Philippe Ramain

Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate

BACKGROUND The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. RESULTS Here, we describe new alleles of Notch that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. We show that the loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. CONCLUSIONS Our results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices.

Interactions between chip and the achaete/scute-daughterless heterodimers are required for pannier-driven proneural patterning.

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip was previously identified as a cofactor of the dorsal selector Apterous, and we show here that both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax.

[16] Nuclease digestion of transcriptionally active chromatin

Publisher Summary This chapter discusses the various nuclease digestion of transcriptionally active chromatin. Active chromatin is less compact than bulk chromatin, and this is reflected in its increased accessibility to enzymes. This can be seen at several levels. First, it compare the structure surrounding a specific gene in terms of its nuclease sensitivity in a cell type where there can be no expression to a cell type where there has been, is, or will be expression of that gene. In general, the structure will be more sensitive in the latter case, even when gene expression is not evident. Second, further changes are associated with gene activity and expression during cell development that are due to modifications in chromatin structure of flanking sequences and increased sensitivity in coding sequences, probably reflecting changes in nucleosomal structure during the passage of polymerase. A number of different situations have been described when filters are probed with DNA from regions of active chromatin. In general, there is an important increase in the general sensitivity to micrococcal nuclease. In certain cases, the regular ladder is disrupted and bands of novel size are detected. In other cases, a repeat structure is found but displaced when compared with that of the bulk chromatin.

Changes in the chromatin structure of Drosophila glue genes accompany developmental cessation of transcription in wild type and transformed strains

Three Drosophila salivary gland glue genes show a dramatic transition in their DNAse I hypersensitive sites during the short period between the late third instar and the white prepupa, which correlates with the cessation of their transcription. In culture cells, where the genes are inactive, there is a chromatin configuration similar to that of prepupal salivary glands. In two transformed fly strains where the sgs3 gene is active at new chromosomal sites, including one in which 2.6 kb of sgs3 upstream sequences have been inverted, the same DNAase I hypersensitive sites and developmental transitions are seen over the same DNA regions. These results, together with the analysis of transformants carrying rearranged sgs3 genes, suggest that there is at least one distal DNAase I hypersensitive site associated with an element of regulation which may be exchanged between sgs genes.

Toutatis, a TIP5-related protein, positively regulates Pannier function during Drosophila neural development

The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of the achaete-scute (ac-sc) complex. Chip associates both with Pnr and with the (Ac-Sc)-Daughterless heterodimer bound to the ac-sc promoters to give a proneural complex that facilitates enhancer-promoter communication during development. Using a yeast two-hybrid screening, we have identified Toutatis (Tou), which physically interacts with both Pnr and Chip. Loss-of-function and gain-of-function experiments indicate that Tou cooperates with Pnr and Chip during neural development. Tou shares functional domains with chromatin remodelling proteins, including TIP5 (termination factor TTFI-interacting protein 5) of NoRC (nucleolar remodelling complex), which mediates repression of RNA polymerase 1 transcription. In contrast, Tou acts positively to activate proneural gene expression. Moreover, we show that Iswi associates with Tou, Pnr and Chip, and is also required during Pnr-driven neural development. The results suggest that Tou and Iswi may belong to a complex that directly regulates the activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling.

pannier encodes two structurally related isoforms that are differentially expressed during Drosophila development and display distinct functions during thorax patterning

Previous studies have shown that the pannier (pnr) gene of Drosophila encodes a GATA transcription factor which is involved in various biological processes, including heart development, dorsal closure during embryogenesis as well as neurogenesis and regulation of wingless (wg) expression during imaginal development. We demonstrate here that pnr encodes two highly related isoforms that share functional domains but are differentially expressed during development. Moreover, we describe two genomic regions of the pnr locus that drive expression of a reporter in transgenic flies in patterns that recapitulate essential features of the expression of the isoforms, suggesting that these regions encompass crucial regulatory elements. These elements contain, in particular, sequences mediating regulation of expression by Decapentaplegic (Dpp) signaling, during both embryogenesis and imaginal development. Analysis of pnr alleles reveals that the isoforms differentially regulate expression of both wg and proneural achaete/scute (as/sc) targets during imaginal development. Pnr function has been demonstrated to be necessary both for activation of wg and, together with U-shaped (Ush), for its repression in the dorsal-most region of the presumptive notum. Expression of the isoforms define distinct longitudinal domains and, in this regard, we importantly show that the dual function of pnr during regulation of wg is achieved by one isoform repressing expression of the morphogen in the dorsal-most region of the disc while the other laterally promotes activation of the notal wg expression. Our study provides novel insights into pnr function during Drosophila development and extends our knowledge of the roles of prepattern factors during thorax patterning.

Transcriptional interactions between the pannier isoforms and the cofactor U-shaped during neural development in Drosophila.

The pannier (pnr) gene of Drosophila melanogaster encodes two isoforms that belong to the family of GATA transcription factors. The isoforms share an expression domain in the wing discs where they exhibit distinct functions during regulation of the proneural achaete/scute (ac/sc) genes. We previously identified two regions in the pnr locus that drive reporter expression in transgenic lines in patterns that recapitulate the essential features of expression of the two isoforms. Here, we identify promoter regions driving isoform expression, showing that pnr-α regulatory sequences are close to the transcription start site while pnr-β expression requires functional interactions between proximal and distal regulatory elements. We find that the promoter domains necessary for reporter expression also mediate autoregulation of Pnr-β and repression of pnr-α by Pnr-β. The cofactor U-shaped (Ush), which is known to down-regulate the function of Pnr during thorax patterning postranscriptionally, in addition represses pnr-β required for ac/sc activation. Moreover, Ush negatively regulates its own expression, while the pnr isoforms positively regulate ush. Our study uncovers complex transcriptional interactions between the pnr isoforms and the cofactor Ush that may be important for regulation of proneural expression and thorax patterning.

The Drosophila DAXX-Like Protein (DLP) Cooperates with ASF1 for H3.3 Deposition and Heterochromatin Formation.

ABSTRACT Histone variants are nonallelic isoforms of canonical histones, and they are deposited, in contrast to canonical histones, in a replication-independent (RI) manner. RI deposition of H3.3, a histone variant from the H3.3 family, is mediated in mammals by distinct pathways involving either the histone regulator A (HIRA) complex or the death-associated protein (DAXX)/α-thalassemia X-linked mental retardation protein (ATRX) complex. Here, we investigated the function of the Drosophila DAXX-like protein (DLP) by using both fly genetic approaches and protein biochemistry. DLP specifically interacts with H3.3 and shows a prominent localization on the base of the X chromosome, where it appears to act in concert with XNP, the Drosophila homolog of ATRX, in heterochromatin assembly and maintenance. The functional association between DLP and XNP is further supported by a series of experiments that illustrate genetic interactions and the DLP-XNP-dependent localization of specific chromosomal proteins. In addition, DLP both participates in the RI deposition of H3.3 and associates with anti-silencing factor 1 (ASF1). We suggest, in agreement with a recently proposed model, that DLP and ASF1 are part of a predeposition complex, which is recruited by XNP and is necessary to prevent DNA exposure in the nucleus.