We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

Interferons play key roles in mediating antiviral and antigrowth responses and in modulating immune response. The main signaling pathways are rapid and direct. They involve tyrosine phosphorylation and activation of signal transducers and activators of transcription factors by Janus tyrosine kinases at the cell membrane, followed by release of signal transducers and activators of transcription and their migration to the nucleus, where they induce the expression of the many gene products that determine the responses. Ancillary pathways are also activated by the interferons, but their effects on cell physiology are less clear. The Janus kinases and signal transducers and activators of transcription, and many of the interferon-induced proteins, play important alternative roles in cells, raising interesting questions as to how the responses to the interferons intersect with more general aspects of cellular physiology and how the specificity of cytokine responses is maintained.

http://www3.uah.es/farmamol/Public/AnnReviews/PDF/Biochemistry/Interferons.pdf

How cells respond to interferons

STATs (signal transducers and activators of transcription) are a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular polypeptides. Biochemical and molecular genetic explorations have defined a single tyrosine phosphorylation site and, in a dimeric partner molecule, an Src homology 2 (SH2) phosphotyrosine-binding domain, a DNA interaction domain, and a number of protein-protein interaction domains (with receptors, other transcription factors, the transcription machinery, and perhaps a tyrosine phosphatase). Mouse genetics experiments have defined crucial roles for each known mammalian STAT. The discovery of a STAT in Drosophila , and most recently in Dictyostelium discoideum , implies an ancient evolutionary origin for this dual-function set of proteins.

http://science.sciencemag.org/content/sci/277/5332/1630.full.pdf

STATs and Gene Regulation

Interferon-gamma (IFN-gamma) coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. This article reviews the current understanding of IFN-gamma ligand, receptor, signal transduction, and cellular effects with a focus on macrophage responses and to a lesser extent, responses from other cell types that influence macrophage function during infection. The current model for IFN-gamma signal transduction is discussed, as well as signal regulation and factors conferring signal specificity. Cellular effects of IFN-gamma are described, including up-regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafficking. In addition, integration of signaling and response with other cytokines and pathogen-associated molecular patterns, such as tumor necrosis factor-alpha, interleukin-4, type I IFNs, and lipopolysaccharide are discussed.

https://jlb.onlinelibrary.wiley.com/doi/epdf/10.1189/jlb.0603252

Interferon-γ: an overview of signals, mechanisms and functions

A novel transcription factor, T-bet, directs Th1 lineage commitment.

STAT proteins (signal transducers and activators of transcription) activate distinct target genes despite having similar DNA binding preferences. The transcriptional specificity of STAT proteins was investigated on natural STAT binding sites near the interferon-gamma gene. These sites are arranged in multiple copies and required cooperative interactions for STAT binding. The conserved amino-terminal domain of STAT proteins was required for cooperative DNA binding, although this domain was not essential for dimerization or binding to a single site. Cooperative binding interactions enabled the STAT proteins to recognize variations of the consensus site. These sites can be specific for the different STAT proteins and may function to direct selective transcriptional activation.

https://science.sciencemag.org/content/sci/273/5276/794.full.pdf

Cooperative DNA Binding and Sequence-Selective Recognition Conferred by the STAT Amino-Terminal Domain

On the basis of homeo box cross-homology we have isolated the pair-rule gene even-skipped (eve) of Drosophila. The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb. The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes. Using antibodies raised against a beta-galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene. After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes. Both the original and new stripes gradually disappear during germ band elongation. A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia.

/pdf/characterization-and-localization-of-the-even-skipped-4fg9wlmhqy.pdf

Characterization and localization of the even-skipped protein of Drosophila.

A member of a small group of genes in Drosophila that define the segmentation pattern of the early embryo1–5 even-skipped (eve), which plays a key role in a network of interactions among segmentation genes. It appears to control morphogenesis by regulating the expression of the segmentation gene engrailed (en)3,4, and by autoregulating its own expression (M. Frasch and M.L., in preparation). Here we show that these regulatory interactions could occur at the level of transcription as a full-length eve protein binds with high affinity to specific sequences located near the 5' ends of the eve and en genes. The en binding sites contain at least one copy of a 10-base pair consensus sequence: T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve binding sites are relatively G-C rich and do not share obvious similarities with the 10-base pair consensus sequence associated with en. Other homeo box proteins can recognize both classes of eve binding sites, lending support to the proposal that regulatory interactions among homeo box genes involve a competition of different homeo box proteins for similar cis regulatory sequences6,7.

Divergent homeo box proteins recognize similar DNA sequences in Drosophila.

Homeobox proteins as sequence-specific transcription factors.

The pair-rule gene even-skipped (eve) plays a key role in the regulatory hierarchy governing segmentation in Drosophila. Here we describe the use of P-transformation and eve promoter fusions to identify cis elements that regulate the periodic seven-stripe eve pattern. A distal region of the eve promoter, located between -5.9 and -5.2 kb, controls autoregulation. Sequences from this region will induce striped expression of a heterologous hsp70 basal promoter in the presence, but not absence, of endogenous eve+ products. Autoregulatory activity was localized to a 200-bp region of the distal eve promoter. We also provide evidence that individual eve expression stripes are regulated by separate cis sequences. eve promoter sequences located between -4.7 and -3 kb upstream of the transcription start site are important for the initiation of stripe 3, whereas sequences between -1.7 and -0.4 kb are needed for stripes 2 and 7. It is possible that these latter regions are directly regulated by the products of gap genes.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1989.tb03493.x

Timothy Hoey

Papers

Cooperative DNA Binding and Sequence-Selective Recognition Conferred by the STAT Amino-Terminal Domain

Characterization and localization of the even-skipped protein of Drosophila.

Divergent homeo box proteins recognize similar DNA sequences in Drosophila.

Homeobox proteins as sequence-specific transcription factors.

Autoregulatory and gap gene response elements of the even-skipped promoter of Drosophila.