Transcriptional co-activators were originally identified as proteins that act as intermediaries between upstream activators and the basal transcription machinery. The discovery that co-activators such as Tetrahymena and yeast Gcn5, as well as human p300/CBP, pCAF, Src-1, ACTR and TAFII250, can acetylate histones suggests that activators may be involved in targeting acetylation activity to promoters. Several histone deacetylases have been linked to transcriptional co-repressor proteins, suggesting that the action of both acetylases and deacetylases is important in the regulation of many genes. Here we demonstrate the binding of two native yeast histone acetyltransferase (HAT) complexes to the herpesvirus VP16 activation domain and the yeast transcriptional activator Gcn4, and show that it is their interaction with the VP16 activation domain that targets Gal4-VP16-bound nucleosomes for acetylation. We find that Gal4-VP16-driven transcription from chromatin templates is stimulated by both HAT complexes in an acetyl CoA-dependent reaction. Our results demonstrate the targeting of native HAT complexes by a transcription-activation domain to nucleosomes in order to activate transcription.

A similar gene network was found to control chick myogenesis, in which Six1, Eya2 and Dach2 synergistically regulate the expression of myogenic genes such as myogenin and MyoD (Heanue et Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wildtype otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1-and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.

Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra. The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides. Although polyhedra have been extensively characterized since the early 1900s, their atomic organization remains elusive. Here we describe the 2 A crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5-12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination. We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen-antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications such as microarrays and biopesticides.

Opn5 (neuropsin) belongs to an independent group separated from the other six groups in the phylogenetic tree of opsins, for which little information of absorption characteristics and molecular properties of the members is available. Here we show that the chicken Opn5 (cOpn5m) is a UV-sensitive bistable pigment that couples with Gi subtype of G protein. The recombinant expression of cOpn5m in HEK 293s cells followed by the addition of 11-cis-and all-trans-retinal produced UV light-absorbing and visible lightabsorbing forms, respectively. These forms were interconvertible by UV and visible light irradiations, respectively, indicating that cOpn5m is a bistable pigment. The absorption maxima of these forms were estimated to be 360 and 474 nm, respectively. The GTPγS binding assay clearly showed that the visible light-absorbing form having all-trans-retinal activates Gi type of G protein, whereas no Gt or Gq activation ability was observed. Immunohistochemical studies using an antibody against cOpn5m clearly showed that this pigment is localized within some types of amacrine cells and some cells in the ganglion cell layer of the retinas, the vast majority of cells in the pineal gland and serotonin-positive cells in the paraventricular organ. Because cOpn5m is the only UV-sensitive opsin among the opsins found so far in chicken, this study provides the molecular basis for UV reception in chicken.G protein-coupled receptor | nonvisual photoreception | phototransduction

The members of the Six gene family were identified as homologues of Drosophila sine oculis which is essential for compound-eye formation. The Six proteins are characterized by the Six domain and the Six-type homeodomain, both of which are essential for specific DNA binding and for cooperative interactions with Eya proteins. Mammals possess six Six genes which can be subdivided into three subclasses, and mutations of Six genes have been identified in human genetic disorders. Characterization of Six genes from various animal phyla revealed the antiquity of this gene family and roles of its members in several different developmental contexts. Some members retain conserved roles as components of the Pax-Six-Eya-Dach regulatory network, which may have been established in the common ancestor of all bilaterians as a toolbox controlling cell proliferation and cell movement during embryogenesis. Gene duplications and cis-regulatory changes may have provided a basis for diverse functions of Six genes in different animal lineages.

Phox2b protein is a specific marker for neurons in the parafacial region of the ventral medulla, which are proposed to play a role in central chemoreception and postnatal survival. Mutations of PHOX2B cause congenital central hypoventilation syndrome. However, there have been no reports concerning electrophysiological characteristics of these Phox2b-expressing neurons in the parafacial region of the neonate immediately after birth. This region overlaps with the parafacial respiratory group (pFRG) composed predominantly of preinspiratory (Pre-I) neurons that are involved in respiratory rhythm generation. We studied (1) whether pFRG neurons are Phox2b immunoreactive or not and (2) whether they show intrinsic CO 2 chemosensitivity. We found that most pFRG/Pre-I neurons were Phox2b immunoreactive and depolarized upon increase in CO 2 concentration under condition of action potential-dependent synaptic transmission blockade by tetrodotoxin. We also confirmed that these pFRG neurons expressed neurokinin-1 receptor. They were tyrosine hydroxylase negative and presumed to be glutamatergic. Our findings suggest that Phox2b-expressing parafacial neurons play a role in respiratory rhythm generation as well as central chemoreception and thus are essential for postnatal survival.

The sodium pump is the enzyme responsible for the maintenance of Na+ and K+ gradients across the cell membrane. Four isoforms of the catalytic alpha subunit have been identified, but their individual roles remain essentially unknown. To investigate the necessary functions of the alpha2 subunit in vivo, we generated and analyzed mice defective in the alpha2 subunit gene. Mice homozygous for the alpha2 mutation died just after birth and displayed selective neuronal apoptosis in the amygdala and piriform cortex. In these regions, high expression of c-Fos before apoptosis indicated neural hyperactivity, and re-uptake of glutamic acid and GABA into P2 fraction containing crude synaptosome was impaired. These results indicate that the alpha2 subunit plays a critical role regulating neural activity in the developing amygdala and piriform cortex. Further supporting a role of the alpha2 subunit in the function of the amygdala, heterozygous adult mice showed augmented fear/anxiety behaviors and enhanced neuronal activity in the amygdala and piriform cortex after conditioned fear stimuli.

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, "early neurogenesis" occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows "established neurogenesis," in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1-/-). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1-/- at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1-/-. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.