Transcriptional Specificity of Drosophila Dysfusion and the Control of Tracheal Fusion Cell Gene Expression

Abstract

The Drosophila Dysfusion basic-helix-loop-helix-PAS (bHLH-PAS) protein controls the transcription of genes that mediate tracheal fusion. Dysfusion is highly related to the mammalian Nxf protein that has been implicated in nervous system gene regulation. Toward the goal of understanding how Dysfusion controls fusion cell gene expression, the biochemical properties of Dysfusion were investigated using protein interaction experiments, cell culture-based transcription assays, and in vivo transgenic analyses. Dysfusion dimerizes with the Tango bHLH-PAS protein, and together they act as a DNA binding transcriptional activator. Dysfusion/Tango binds multiple NCGTG binding sites, with the following preference: TCGTG > GCGTG > ACGTG > CCGTG. This binding site promiscuity differs from the restricted binding site preferences of other bHLH-PAS/Tango heterodimers. However, it is identical to the binding site preferences of mammalian Nxf/Arnt, indicating that the specificity is evolutionarily conserved. Germ line transformation experiments using a fragment of the CG13196 Dysfusion target gene allowed identification of a fusion cell enhancer. Experiments in which NCGTG sites were mutated individually and in combination revealed that TCGTG sites were required for fusion cell expression but that the single ACGTG and GCGTG sites present were not. Finally, a reporter transgene containing four tandemly arranged TCGTG elements has strong expression in tracheal fusion cells. Transgenic misexpression of dysfusion further revealed that Dysfusion has the ability to activate transcription in multiple cell types, although it does this most effectively in tracheal cells and can only function at midembryogenesis and later.

The Drosophila jing gene is a downstream target in the Trachealess/Tango tracheal pathway

“…1a). Sim/Tgo and Trh/Tgo heterodimers selectively bind CMEs of the sequence ACGTG (Emmons et al 1999;Jiang and Crews 2007). The sequences of CME1 showed a high degree of conservation in four Drosophila sibling species suggesting a functional requirement ( Fig.…”

“…Additional consensus DNA-binding sites are present for bHLH-PAS transcription factors including heterodimers of Dys and Tgo Crews 2003, 2007). Dys/Tgo heterodimers require TCGTG sites to activate gene expression, in vivo (Jiang and Crews 2007), of which there is one in the jing1.5 regulatory region and one in the adjacent jing1.3 element ( Fig. 1a and b).…”

“…The btl regulatory region was weakly immunoprecipitated from cells expressing only Tgo which usually requires a heterodimeric state to bind CMEs and activate transcription (Fig. 6a, lane 4; Sonnenfeld et al 1997;Ohshiro and Saigo 1997;Emmons et al 1999;Jiang and Crews 2007). However, only Trh/ Tgo heterodimers were able to associate with chromatin containing jing1.5 (Fig.…”

“…The Drifter/Vvl POU DNAbinding site was gathered from Ryan and Rosenfeld (1997), Anderson et al (1996), andCertel et al (1996). Dys/Tgo and bHLH-PAS DNA-binding sites were obtained from Jiang and Crews (2007).…”

Maximum-entropy network analysis reveals a role for tumor necrosis factor in peripheral nerve development and function

“…This group included netrin-1, which plays a role in axon guidance. The second group included genes related to networks involved in cytoplasmic extension, enervation, and Schwann cell function (Table S3) (30)(31)(32)(33)(34)(35). TNF is more tightly connected with the first group at the more fundamental level of transcriptional control related to NF-B, compared with the cellular effectors represented in the second group, suggesting that TNF is a network node that intersects with these 2 groups.…”