The PBAP remodeling complex is required for histone H3.3 replacement at chromatin boundaries and for boundary functions.

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.

Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system.

Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G(i)alpha) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [35S]GTPgammaS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1.

Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to obtain sufficient amounts of these receptors for biochemical and structural studies because they often require posttranslational modifications, especially sugar modification. Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni(2+) affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2mg/larva. The sugar profiling revealed that the N-linked sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc and Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc. This revealed that the protein product was much more homogeneous than the complex-sugar type product obtained by mammalian cell expression. The surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary structure. These results clearly suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for their structural and binding studies, due to the relatively homogeneous N-linked sugar modifications.

Entropically driven MHC class I recognition by human inhibitory receptor leukocyte Ig-like receptor B1 (LILRB1/ILT2/CD85j).

The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHCI-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCI recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses showed that LILRB1-MHCI interactions are entropically driven (-TdeltaS = -9.4 approximately -6.6 kcal mol(-1)) with low heat capacity changes (deltaC(p) = -0.22 approximately -0.10 kcal mol(-1) K(-1)). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility. Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1-MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from "induced-fit" binding, which is associated with large reductions in conformational flexibility, and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells.

MDM2 acts downstream of p53 as an E3 ligase to promote FOXO ubiquitination and degradation.

Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasome-mediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.

Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading.

Epigenetic maintenance of the expression state of the genome is critical for development. Drosophila GAGA factor interacts with FACT and modulates chromatin structure for the maintenance of gene expression. Here we show that the GAGA factor-FACT complex and its binding site just downstream from the white gene are crucial for position effect variegation. Interestingly there is a dip of histone H3 Lys 9 methylation and a peak of H3 Lys 4 methylation at this site. The GAGA factor and FACT direct replacement of histone H3 by H3.3 through association of HIRA at this site, and maintain white expression under the heterochromatin environment. Based on these findings we propose that the GAGA factor and FACT-dependent replacement of Lys 9-methylated histone H3 by H3.3 counteracts the spreading of silent chromatin.

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system.

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.

Cloning CYP2D21 and CYP3A22 cDNAs from liver of miniature pigs.

To compare the identity of the primary structure of drug-metabolizing cytochrome P450 between miniature pigs and humans, two cDNA clones, coding for miniature pig CYP2D21 and CYP3A22, were isolated. The deduced amino acid sequences of CYP2D21 and CYP3A22 were 78.3 and 75.0% identical to human CYP2D6 and CYP3A4, respectively. These values were nearly the same as those of bovine, dog, and some rodent isoforms, and 12.2 to 18.4% lower than those of nonhuman primates such as cynomolgus monkeys, Japanese monkey, and marmosets. These data indicate that miniature pig P450s are genetically not so close as monkey P450s to human P450s as previously expected. The recombinant CYP2D21 enzyme, however, showed bufuralol 1'-hydroxylase activity, suggesting that miniature pig CYP2D21 is capable of metabolizing some of the same substrates associated with human CYP2D6 despite its low identity to human counterparts.

Drosophila FACT contributes to Hox gene expression through physical and functional interactions with GAGA factor.

Chromatin structure plays a critical role in the regulation of transcription. Drosophila GAGA factor directs chromatin remodeling to its binding sites. We show here that Drosophila FACT (facilitates chromatin transcription), a heterodimer of dSPT16 and dSSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions between Trithorax-like encoding GAGA factor and spt16 implicate the GAGA factor-FACT complex in expression of Hox genes Ultrabithorax, Sex combs reduced, and Abdominal-B. Chromatin immunoprecipitation experiments indicated the presence of the GAGA factor-FACT complex in the regulatory regions of Ultrabithorax and Abdominal-B. These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression.