cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg.

We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development.

Accurate and sensitive quantification of protein-DNA binding affinity.

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Quantitative Analysis of the DNA Methylation Sensitivity of Transcription Factor Complexes.

Although DNA modifications play an important role in gene regulation, the underlying mechanisms remain elusive. We developed EpiSELEX-seq to probe the sensitivity of transcription factor binding to DNA modification in vitro using massively parallel sequencing. Feature-based modeling quantifies the effect of cytosine methylation (5mC) on binding free energy in a position-specific manner. Application to the human bZIP proteins ATF4 and C/EBPß and three different Pbx-Hox complexes shows that 5mCpG can both increase and decrease affinity, depending on where the modification occurs within the protein-DNA interface. The TF paralogs tested vary in their methylation sensitivity, for which we provide a structural rationale. We show that 5mCpG can also enhance in vitro p53 binding and provide evidence for increased in vivo p53 occupancy at methylated binding sites, correlating with primed enhancer histone marks. Our results establish a powerful strategy for dissecting the epigenomic modulation of protein-DNA interactions and their role in gene regulation.

Yorkie promotes transcription by recruiting a histone methyltransferase complex.

Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. We report that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. We show that Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr) methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie's ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie's mammalian homolog YAP. Our results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification.

Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc.

The accurate control of cell proliferation and survival is critical for animal development. The Hippo tumor suppressor pathway regulates both of these parameters by controlling the nuclear availability of the transcriptional coactivator Yorkie (Yki), which regulates downstream target genes together with Scalloped (Sd), a DNA-binding protein. Here we provide evidence that Yki can also regulate target genes in conjunction with Homothorax (Hth) and Teashirt (Tsh), two DNA-binding transcription factors expressed in the uncommitted progenitor cells of the Drosophila eye imaginal disc. Clonal analyses demonstrate that Hth and Tsh promote cell proliferation and protect eye progenitor cells from apoptosis. Genetic epistasis experiments suggest that Hth and Tsh execute these functions with Yki, in part by up-regulating the microRNA bantam. A physical interaction between Hth and Yki can be detected in cell culture, and we show that Hth and Yki are bound to a DNA sequence approximately 14 kb upstream of the bantam hairpin in eye imaginal disc cells, arguing that this regulation is direct. These data suggest that the Hippo pathway uses different DNA-binding transcription factors depending on the cellular context. In the eye disc, Hth and Tsh provide spatial information to this pathway, promoting cell proliferation and survival in the progenitor domain.

Molecular integration of wingless, decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development.

The development of the Drosophila leg requires both Decapentaplegic (Dpp) and Wingless (Wg), two signals that establish the proximo-distal (PD) axis by activating target genes such as Distalless (Dll). Dll expression in the leg depends on a Dpp- and Wg-dependent phase and a maintenance phase that is independent of these signals. Here, we show that accurate Dll expression in the leg results from the synergistic interaction between two cis-regulatory elements. The Leg Trigger (LT) element directly integrates Wg and Dpp inputs and is only active in cells receiving high levels of both signals. The Maintenance (M) element is able to maintain Wg- and Dpp-independent expression, but only when in cis to LT. M, which includes the native Dll promoter, functions as an autoregulatory element by directly binding Dll. The "trigger-maintenance" model describes a mechanism by which secreted morphogens act combinatorially to induce the stable expression of target genes.

Logic of Wg and Dpp induction of distal and medial fates in the Drosophila leg.

Drosophila leg development requires the cooperation of two secreted signals, Decapentaplegic (Dpp) and Wingless (Wg), to form the proximodistal (PD) axis. Wg and Dpp are also required to pattern the dorsoventral (DV) axis of the leg. Here, we show that Distalless (Dll) and dachshund (dac), genes expressed at different positions along the PD axis, are activated by Wg signaling and repressed by Brinker (Brk), a transcriptional repressor in the Dpp pathway. The levels of both Brk and Wg determine which of these PD genes is activated. Surprisingly, Brk does not play a role in DV axis specification in the leg, suggesting that Dpp uses two distinct mechanisms for generating the PD and DV axes. Based on these results, we present a model for how Dpp and Wg, which are present as dorsal and ventral gradients, respectively, induce nearly circular domains of gene expression along the PD axis.

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling.

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. Here, we describe the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. We propose a model in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP.

The t(8;21) translocation converts AML1 into a constitutive transcriptional repressor.

The human translocation (t8;21) is associated with approximately 12% of the cases of acute myelogenous leukemia. Two genes, AML1 and ETO, are fused together at the translocation breakpoint, resulting in the expression of a chimeric protein called AML1-ETO. AML1-ETO is thought to interfere with normal AML1 function, although the mechanism by which it does so is unclear. Here, we have used Drosophila genetics to investigate two models of AML1-ETO function. In the first model, AML1-ETO is a constitutive transcriptional repressor of AML1 target genes, regardless of whether they are normally activated or repressed by AML1. In the second model, AML1-ETO dominantly interferes with AML1 activity by, for example, competing for a common co-factor. To discriminate between these models, the effects of expressing AML1-ETO were characterized and compared with loss-of-function phenotypes of lozenge (lz), an AML1 homolog expressed during Drosophila eye development. We also present results of genetic interaction experiments with AML1 co-factors that are not consistent with AML1-ETO behaving as a dominant-negative factor. Instead, our data suggest that AML1-ETO acts as a constitutive transcriptional repressor.

Differing strategies for the establishment and maintenance of teashirt and homothorax repression in the Drosophila wing.

Secreted signaling molecules such as Wingless (Wg) and Decapentaplegic (Dpp) organize positional information along the proximodistal (PD) axis of the Drosophila wing imaginal disc. Responding cells activate different downstream targets depending on the combination and level of these signals and other factors present at the time of signal transduction. Two such factors, teashirt (tsh) and homothorax (hth), are initially co-expressed throughout the entire wing disc, but are later repressed in distal cells, permitting the subsequent elaboration of distal fates. Control of tsh and hth repression is, therefore, crucial for wing development, and plays a role in shaping and sizing the adult appendage. Although both Wg and Dpp participate in this control, their specific contributions remain unclear. In this report, we analyze tsh and hthregulation in the wing disc, and show that Wg and Dpp act independently as the primary signals for the repression of tsh and hth, respectively. In cells that receive low levels of Dpp, hth repression also requires Vestigial (Vg). Furthermore, although Dpp is required continuously for hth repression throughout development, Wg is only required for the initiation of tsh repression. Instead, the maintenance of tsh repression requires Polycomb group (PcG) mediated gene silencing, which is dispensable for hth repression. Thus, despite their overall similar expression patterns, tsh and hth repression in the wing disc is controlled by two very different mechanisms.

Boca, an endoplasmic reticulum protein required for wingless signaling and trafficking of LDL receptor family members in Drosophila.

The maturation of cell surface receptors through the secretory pathway often requires chaperones that aid in protein folding and trafficking from one organelle to another. Here we describe boca, an evolutionarily conserved gene in Drosophila melanogaster, which encodes an endoplasmic reticulum protein that is specifically required for the intracellular trafficking of members of the low-density lipoprotein family of receptors (LDLRs). Two LDLRs in flies, Arrow, which is required for Wingless signal transduction, and Yolkless, which is required for yolk protein uptake during oogenesis, both require boca function. Consequently, boca is an essential component of the Wingless pathway but is more generally required for the activities of multiple LDL receptor family members.

Engrailed cooperates with extradenticle and homothorax to repress target genes in Drosophila.

Engrailed is a key transcriptional regulator in the nervous system and in the maintenance of developmental boundaries in Drosophila, and its vertebrate homologs regulate brain and limb development. Here, we show that the functions of both of the Hox cofactors Extradenticle and Homothorax play essential roles in repression by Engrailed. Mutations that remove either of them abrogate the ability of Engrailed to repress its target genes in embryos, both cofactors interact directly with Engrailed, and both stimulate repression by Engrailed in cultured cells. We suggest a model in which Engrailed, Extradenticle and Homothorax function as a complex to repress Engrailed target genes. These studies expand the functional requirements for extradenticle and homothorax beyond the Hox proteins to a larger family of non-Hox homeodomain proteins.