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

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.

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-ß 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-ß and repression of pnr-α by Pnr-ß. The cofactor U-shaped (Ush), which is known to down-regulate the function of Pnr during thorax patterning postranscriptionally, in addition represses pnr-ß 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.

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.

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.

Enhancer-promoter communication mediated by Chip during Pannier-driven proneural patterning is regulated by Osa.

The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. We show here that this communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes and we show that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. Our study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication.