Susumu Hirose

Transcription of bxd Noncoding RNAs Promoted by Trithorax Represses Ubx in cis by Transcriptional Interference

Much of the genome is transcribed into long noncoding RNAs (ncRNAs). Previous data suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex (BX-C) prevent silencing of Ultrabithorax (Ubx) and recruit activating proteins of the trithorax group (trxG) to their maintenance elements (MEs). We found that, surprisingly, Ubx and several bxd ncRNAs are expressed in nonoverlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. Our data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. We propose that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription.

Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication

Chromatin is suppressive in nature to cellular enzymes that metabolize DNA, mainly due to the inherent inaccessibility of the DNA template. Despite extensive understanding of the involvement of chromatin‐modifying factors in transcription, roles of related activities in DNA replication remain largely elusive. Here, we show that the heterodimeric transcriptional elongation factor FACT (facilitates chromatin transcription) is functionally linked to DNA synthesis. Its involvement in DNA replication is partly mediated by the stable association with the replicative helicase complex, MCM, and further by the coexistence with MCM on replication origin. Furthermore, relying on its nucleosome‐reorganizing activity, FACT can facilitate chromatin unwinding by the MCM complex, which is otherwise inert on the nucleosomal template. As a consequence, the physical and functional interaction between FACT and MCM is an important determinant in the proper initiation of DNA replication and S phase in vivo. Together, our findings identify FACT as an integral and conserved component of the endogenous replication machinery, and support a model in which the concerted action of helicase and chromatin‐modifying activities promotes chromosome replication.

Control of imaginal cell development by the escargot gene of Drosophila.

Mutations in the escargot (esg) locus, which codes for a zinc-finger-containing protein with similarity to the product of the snail gene, cause a variety of defects in adult structures such as loss of abdominal cuticle and malformation of the wings and legs. esg RNA is expressed in wing, haltere, leg and genital imaginal discs and in abdominal histoblast nests in the embryo. Expression in imaginal tissues is also found in third instar larvae. In esg mutant larvae, normally diploid abdominal histoblasts replicate their DNA without cell division and become similar in appearance to the polytene larval epidermal cells. A similar phenotype was also found in imaginal discs of larvae mutant for both esg and the Drosophila raf gene. These results suggest that one of the normal functions of esg may be the maintenance of diploidy in imaginal cells.

Yeast Coactivator MBF1 Mediates GCN4-Dependent Transcriptional Activation

ABSTRACT Transcriptional coactivators play a crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery. The coactivator multiprotein bridging factor 1 (MBF1) was originally identified as a bridging molecule that connects theDrosophila nuclear receptor FTZ-F1 and TATA-binding protein (TBP). The MBF1 sequence is highly conserved across species fromSaccharomyces cerevisiae to human. Here we provide evidence acquired in vitro and in vivo that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4 and TBP. These findings indicate that the coactivator MBF1 functions by recruiting TBP to promoters where DNA-binding regulators are bound.

The conserved nuclear receptor Ftz-F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans

Nuclear receptors are essential players in the development of all metazoans. The nematode Caenorhabditis elegans possesses more than 200 putative nuclear receptor genes, several times more than the number known in any other organism. Very few of these transcription factors are conserved with components of the steroid response pathways in vertebrates and arthropods. Ftz‐F1, one of the evolutionarily oldest nuclear receptor types, is required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila.

The Role of Human MBF1 as a Transcriptional Coactivator

Multiprotein bridging factor 1 (MBF1) is a coactivator which mediates transcriptional activation by interconnecting the general transcription factor TATA element-binding protein and gene-specific activators such as the Drosophilanuclear receptor FTZ-F1 or the yeast basic leucine zipper protein GCN4. The human homolog of MBF1 (hMBF1) has been identified but its function, especially in transcription, remains unclear. Here we report the cDNA cloning and functional analysis of hMBF1. Two isoforms, which we term hMBF1α and hMBF1β, have been identified. hMBF1α mRNA was detected in a number of tissues, whereas hMBF1β exhibited tissue-specific expression. Both isoforms bound to TBP and Ad4BP/SF-1, a mammalian counterpart of FTZ-F1, and mediated Ad4BP/SF-1-dependent transcriptional activation. While hMBF1 was detected in the cytoplasm by immunostaining, coexpression of the nuclear protein Ad4BP/SF-1 with hMBF1 induced accumulation of hMBF1 in the nucleus, suggesting that hMBF1 is localized in the nucleus through its binding to Ad4BP/SF-1. hMBF1 also bound to ATF1, a member of the basic leucine zipper protein family, and mediated its activity as a transcriptional activator. These data establish that the coactivator MBF1 is functionally conserved in eukaryotes.

Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems.

During gene activation, the effect of binding of transcription factors to cis-acting DNA sequences is transmitted to RNA polymerase by means of co-activators. Although co-activators contribute to the efficiency of transcription, their developmental roles are poorly understood. We used Drosophila to conduct molecular and genetic dissection of an evolutionarily conserved but unique co-activator, Multiprotein Bridging Factor 1 (MBF1), in a multicellular organism. Through immunoprecipitation, MBF1 was found to form a ternary complex including MBF1, TATA-binding protein (TBP) and the bZIP protein Tracheae Defective (TDF)/Apontic. We have isolated a Drosophila mutant that lacks the mbf1 gene in which no stable association between TBP and TDF is detectable, and transcription of a TDF-dependent reporter gene is reduced by 80%. Although the null mutants of mbf1 are viable, tdf becomes haploinsufficient in mbf1-deficient background, causing severe lesions in tracheae and the central nervous system, similar to those resulting from a complete loss of tdf function. These data demonstrate a crucial role of MBF1 in the development of tracheae and central nervous system.

Regulation of the EDG84A gene by FTZ-F1 during metamorphosis in Drosophila melanogaster.

The transcription factor FTZ-F1 is a member of the nuclear hormone receptor superfamily and is transiently expressed during the mid- and late prepupal periods in Drosophila melanogaster. A putative pupal cuticle gene, EDG84A, is expressed slightly following FTZ-F1 expression during the prepupal period and carries a strong FTZ-F1 binding site between bases 100 and 92 upstream of its transcription start site. In this study, EDG84A mRNA was found to be prematurely expressed upon heat induction of FTZ-F1 in prepupae carrying the heat shock promoter-FTZ-F1 cDNA fusion gene construct. Transgenic fly lines having the 0.8-kb region of the EDG84A promoter fused to lacZ expressed the reporter gene in a tissue- and stage-specific manner. Base substitutions in the FTZ-F1 binding site within the 0.8-kb promoter abolished expression of lacZ. These results strongly suggest that the EDG84A gene is a direct target of FTZ-F1. Deletion studies of the cis-regulatory region of the EDG84A gene revealed that space-specific expression in imaginal disc-derived epidermis is controlled by the region between bp -408 and -104 from the transcription start site. The region between bp -408 and -194 is necessary to repress expression in a posterior part of the body, while the region between bp -193 and -104 carries a positive element for activation in an anterior part of the body. These results suggest that FTZ-F1 governs expression of the EDG84A gene in conjunction with putative tissue-specific regulators.

Chromatin Remodeling Mediated by Drosophila GAGA Factor and ISWI Activates fushi tarazu Gene Transcription In Vitro

ABSTRACT GAGA factor is known to remodel the chromatin structure in concert with nucleosome-remodeling factor NURF in a Drosophilaembryonic S150 extract. The promoter region of the Drosophila fushi tarazu (ftz) gene carries several binding sites for GAGA factor. Both the GAGA factor-binding sites and GAGA factor per se are necessary for the proper expression of ftz in vivo. We observed transcriptional activation of the ftz gene when a preassembled chromatin template was incubated with GAGA factor and the S150 extract. The chromatin structure within the ftzpromoter was specifically disrupted by incubation of the preassembled chromatin with GAGA factor and the S150 extract. Both transcriptional activation and chromatin disruption were blocked by an antiserum raised against ISWI or by base substitutions in the GAGA factor-binding sites in the ftz promoter region. These results demonstrate that GAGA factor- and ISWI-mediated disruption of the chromatin structure within the promoter region of ftz activates transcription on the chromatin template.

Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy

Intrinsically disordered (ID) regions of proteins are recognized to be involved in biological processes such as transcription, translation, and cellular signal transduction. Despite the important roles of ID regions, effective methods to observe these thin and flexible structures directly were not available. Herein, we use high-speed atomic force microscopy (AFM) to observe the heterodimeric FACT (facilitates chromatin transcription) protein, which is predicted to have large ID regions in each subunit. Successive AFM images of FACT on a mica surface, captured at rates of 5-17 frames per second, clearly reveal two distinct tail-like segments that protrude from the main body of FACT and fluctuate in position. Using deletion mutants of FACT, we identify these tail segments as the two major ID regions predicted from the amino acid sequences. Their mechanical properties estimated from the AFM images suggest that they have more relaxed structures than random coils. These observations demonstrate that this state-of-the-art microscopy method can be used to characterize unstructured protein segments that are difficult to visualize with other experimental techniques.