1.. Comprehensive Drosophila Y2H TF-TF Interactome

(A) Overview of the Y2H screen. (B) Network view of the Y2H TF-TF interactome. (C) Degree distribution of the Y2H interactome. Median degree, 4; mean degree, 6.79. The red vertical line indicates the threshold for calling hub genes (degree, 50). (D) Comparisons of identified interactions with those in MasterNet. See also Figure S1 and Tables S1 and S2.

Figure 2.. Orthogonal Assays Validate Y2H Interactions

Summary of Y2H interactions validated by MITOMI and BiFC (A). Representative examples of positive and negative interactions assayed by (B) MITOMI and (C) BiFC. The scale bars in (B) are 250 μm; the scale bars in (C) are 50 mm. The depicted BiFC interactions (C) were tested in Drosophila eye antennal discs. See also Figure S2 and Tables S3 and S4.

Figure 3.. Examples of TF-TF Co-binding and Indirect Binding in Genomic Regions

(A) Schematic representation of Cyc-Foxo co-binding on DNA. (B) Cyc-Foxo co-binding is observed in the promoter region of vrille. GOMER tracks indicate motif enrichment for each TF. (C) Schematic representation of Hr78 indirect DNA binding through Hairy. (D) Hr78 indirect binding through Hairy is observed in the promoter region of kayak. GOMER tracks indicate motif enrichment for each TF. “S14 Regions” track indicates open chromatin regions derived from DNase-seq experiments performed at 10–11 h post-hatch (Li et al., 2011) (retrieved from the University of California, Santa Cruz [UCSC] Genome Browser), corresponding to the co-expressed time points of the 2 TFs.

Figure 4.. Integrated Fly TF-TF Interactome Identifies Candidate Human TFs Involved in Congenital Heart Disease and Eye Development

(A) The Drosophila TF-TF interactome can be used to identify candidate heart development and CHD genes. Nodes: Drosophila TFs; edges: PPIs. Nodes with human orthologs that are annotated as CHD associated in HGMD (pink nodes) and their first neighbors (blue nodes) that are expressed in Drosophila heart tissue are shown. Unconnected components arose due to this tissue expression requirement. Square: the Drosophila gene and its human ortholog(s) are involved in CHD and heart development based on a literature search; triangle: the Drosophila gene is involved in heart development; hexagon: the Drosophila gene has a human ortholog involved in heart development; circle: neither the Drosophila gene nor human ortholog(s) are known to be involved in heart development. Node sizes roughly reflect node degrees. Orange edges indicate newly discovered interactions in this study. Human ortholog gene names are given in parentheses below the Drosophila gene name. (B) The Drosophila TF-TF interactome can be used to identify candidate eye development genes. Nodes: Drosophila TFs; edges: PPIs. Nodes associated with eye development in Drosophila (based on Kumar, 2009; purple nodes) and their first neighbors (green nodes) are shown. Triangle: the Drosophila gene has eye development-related GO terms; hexagon: human, rat, mouse, or zebrafish (henceforth, vertebrate) ortholog of the Drosophila gene shown has eye development-related GO terms; square: the Drosophila gene (shown) and its vertebrate ortholog(s) have eye development-relatedGO terms; circle: neither the Drosophilagene nor vertebrate ortholog(s) have GO terms pertaining to eye development. Orange edges indicate newly discovered interactions in this study. Node sizes roughly reflect node degrees.