Plant orthologs of p300/CBP: conservation of a core domain in metazoan p300/CBP acetyltransferase-related proteins

p300 and CBP participate as transcriptional coregulators in the execution of a wide spectrum of cellular gene expression programs controlling cell differentiation, growth and homeostasis. Both proteins act together with sequence-specific transcription factors to modify chromatin structure of target genes via their intrinsic acetyltransferase activity directed towards core histones and some transcription factors. So far, p300-related proteins have been described in animals ranging from Drosophila and Caenorhabditis elegans to humans. In this report, we describe p300/CBP-like polypeptides in the plant Arabidopsis thaliana. Interestingly, homology between animal and plant p300/CBP is largely restricted to a C-terminal segment, about 600 amino acids in length, which encompasses acetyltransferase and E1A-binding domains. We have examined whether this conservation in sequence is paralleled by a conservation in function. The same amino acid residues critical for acetyltransferase activity in human p300 are also critical for the function of one of the plant orthologs. Remarkably, plant proteins bind to the adenovirus E1A protein in a manner recapitulating the binding specificity of mammalian p300/CBP. The striking conservation of an extended segment of p300/CBP suggests that it may constitute a functional entity fulfilling functions that may be essential for all metazoan organisms.

Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus.

We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.

Two mitochondrial group I introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase.

Mitochondrial genes for cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) of the sea anemone Metridium senile (phylum Cnidaria) each contain a group I intron. This is in contrast to the reported absence of introns in all other metazoan mtDNAs so far examined. The ND5 intron is unusual in that it ends with A and contains two genes (ND1 and ND3) encoding additional subunits of NADH dehydrogenase. Correctly excised ND5 introns are not circularized but are precisely cleaved near their 3' ends and polyadenylylated to provide bicistronic transcripts of ND1 and ND3. COI introns, which encode a putative homing endonuclease, circularize, but in a way that retains the entire genome-encoded intron sequence (other group I introns are circularized with loss of a short segment of the intron 5' end). Introns were detected in the COI and ND5 genes of other sea anemones, but not in the COI and ND5 genes of other cnidarians. This suggests that the sea anemone mitochondrial introns may have been acquired relatively recently.

Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere.

In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

Testing the Cambrian explosion hypothesis by using a molecular dating technique

Molecular studies have the potential to shed light on the origin of the animal phyla by providing independent estimates of the divergence times, but have been criticized for failing to account adequately for variation in rate of evolution. A method of dating divergence times from molecular data addresses the criticisms of earlier studies and provides more realistic, but wider, confidence intervals. The data are not compatible with the Cambrian explosion hypothesis as an explanation for the origin of metazoan phyla, and provide additional support for an extended period of Precambrian metazoan diversification.

Molecular evolution of integrins: Genes encoding integrin β subunits from a coral and a sponge

The integrin family of cell surface receptors is strongly conserved in higher animals, but the evolutionary history of integrins is obscure. We have identified and sequenced cDNAs encoding integrin β subunits from a coral (phylum Cnidaria) and a sponge (Porifera), indicating that these proteins existed in the earliest stages of metazoan evolution. The coral βCn1 and, especially, the sponge βPo1 sequences are the most divergent of the “β1-class” integrins and share a number of features not found in any other vertebrate or invertebrate integrins. Perhaps the greatest difference from other β subunits is found in the third and fourth repeats of the cysteine-rich stalk, where the generally conserved spacings between cysteines are highly variable, but not similar, in βCn1 and βPo1. Alternatively spliced cDNAs, containing a stop codon about midway through the full-length translated sequence, were isolated from the sponge library. These cDNAs appear to define a boundary between functional domains, as they would encode a protein that includes the globular ligand-binding head but would be missing the stalk, transmembrane, and cytoplasmic domains. These and other sequence comparisons with vertebrate integrins are discussed with respect to models of integrin structure and function.

Photorepair mutants of Arabidopsis

UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6–4) pyrimidinone dimer (6–4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6–4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6–4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6–4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.

Functional domains for assembly of histones H3 and H4 into the chromatin of Xenopus embryos

Histones H3 and H4 have a well defined structural role in the nucleosome and an established role in the regulation of transcription. We have made use of a microinjection strategy using Xenopus embryos to define the minimal structural components of H3 and H4 necessary for nucleosome assembly into metazoan chromosomes in vivo. We find that both the N-terminal tail of H4, including all sites of acetylation, and the C-terminal α-helix of the H4 histone fold domain are dispensable for chromatin assembly. The N-terminal tail and an N-terminal α-helix of H3 are also dispensable for chromatin assembly. However, the remainder of the H3 and H4 histone folds are essential for incorporation of these proteins into chromatin. We suggest that elements of the histone fold domain maintain both nucleosomal integrity and have distinct functions essential for cell viability.

The structural basis for the oriented assembly of a TBP/TFB/promoter complex

Recently the definition of the metazoan RNA polymerase II and archaeal core promoters has been expanded to include a region immediately upstream of the TATA box called the B recognition element (BRE), so named because eukaryal transcription factor TFIIB and its archaeal orthologue TFB interact with the element in a sequence-specific manner. Here we present the 2.4-Å crystal structure of archaeal TBP and the C-terminal core of TFB (TFBc) in a complex with an extended TATA-box-containing promoter that provides a detailed picture of the stereospecific interactions between the BRE and a helix–turn–helix motif in the C-terminal cyclin repeat of TFBc. This interaction is important in determining the level of basal transcription and explicitly defines the direction of transcription.

GERp95, a Membrane-associated Protein that Belongs to a Family of Proteins Involved in Stem Cell Differentiation

A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin

Coelomocytes, the heterogeneous population of sea urchin putative immune cells, were found to express a complex set of transcripts featuring scavenger receptor cysteine-rich (SRCR) repeats. SRCR domains define a metazoan superfamily of proteins, many of which are implicated in development and regulation of the immune system of vertebrates. Coelomocytes transcribe multiple SRCR genes from among a multigene family encoding an estimated number of 1,200 SRCR domains in specific patterns particular to each individual. Transcription levels for given SRCR genes may range from pronounced to undetectable, yet all tested animals harbor the genomic loci encoding these genes. Analysis of several SRCR genes revealed multiple loci corresponding to each type. In the case of one SRCR type, a cluster of at least three genes was detected within a 133-kb bacterial artificial chromosome insert, and conserved as well as unique regions were identified in sequences of three genomic clones derived from a single animal. Array hybridizations with repeated samples of coelomocyte messages revealed substantial alterations in levels of expression of many SRCR genes, with fluctuations of up to 10-fold in 1 week and up to 30-fold over a period of 3 months. This report is the first demonstration of genomic and transcriptional complexity in molecules expressed by invertebrate coelomocytes. The mechanisms controlling SRCR gene expression and the functional significance of this dynamic system await elucidation.

Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae

The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete. Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts. Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae. The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.

Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa?

Homeodomain proteins are transcription factors that play a critical role in early development in eukaryotes. These proteins previously have been classified into numerous subgroups whose phylogenetic relationships are unclear. Our phylogenetic analysis of representative eukaryotic sequences suggests that there are two major groups of homeodomain proteins, each containing sequences from angiosperms, metazoa, and fungi. This result, based on parsimony and neighbor-joining analyses of primary amino acid sequences, was supported by two additional features of the proteins. The two protein groups are distinguished by an insertion/deletion in the homeodomain, between helices I and II. In addition, an amphipathic alpha-helical secondary structure in the region N terminal of the homeodomain is shared by angiosperm and metazoan sequences in one group. These results support the hypothesis that there was at least one duplication of homeobox genes before the origin of angiosperms, fungi, and metazoa. This duplication, in turn, suggests that these proteins had diverse functions early in the evolution of eukaryotes. The shared secondary structure in angiosperm and metazoan sequences points to an ancient conserved functional domain.