Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I-HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.

Two forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway.

The development of a complex organ requires the specification of appropriate numbers of each of its constituent cell types, as well as their proper differentiation and correct positioning relative to each other. During Drosophila cardiogenesis, all three of these processes are controlled by jumeau (jumu) and Checkpoint suppressor homologue (CHES-1-like), two genes encoding forkhead transcription factors that we discovered utilizing an integrated genetic, genomic, and computational strategy for identifying genes expressed in the developing Drosophila heart. Both jumu and CHES-1-like are required during asymmetric cell division for the derivation of two distinct cardiac cell types from their mutual precursor and in symmetric cell divisions that produce yet a third type of heart cell. jumu and CHES-1-like control the division of cardiac progenitors by regulating the activity of Polo, a kinase involved in multiple steps of mitosis. This pathway demonstrates how transcription factors integrate diverse developmental processes during organogenesis.

Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors.

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs - including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) - to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, our results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression.