Molecular mechanism underlying the regulatory specificity of aDrosophilahomeodomain protein that specifies myoblast identity

Abstract

A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein-binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs that have mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To understand better the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou-binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity.

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

“…To test this hypothesis, we examined 18 such regions for transcriptional activity using transgenic reporter assays (Dataset S1; Fig. S2) (15,16). Although only 2 of these regions were active in FCMs (Fig.…”

“…Raw sequencing data were obtained with an Illumina HiSeq-2000 sequencer and deposited in the Gene Expression Omnibus as GSE38402. PBM assays (15), classifier training (16), and analysis of transgenic reporter constructs (15, 16) were performed as described.…”

Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements

“…We used the TF binding prediction tool PROMO (28) to computationally analyze the catTFRE sequence and find 132 "accidental" TF binding sites for human TFs and more than 300 additional TF binding sites for TFs of other species (Dataset S2). It has been known that each family of TFs binds a specific consensus sequence, but there are clear differences among members of a family (29)(30)(31)(32). Our simplified generic design of TFRE may not reveal the subtle differences in DNA binding among members of a family.…”

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of <em>Drosophila</em> Embryos

“…The only tissue-specific transcriptomic approaches conducted so far in this model have been performed using cell or nuclear sorting and have only been able to study steady-state transcriptome [27][28][29][30][31] , creating a need for methods dedicated to tissue/cell-specific translatome profiling in the fly embryo. Here we report the first TRAP protocol dedicated to Drosophila embryos.…”