Akirin Links Twist-Regulated Transcription with the Brahma Chromatin Remodeling Complex during Embryogenesis

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein–protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery

The PDZ Protein Canoe/AF-6 Links Ras-MAPK, Notch and Wingless/Wnt Signaling Pathways by Directly Interacting with Ras, Notch and Dishevelled

Over the past few years, it has become increasingly apparent that signal transduction pathways are not merely linear cascades; they are organized into complex signaling networks that require high levels of regulation to generate precise and unique cell responses. However, the underlying regulatory mechanisms by which signaling pathways cross-communicate remain poorly understood. Here we show that the Ras-binding protein Canoe (Cno)/AF-6, a PDZ protein normally associated with cellular junctions, is a key modulator of Wingless (Wg)/Wnt, Ras-Mitogen Activated Protein Kinase (MAPK) and Notch (N) signaling pathways cross-communication. Our data show a repressive effect of Cno/AF-6 on these three signaling pathways through physical interactions with Ras, N and the cytoplasmic protein Dishevelled (Dsh), a key Wg effector. We propose a model in which Cno, through those interactions, actively coordinates, at the membrane level, Ras-MAPK, N and Wg signaling pathways during progenitor specification

A Mutational Analysis of the Period Locus of Drosophila Melanogaster

The period (per) gene of Drosophila melanogaster is fundamentally involved in the generation of biological rhythms. Three classes of per mutations which alter circadian periodicity have been identified: pers mutants have circadian behavioral rhythms of 19h instead of 24h; per mutants have long period rhythms of 28h; and per0 mutants have no detectable circadian rhythms. Steps have been taken to gather more information about per\u27s role in the construction or maintenance of biological clocks. By analyzing transformed Drosophila lines, the amount of per product was found to be integral to the pace of the clock. Absence of. the per product leads to arrhythmicity; more per product shortens the period length; less per product lengthens period. In addition, single amino acid changes in the per product can mimic these results. DNA sequence analysis has revealed that in per0 flies, a single nucleotide change resulted in a translational stop codon and hence a truncated protein. A valine-to-aspartic acid change in the per1 mutants lengthens period. Likewise the shortened period length in pers mutants is a result of a serine-to-asparagine substitution. These combined studies suggest that per1 and pers mutants produce hypoactive and hyperactive per proteins, respectively. Using the sequence analysis of the mutants as a starting point, further amino acid changes in per were created, introduced back into the fly, and then evaluated for effects on biological rhythms. Five out of six amino acid changes near the pers mutation also gave short period lengths. These results suggest that the region near the permutation acts as a domain to restrain per function and thereby slows the clock. Further insight into the nature of per function was obtained through a cell level assay. The per mutations have a significant effect on intercellular communication in the salivary gland cells of third instar larvae. Dye transfer and electrophysiological experiments indicate that gap junction conductances varies inversely with the period of the behavioral rhythms. Such alterations in communication in the nervous system may explain how per influences biological rhythms. Lastly, a detailed localization study of the per gene products during embryogenesis shows that it is expressed in particular cells in the brain and ventral nerve cord. This information should make it possible to localize the focus of per\u27s clock function to specific cells

Akirin: A Context-Dependent Link Between Transcription and Chromatin Remodeling

Embryonic patterning relies upon an exquisitely timed program of gene regulation. While the regulation of this process via the action of transcription factor networks is well understood, new lines of study have highlighted the importance of a concurrently regulated program of chromatin remodeling during development. Chromatin remodeling refers to the manipulation of the chromatin architecture through rearrangement, repositioning, or restructuring of nucleosomes to either favor or hinder the expression of associated genes. While the role of chromatin remodeling pathways during tumor development and cancer progression are beginning to be clarified, the roles of these pathways in the course of tissue specification, morphogenesis and patterning remains relatively unknown. Further, relatively little is understood as to the mechanism whereby developmentally critical transcription factors coordinate with chromatin remodeling factors to optimize target gene loci for gene expression. Such a mechanism might involve direct transcription factor/chromatin remodeling factor interactions, or could likely be mediated via an unknown intermediary. Our group has identified the relatively unknown protein Akirin as a putative member of this latter group: a secondary cofactor that serves as an interface between a developmentally critical transcription factor and the chromatin remodeling machinery. This role for the Akirin protein suggests a novel regulatory mode for regulating gene expression during development

Whole-Genome Analysis of Muscle Founder Cells Implicates the Chromatin Regulator Sin3A in Muscle Identity

Skeletal muscles are formed in numerous shapes and sizes, and this diversity impacts function and disease susceptibility. To understand how muscle diversity is generated, we performed gene expression profiling of two muscle subsets from Drosophila embryos. By comparing the transcriptional profiles of these subsets, we identified a core group of founder cell-enriched genes. We screened mutants for muscle defects and identified functions for Sin3A and 10 other transcription and chromatin regulators in the Drosophila embryonic somatic musculature. Sin3A is required for the morphogenesis of a muscle subset, and Sin3A mutants display muscle loss and misattachment. Additionally, misexpression of identity gene transcription factors in Sin3A heterozygous embryos leads to direct transformations of one muscle into another, whereas overexpression of Sin3A results in the reverse transformation. Our data implicate Sin3A as a key buffer controlling muscle responsiveness to transcription factors in the formation of muscle identity, thereby generating tissue diversity

The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation

<div><p>The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for <i>Drosophila</i> myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.</p></div

Dia localization at the fusion site is dependent on FC/FCM recognition and adhesion, but independent of regulators of Arp2/3.

<p>Stage 15 embryos stained with phalloidin (<b>i.</b>), antibodies against Dia (<b>ii.</b>), and Myosin Heavy Chain (<b>iii.</b>, MHC). Phalloidin labels F-actin (focus and sheath) at the fusion site; MHC identifies myoblasts. FCM (magenta) and FC/Myotube (turquoise). <b>A-iv.</b> Dia localization in FCM and FC/myotube in a wild-type embryo during myoblast fusion. Dia accumulates at the fusion site. The averaged fluorescence intensity curve (Aiv, n = 5) in wild-type embryos confirms Dia colocalization with actin. <b>B-iv.</b> In <i>sns</i> mutants, no F-actin focus is formed and no specific accumulation of actin or Dia are observed. Average fluorescence intensity curve of <i>sns</i> mutant embryos (Biv, n = 5) supports that Dia does not accumulate at the fusion site and is cytoplasmic. <b>C-E-iv.</b> In <i>rac</i>, <i>mbc</i>, and <i>kette</i> mutants, SCAR activity is lost, an enlarged focus is observed at the fusion site, and Dia is enriched at the fusion site. Fluorescence intensity curves confirm Dia and actin colocalization in <i>rac</i> (Civ), <i>mbc</i> (Div), and <i>kette</i> (Eiv) mutants (n = 5/genotype). <b>F-iv.</b> In <i>loner</i> mutant embryos, Dia accumulates at the F-actin focus, as confirmed by the fluorescence intensity curves (n = 5). <b>G-I-iv.</b> In <i>blow</i>, <i>sltr(Dwip)</i> and <i>wsp</i> mutants, where WASp-mediated actin remodeling is lost, Dia accumulation at the fusion site is unaffected. Fluorescence intensity curves confirm the colocalization of Dia and F-actin in <i>blow</i> (Giv), <i>sltr(Dwip)</i> (Hiv), <i>and wsp</i> (Iiv) mutants (n = 5/genotype). Scale bar: 2.5μM.</p

Constitutively active Dia alters the F-actin structure at the fusion site and regulates localization of Arp2/3 regulators.

<p><b>A-B.</b> Stage 14 embryos stained for F-actin (phalloidin, white) and for Dia::GFP or DiaΔDAD::GFP (GFP antibody, white); FCM (magenta), FC/Myotube (turquoise). Scale bar: 5μM. <b>A.</b> Control <i>DMef2-Gal4>UAS-dia</i>::<i>GFP</i> myoblasts show colocalization of Dia::GFP and the F-actin focus at the fusion site. <b>B.</b><i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> myoblasts show that F-actin does not form a well-defined focus at the fusion site but appears diffuse. DiaΔDAD::GFP localizes to the plasma membrane and is enriched at cell contact sites. <b>C.</b> Fluorescent intensity curves confirm the distribution of F-actin in embryos expressing Dia::GFP and DiaΔDAD::GFP. <b>D-E.</b> Stage 15 embryos stained for F-actin (phalloidin) and DiaΔDAD::HA (antibodies against HA) showing FCM (magenta) and FC/Myotube (turquoise). Scale bar: 5μM. <b>D.</b><i>DMef2-Gal4</i> driven expression of DiaΔDAD::HA in <i>kette</i>^<i>J4-48</i> mutant background. The morphology of the F-actin focus at the fusion site appears similar to <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> embryos. While F-actin localizes at the cell cortex, it spreads out at the fusion site and does not make a concentrated focus. <b>E.</b><i>DMef2-Gal4</i> driven expression of DiaΔDAD::GFP in <i>sltr</i>^<i>s1946</i> mutant background. Similar to expression of DiaCA alone, the F-actin localizes at the cell cortex and spreads out at the fusion site. <b>F-G.</b> Stage 14 embryos stained for F-actin (phalloidin), Dia<i>ΔDAD</i>::GFP (GFP antibody) and SCAR or WASp. Scale bar: 5μM. <b>F.</b> SCAR localization in control and <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> embryos. In control embryos, SCAR accumulates at the fusion site, as confirmed by the fluorescent intensity curves. When expressing DiaΔDAD::GFP, SCAR loses its characteristic concentration at the fusion site and becomes found throughout the cytoplasm. The multiple peaks in the SCAR fluorescent intensity curve confirm SCAR’s change in localization. <b>G.</b> Localization of WASp in control and <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> embryo. In control embryos, WASp accumulates at the fusion site, as confirmed by the fluorescent intensity curves. When expressing DiaΔDAD::GFP, WASp displays a more diffused localization. Fluorescent intensity curves confirm the broader distribution of WASp signal in relation to the controls.</p