Susumu Hirose

Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. Here, we show that Drosophila Mbf1 associates with E(z) mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. Our results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing.

Hedgehog (Hh) signaling pathway and Cyclin E are key players in cell proliferation and organ development. Hyperactivation of hh and cyclin E has been linked to several types of cancer. However, coordination of the expression of hh and cyclin E was not well understood. Here we show that an evolutionarily conserved transcription factor Apontic (Apt) directly activates hh and cyclin E through its binding site in the promoter regions of hh and cyclin E. This Apt-dependent proper expression of hh and cyclin E is required for cell proliferation and development of the Drosophila wing. Furthermore, Fibrinogen silencer-binding protein (FSBP), a mammalian homolog of Apt, also positively regulates Sonic hh (Shh), Desert hh (Dhh), Cyclin E1 (CCNE1) and Cyclin E2 (CCNE2) in cultured human cells, suggesting evolutionary conservation of the mechanism. Apt-mediated expression of hh and cyclin E can direct proliferation of Hh-expressing cells and simultaneous growth, patterning and differentiation of Hh-recipient cells. The discovery of the simultaneous expression of Hh and principal cell-cycle regulator Cyclin E by Apt implicates insight into the mechanism by which deregulated hh and cyclin E promotes tumor formation.

Hedgehog (Hh) signaling pathway and Cyclin E are key players in cell proliferation and development. Hyperactivation of hh and cyclin E. has been linked to several types of cancer. However, transcriptional regulation of hh and cyclin E. are not well understood. Here we show that an evolutionarily conserved transcription factor Apontic (Apt) is an activator of hh and cyclin E. in Drosophila . Apt directly promotes the expression of hh and cyclin E. through its binding site in the promoter regions of hh and cyclin E. during wing development. This Apt-dependent proper expression of hh and cyclin E. is required for cell proliferation and development of the wing. Apt-mediated expression of hh and cyclin E. can direct proliferation of Hh-expressing cells and simultaneous growth, patterning and differentiation of Hh-recipient cells. The discovery of the coordinated expression of Hh and principal cell-cycle regulator Cyclin E. by Apt implicates insight into the mechanism by which deregulated hh and cyclin E promotes tumor formation. Summary statement: We identified a novel role for Apontic as an important common regulator of the transcription of hedgehog and cyclin E. Our study provides important insights into the mechanism of organ development.

Establishment and maintenance of epigenetic memories are essential for development. Replacement of canonical histone H3 by its variant H3.3 has been implicated in cellular memory. Drosophila sequence-specific DNA-binding protein GAGA factor and a chromatin factor FACT direct H3.3 replacement in conjunction with H3.3-specific chaperone HIRA at chromatin boundaries to counteract the spreading of silent chromatin. However, little is known about which ATP-driven chromatin remodeling factor is responsible for the H3.3 replacement at chromatin boundaries. Here, we report that GAGA factor associates with the Polybromo-associated Brm (PBAP) remodeling complex, which consists of many Trithorax group proteins, and recruits this complex to chromatin boundaries d1 (which is downstream of w), the Fab-7 DNase-hypersensitive site (HS) 1 of Abd-B and the bxd region of Ubx. Trl-encoding GAGA factor, brm and polybromo/bap180 mutations compromise the H3.3 replacement and boundary functions in a synergistic manner. Furthermore, Polybromo is necessary for generation of the DNase HS at d1, and HIRA functions to restore the alteration. Taken together, we propose that FACT and PBAP complexes are recruited to chromatin boundaries in a GAGA factor-dependent manner, and are needed for H3.3 replacement to execute boundary functions. Our results provide new insight into the function of the trithorax group during development.

Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. Here, we show that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. We propose that Midline controls axon pathfinding through coordinating the two guidance systems.

Human remodeling and spacing factor (RSF) consists of a heterodimer of Rsf-1 and hSNF2H, a counterpart of Drosophila ISWI. RSF possesses not only chromatin remodeling activity but also chromatin assembly activity in vitro. While no other single factor can execute the same activities as RSF, the biological significance of RSF remained unknown. To investigate the in vivo function of RSF, we generated a mutant allele of Drosophila Rsf-1 (dRsf-1). The dRsf-1 mutant behaved as a dominant suppressor of position effect variegation. In dRsf-1 mutant, the levels of histone H3K9 dimethylation and histone H2A variant H2Av were significantly reduced in an euchromatic region juxtaposed with heterochromatin. Furthermore, using both genetic and biochemical approaches, we demonstrate that dRsf-1 interacts with H2Av and the H2Av-exchanging machinery Tip60 complex. These results suggest that RSF contributes to histone H2Av replacement in the pathway of silent chromatin formation. As DNA is packaged into chromatin in the nucleus, every DNA transaction requires alteration of the chromatin structure. RSF, a heterodimer of Rsf-1 and ISWI/SNF2H, is a unique chromatin remodeling factor that can assemble regularly spaced nucleosome arrays without the aid of histone chaperons, but its biological function is not clear. Using Drosophila melanogaster as a model organism, we investigated the in vivo role of RSF in gene expression. The loss of RSF function reduces the levels of histone variant H2Av and histone H3-K9 methylation, and suppresses silencing of transcription in an euchromatic region neighboring the centromeric heterochromatin. We also observed that Rsf-1 interacts with histone H2Av and the H2Av-exchanging machinery Tip60 complex. Based on these findings, we propose that RSF plays a role in silent chromatin formation by promoting histone H2Av replacement.

DNA supercoiling factor (SCF) generates unconstrained negative supercoils of DNA in conjunction with eukaryotic topoisomerase II. In Drosophila melanogaster, SCF localizes to puffs on polytene chromosomes and is required for dosage compensation via hypertranscripton of genes on the male X chromosome. The present study investigated the role of SCF on autosomes. Although RNAi knockdown of scf results in male lethality, some escapers showed anterior homeotic transformation of the male sixth abdominal segment, similar to that arising from reduced expression of Abdominal-B (Abd-B). Heterozygotes for an scf mutant allele (scf(1)) displayed suppression of Pc mutation-dependent posterior transformation and enhancement of anterior transformation caused by trxG mutations. The level of Abd-B mRNA decreased in scf(1) embryos compared with wild-type. Tiling array experiments showed the presence of significant SCF signals in an Abd-B promoter region. Expression from the basal Abd-B promoter on a transgene was reduced in scf(1) embryos compared with wild-type. Collectively, these results demonstrate that SCF occupies the promoter region of Abd-B and activates expression for the proper formation of abdominal segments. Furthermore, preferential occupancy of SCF around transcription start sites of many active genes suggests a role for the factor in positive regulation of promoters.

To elucidate the transcriptional regulation in eukaryotic genome network, it is important to understand coevolution of transcription factors, transcriptional coactivators, and TATA-box–binding protein (TBP). In this study, coevolution of transcriptional coactivator multiprotein-bridging factor 1 and its interacting target TBP was first evaluated experimentally by examining if compensatory amino acid changes took place at interacting sites of both proteins. The experiments were conducted by identifying interaction sites and comparing the amino acids at these sites among different organisms. Here, we provide evidence for compensatory changes of transcription coactivator and its interacting target, presenting the 1st report that transcription coactivator may have undergone coevolution with TBP.