Cell cycle expression of polarity genes features Rb targeting of Vang.

Specification of cellular polarity is vital to normal tissue development and function. Pioneering studies in Drosophila and C. elegans have elucidated the composition and dynamics of protein complexes critical for establishment of cell polarity, which is manifest in processes such as cell migration and asymmetric cell division. Conserved throughout metazoans, planar cell polarity (PCP) genes are implicated in disease, including neural tube closure defects associated with mutations in VANGL1/2. PCP protein regulation is well studied; however, relatively little is known about transcriptional regulation of these genes. Our earlier study revealed an unexpected role for the fly Rbf1 retinoblastoma corepressor protein, a regulator of cell cycle genes, in transcriptional regulation of polarity genes. Here we analyze the physiological relevance of the role of E2F/Rbf proteins in the transcription of the key core polarity gene Vang. Targeted mutations to the E2F site within the Vang promoter disrupts binding of E2F/Rbf proteins in vivo, leading to polarity defects in wing hairs. E2F regulation of Vang is supported by the requirement for this motif in a reporter gene. Interestingly, the promoter is repressed by overexpression of E2F1, a transcription factor generally identified as an activator. Consistent with the regulation of this polarity gene by E2F and Rbf factors, expression of Vang and other polarity genes is found to peak in G2/M phase in cells of the embryo and wing imaginal disc, suggesting that cell cycle signals may play a role in regulation of these genes. These findings suggest that the E2F/Rbf complex mechanistically links cell proliferation and polarity.

Transcriptional Regulation of INSR, the Insulin Receptor Gene.

The insulin receptor gene encodes an evolutionarily conserved signaling protein with a wide spectrum of functions in metazoan development. The insulin signaling pathway plays key roles in processes such as metabolic regulation, growth control, and neuronal function. Misregulation of the pathway features in diabetes, cancer, and neurodegenerative diseases, making it an important target for clinical interventions. While much attention has been focused on differential pathway activation through ligand availability, sensitization of overall signaling may also be mediated by differential expression of the insulin receptor itself. Although first characterized as a "housekeeping" gene with stable expression, comparative studies have shown that expression levels of the human INSR mRNA differ by tissue and in response to environmental signals. Our recent analysis of the transcriptional controls affecting expression of the Drosophila insulin receptor gene indicates that a remarkable amount of DNA is dedicated to encoding sophisticated feedback and feed forward signals. The human INSR gene is likely to contain a similar level of transcriptional complexity; here, we summarize over three decades of molecular biology and genetic research that points to a still incompletely understood regulatory control system. Further elucidation of transcriptional controls of INSR will provide the basis for understanding human genetic variation that underlies population-level physiological differences and disease.

Regulation of cell polarity determinants by the Retinoblastoma tumor suppressor protein.

In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical-basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis.

Role of Drosophila retinoblastoma protein instability element in cell growth and proliferation.

The RB tumor suppressor, a regulator of the cell cycle, apoptosis, senescence, and differentiation, is frequently mutated in human cancers. We recently described an evolutionarily conserved C-terminal "instability element" (IE) of the Drosophila Rbf1 retinoblastoma protein that regulates its turnover. Misexpression of wild-type or non-phosphorylatable forms of the Rbf1 protein leads to repression of cell cycle genes. In contrast, overexpression of a defective form of Rbf1 lacking the IE (ΔIE), a stabilized but transcriptionally less active form of the protein, induced ectopic S phase in cell culture. To determine how mutations in the Rbf1 IE may induce dominant effects in a developmental context, we assessed the impact of in vivo expression of mutant Rbf1 proteins on wing development. ΔIE expression resulted in overgrowth of larval wing imaginal discs and larger adult wings containing larger cells. In contrast, a point mutation in a conserved lysine of the IE (K774A) generated severely disrupted, reduced wings. These contrasting effects appear to correlate with control of apoptosis; expression of the pro-apoptotic reaper gene and DNA fragmentation measured by acridine orange stain increased in flies expressing the K774A isoform and was suppressed by expression of Rbf1ΔIE. Intriguingly, cancer associated mutations affecting RB homologs p130 and p107 may similarly induce dominant phenotypes.

Transcriptional repression: conserved and evolved features.

The regulation of gene expression by transcriptional repression is an ancient and conserved mechanism that manifests itself in diverse ways. Here we summarize conserved pathways for transcriptional repression prevalent throughout all forms of life, as well as indirect mechanisms that appear to have originated in eukaryotes, consistent with the unique chromatin environment of eukaryotic genes. The direct interactions between transcriptional repressors and the core transcriptional machinery in bacteria and archaea are sufficient to generate a sophisticated suite of mechanisms that provide flexible control. These direct interactions contrast with the activity of corepressors, which provide an additional regulatory control in eukaryotes. Their modulation of chromatin structure represents an indirect pathway to downregulate transcription, and their diversity and modulation provide additional complexity suited to the requirements of elaborate eukaryotic repression patterns. New findings indicate that corepressors are not necessarily restricted to generating a single stereotypic output, but can rather exhibit diverse functional responses depending on the context in which they are recruited, providing a hitherto unsuspected additional source of diversity in transcriptional control. Mechanisms within eukaryotes appear to be highly conserved, with novel aspects chiefly represented by addition of lineage-specific corepressor scaffolds that provide additional opportunities for recruiting the same core machinery.

Groucho corepressor functions as a cofactor for the Knirps short-range transcriptional repressor.

Despite the pervasive roles for repressors in transcriptional control, the range of action of these proteins on cis regulatory elements remains poorly understood. Knirps has essential roles in patterning the Drosophila embryo by means of short-range repression, an activity that is essential for proper regulation of complex transcriptional control elements. Short-range repressors function in a local fashion to interfere with the activity of activators or basal promoters within approximately 100 bp. In contrast, long-range repressors such as Hairy act over distances >1 kb. The functional distinction between these two classes of repressors has been suggested to stem from the differential recruitment of the CtBP corepressor to short-range repressors and Groucho to long-range repressors. Contrary to this differential recruitment model, we report that Groucho is a functional part of the Knirps short-range repression complex. The corepressor interaction is mediated via an eh-1 like motif present in the N terminus and a conserved region present in the central portion of Knirps. We also show that this interaction is important for the CtBP-independent repression activity of Knirps and is required for regulation of even-skipped. Our study uncovers a previously uncharacterized interaction between proteins previously thought to function in distinct repression pathways, and indicates that the Groucho corepressor can be differentially harnessed to execute short- and long-range repression.

Gene regulation: boundaries within limits.

Quantitative measurements of the Hunchback transcription factor in Drosophila embryos show that its target genes can respond with a high degree of precision to the exact level of the protein, simulating a continuous, analog readout, while other target genes show a combinatorial effect, resembling a Boolean logic element.

An ELISA-based method for measurement of food-specific IgE antibody in mouse serum: an alternative to the passive cutaneous anaphylaxis assay.

Passive cutaneous anaphylaxis (PCA) assay has been a gold standard method to measure allergen-specific IgE antibody (ASIgE Ab) levels in allergy mouse models. Many factors including stringent guidelines for laboratory animal use make PCA a difficult choice. Therefore, alternative methods are needed that can be readily applied for measurement of specific IgE antibody levels in mouse serum. Herein we describe a novel ELISA-based method that is more sensitive in comparison to PCA, IgE isotype-specific (because it has little cross-reactivity with IgG1 or IgG2a isotype) and highly reproducible (<10% inter- or intra-assay variation). Furthermore, we demonstrate the utility of this assay to measure specific IgE Ab against a variety of food extracts including chicken egg, peanut, almond, filbert/hazelnut and sweet potato. These findings are of particular interest to those who are seeking (i) to measure food-extract-specific IgE antibody in animal models and (ii) an alternative to the animal-based PCA method to measure mouse IgE antibodies.