Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network

Abstract

Contemporary high-throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs, and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including ChIP sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF cooccupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous cooccupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmdbound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide insights into the transcriptional codes regulating muscle gene expression and offer a generalizable approach for similar studies in other systems.

Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

“…These TFs spanned multiple families, including ZFPs. We performed a similar assessment of our new ZFP recognition motifs using recently published ChIP data for nine factors (Chinmo, Disco, Lmd, Pho, Phol, Sens, Shn, Sna, and Ttk) (MacArthur et al 2009;Schuettengruber et al 2009;Negre et al 2010;Busser et al 2012). We evaluated binding potential to each genomic segment using Stubb scores, which reflect motif frequency (Hallikas et al 2006;Robasky and Bulyk 2011 (Kazemian et al 2010(Kazemian et al , 2011.…”

Jeb/Alk signalling regulates the Lame duck GLI family transcription factor in theDrosophilavisceral mesoderm

“…Modulation of Lmd activity occurs at the post-transcriptional level, in part by regulation of its subcellular localisation (Duan and Nguyen, 2006). Recent analysis has identified genomic regions bound by Lmd that are also co-occupied by key regulators such as Twist, Tinman and Mef2 combinatorially regulating the expression of FCM-and FC-specific genes in the embryonic mesoderm (Busser et al, 2012). Interestingly, recent work indicates a role for the E3 ubiquitin ligase Mind bomb 2 (Mib2) in the regulation of Lmd protein levels (Carrasco-Rando and Ruiz-Gómez, 2008).…”

An undergraduate laboratory class using CRISPR/Cas9 technology to mutate drosophila genes

“…Firstly, we determined if the Deficiency/Mutant offspring were either lethal, or if they showed a behavioral deficit consistent with the known expression pattern of the gene. Specifically, we predicted that knockouts of Act57B , CG1674 , CG9336 and CG14687 would be lethal based upon their broad expression in the mesoderm during embryonic development [16–19]. By contrast, we predicted that knockout of TpnC4 would be flightless based upon its expression in the adult flight muscles [20], and that knockout of Act79B would result in failure to jump, based upon its expression in the adult jump muscles ([21,22].…”