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1.  CDK8-Cyclin C Mediates Nutritional Regulation of Developmental Transitions through the Ecdysone Receptor in Drosophila 
PLoS Biology  2015;13(7):e1002207.
The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval–pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval–pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval–pupal transition.
During the larval-pupal transition in Drosophila, CDK8-CycC helps to link nutrient intake to development by activating ecdysone receptor-dependent transcription and to fat metabolism by inhibiting SREBP-activated gene expression.
Author Summary
Arthropods are estimated to account for over 80% of animal species on earth. Characterized by their rigid exoskeletons, juvenile arthropods must periodically shed their thick outer cuticles by molting in order to grow. The steroid hormone ecdysone plays an essential role in regulating the timing of developmental transitions, but exactly how ecdysone and its receptor EcR activates transcription correctly after integrating nutritional and developmental cues remains unknown. Our developmental genetic analyses of two Drosophila mutants, cdk8 and cycC, show that they are lethal during the prepupal stage, with aberrant accumulation of fat and a severely delayed larval–pupal transition. As we have reported previously, CDK8-CycC inhibits fat accumulation by directly inactivating SREBP, a master transcription factor that controls the expression of lipogenic genes, which explains the abnormal fat accumulation in the cdk8 and cycC mutants. We find that CDK8 and CycC are required for EcR to bind to its target genes, serving as transcriptional cofactors for EcR-dependent gene expression. The expression of EcR target genes is compromised in cdk8 and cycC mutants and underpins the retarded pupariation phenotype. Starvation of feeding larvae precociously up-regulates CDK8 and EcR, prematurely down-regulates SREBP activity, and leads to early pupariation, whereas re-feeding starved larvae has opposite effects. Taken together, these results suggest that CDK8 and CycC play important roles in coordinating nutrition intake with fat metabolism by directly inhibiting SREBP-dependent gene expression and regulating developmental timing by activating EcR-dependent transcription in Drosophila.
PMCID: PMC4519132  PMID: 26222308
2.  Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications 
Models for genetic regulation and cell fate specification characteristically assume that gene regulatory networks (GRNs) are essentially deterministic and exhibit multiple stable states specifying alternative, but pre-figured cell fates. Mounting evidence shows, however, that most eukaryotic precursor RNAs undergo alternative splicing (AS) and that the majority of transcription factors contain intrinsically disordered protein (IDP) domains whose functionalities are context dependent as well as subject to post-translational modification (PTM). Consequently, many transcription factors do not have fixed cis-acting regulatory targets, and developmental determination by GRNs alone is untenable. Modeling these phenomena requires a multi-scale approach to explain how GRNs operationally interact with the intra- and intercellular environments. Evidence shows that AS, IDP, and PTM complicate gene expression and act synergistically to facilitate and promote time- and cell-specific protein modifications involved in cell signaling and cell fate specification and thereby disrupt a strict deterministic GRN-phenotype mapping. The combined effects of AS, IDP, and PTM give proteomes physiological plasticity, adaptive responsiveness, and developmental versatility without inefficiently expanding genome size. They also help us understand how protein functionalities can undergo major evolutionary changes by buffering mutational consequences.
PMCID: PMC4341551  PMID: 25767796
cell fate specification; combinatorial transcription regulation; eukaryotes; evolution; development; gene regulatory networks; multi-stable dynamical systems; protein structure
Journal of molecular biology  2009;390(4):760-774.
In developing bilaterans, the Hox transcription factor family regulates batteries of downstream genes to diversify serially repeated units. Given Hox homeodomains bind a wider array of DNA binding sites in vitro than are regulated by the full-length protein in vivo, regions outside the homeodomain must aid DNA site selection. Indeed, we find affinity for disparate DNA sequences varies less than 3-fold for the homeodomain isolated from the Drosophila Hox protein Ultrabithorax Ia (UbxHD), whereas for the full-length protein (UbxIa) affinity differs by more than 10-fold. The rank order of preferred DNA sequences also differs, further demonstrating distinct DNA binding preferences. The increased specificity of UbxIa can be partially attributed to the I1 region, which lies adjacent to the homeodomain and directly impacts binding energetics. Each of three segments within I1 – the Extradenticle-binding YPWM motif, the 6 amino acids immediately N-terminal to this motif, and the 8 amino acids abutting the YPWM C-terminus – uniquely contribute to DNA specificity. Combination of these regions synergistically modifies DNA binding to further enhance specificity. Intriguingly, the presence of the YPWM motif in UbxIa inhibits DNA binding only to Ubx•Extradenticle heterodimer binding sites, potentially functioning in vivo to prevent Ubx monomers from binding and misregulating heterodimer target genes. However, removal of the surrounding region allows the YPWM motif to also inhibit binding to Hox-only recognition sequences. Despite a modular domain design for Hox proteins, these results suggest that multiple Hox protein regions form a network of regulatory interactions that coordinate context-and gene-specific responses. Since most non-homeodomain regions are not conserved between Hox family members, these regulatory interactions have the potential to diversify binding by the highly homologous Hox homeodomains.
PMCID: PMC2739810  PMID: 19481089
Hox; DNA binding; homeodomain; YPWM; hexapeptide motif
4.  The Intrinsically Disordered Regions of the Drosophila melanogaster Hox Protein Ultrabithorax Select Interacting Proteins Based on Partner Topology 
PLoS ONE  2014;9(10):e108217.
Interactions between structured proteins require a complementary topology and surface chemistry to form sufficient contacts for stable binding. However, approximately one third of protein interactions are estimated to involve intrinsically disordered regions of proteins. The dynamic nature of disordered regions before and, in some cases, after binding calls into question the role of partner topology in forming protein interactions. To understand how intrinsically disordered proteins identify the correct interacting partner proteins, we evaluated interactions formed by the Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx), which contains both structured and disordered regions. Ubx binding proteins are enriched in specific folds: 23 of its 39 partners include one of 7 folds, out of the 1195 folds recognized by SCOP. For the proteins harboring the two most populated folds, DNA-RNA binding 3-helical bundles and α-α superhelices, the regions of the partner proteins that exhibit these preferred folds are sufficient for Ubx binding. Three disorder-containing regions in Ubx are required to bind these partners. These regions are either alternatively spliced or multiply phosphorylated, providing a mechanism for cellular processes to regulate Ubx-partner interactions. Indeed, partner topology correlates with the ability of individual partner proteins to bind Ubx spliceoforms. Partners bind different disordered regions within Ubx to varying extents, creating the potential for competition between partners and cooperative binding by partners. The ability of partners to bind regions of Ubx that activate transcription and regulate DNA binding provides a mechanism for partners to modulate transcription regulation by Ubx, and suggests that one role of disorder in Ubx is to coordinate multiple molecular functions in response to tissue-specific cues.
PMCID: PMC4186791  PMID: 25286318
5.  Media composition influences yeast one- and two-hybrid results 
Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.
PMCID: PMC3177868  PMID: 21843345
Yeast one-hybrid; yeast two-hybrid; protein-protein interaction; accuracy; false positive; false negative

Results 1-5 (5)