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

Repression by Notch is required before Wingless signalling during muscle progenitor cell development in Drosophila

AbstractThe larval muscles of Drosophila arise from the fusion of muscle founder cells, which give each individual muscle its identity, with myoblasts (reviewed in [1]). Muscle founder cells arise from the asymmetric division of muscle progenitor cells, each of which develops from a group of cells in the somatic mesoderm that express lethal of scute[2]. All the cells in a cluster can potentially form muscle progenitors, but owing to lateral inhibition, only one or two develop as such [2–5]. Muscle progenitors, and the subsequent founder cells, then express transcription factors such as Krüppel, S59 and Even-skipped, which confer identity on the muscle [6–8]. Definition of some muscle progenitors, including three groups that express S59, depends on Wingless signalling [9]. Lateral inhibition requires Delta signalling through Notch and the transcription factor Suppressor of Hairless [3–5]. As the Wingless and lateral-inhibition signals are sequential [8], one might expect that muscle progenitors would fail to develop in the absence of Wingless signalling, regardless of the presence or absence of lateral-inhibition signalling. Here, we examine the development of the S59-expressing muscle progenitor cells in mutant backgrounds in which both Wingless signalling and lateral inhibition are disrupted. We show that progenitor cells failed to develop when both these processes were disrupted. Our analysis also reveals a repressive function of Notch, required before or concurrently with Wingless signalling, which is unrelated to its role in lateral inhibition

Mechanical positioning of multiple nuclei in muscle cells.

Many types of large cells have multiple nuclei. In skeletal muscle fibers, the nuclei are distributed along the cell to maximize their internuclear distances. This myonuclear positioning is crucial for cell function. Although microtubules, microtubule associated proteins, and motors have been implicated, mechanisms responsible for myonuclear positioning remain unclear. We used a combination of rough interacting particle and detailed agent-based modeling to examine computationally the hypothesis that a force balance generated by microtubules positions the muscle nuclei. Rather than assuming the nature and identity of the forces, we simulated various types of forces between the pairs of nuclei and between the nuclei and cell boundary to position the myonuclei according to the laws of mechanics. We started with a large number of potential interacting particle models and computationally screened these models for their ability to fit biological data on nuclear positions in hundreds of Drosophila larval muscle cells. This reverse engineering approach resulted in a small number of feasible models, the one with the best fit suggests that the nuclei repel each other and the cell boundary with forces that decrease with distance. The model makes nontrivial predictions about the increased nuclear density near the cell poles, the zigzag patterns of the nuclear positions in wider cells, and about correlations between the cell width and elongated nuclear shapes, all of which we confirm by image analysis of the biological data. We support the predictions of the interacting particle model with simulations of an agent-based mechanical model. Taken together, our data suggest that microtubules growing from nuclear envelopes push on the neighboring nuclei and the cell boundaries, which is sufficient to establish the nearly-uniform nuclear spreading observed in muscle fibers

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