Predicting in vivo protein peptide interactions with random phage display.

Binding sites in protein complexes occasionally map to small peptides within one or more proteins. Random peptide display methods simulate binding interactions by providing all possible peptide combinations with an equal opportunity to bind a protein of interest. The natural substrates for the protein are typically known in advance. However, it is often the case that such substrates are identified as putative partner proteins by using in vivo methods such as yeast two hybrid screening. Unfortunately, such methods often produce lengthy datasets of protein sequences and offer little mechanistic insight into how such interactions might take place in vivo. Here, we review an approach that addresses this problem. First, sequence alignment tools identify and characterize blocks of conserved sequences among peptides recovered during random peptide display. Next, searching programs detect similar blocks of conserved sequences within naturally occurring proteins to predict partner proteins. Finally, the significance of an interaction is tested using site specific mutagenesis, binding competition or co-immunoprecipitation experiments. This strategy should become increasingly powerful with the growing popularity of interaction studies, sequencing projects and microarray analyses in modern biology.

The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins.

Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. We have characterized the subnuclear localization patterns of two newly discovered Drosophila HP1-like proteins (HP1b and HP1c), comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. We expressed truncated and chimeric HP1 proteins in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. We conclude that sequence variations within the hinge and shadow account for HP1 targeting distinctions. We propose that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins.

The HP1 chromo shadow domain binds a consensus peptide pentamer.

Heterochromatin-associated protein 1 (HP1) is thought to affect chromatin structure through interactions with other proteins in heterochromatin. Chromo domains located near the amino (amino chromo) and carboxy (chromo shadow) termini of HP1 may mediate such interactions, as suggested by domain swapping, in vitro binding and 3D structural studies . Several HP1-associated proteins have been reported, providing candidates that might specifically complex with the chromo domains of HP1. However, such association studies provide little mechanistic insight and explore only a limited set of potential interactions in a largely non-competitive setting. To determine how chromo domains can selectively interact with other proteins, we probed random peptide phage display libraries using chromo domains from HP1. Our results demonstrate that a consensus pentapeptide is suffident for specific interaction with the HP1 chromo shadow domain. The pentapeptide is found in the amino acid sequence of reported HP1-associated proteins, including the shadow domain itself. Peptides that bind the shadow domain also disrupt shadow domain dimers. Our results suggest that HP1 dimerization, which is thought to mediate heterochromatin compaction and cohesion, occurs via pentapeptide binding. In general, chromo domains may function by avidly binding short peptides at the surface of chromatin-associated proteins.

Excision of micronuclear-specific DNA requires parental expression of pdd2p and occurs independently from DNA replication in Tetrahymena thermophila.

Elimination of germ-line DNA segments is an essential step in the somatic development of many organisms and in the terminal differentiation of several specialized cell types. In binuclear ciliates, including Tetrahymena thermophila, DNA elimination occurs during the conversion of the germ-line micronuclear genome into the somatic genome of the new macronucleus. Little is known about molecular determinants and regulatory mechanisms involved in this process. Pdd2p is one of a small set of Tetrahymena polypeptides whose time of synthesis, nuclear localization, and physical association with sequences destined for elimination suggest an involvement in the DNA elimination process. In this study, we report that loss of parental expression of Pdd2p leads to the perturbation of several DNA rearrangement processes in developing zygotic macronuclei, including excision of internal eliminated sequences, excision of chromosome breakage sequences, and endoreplication of the new macronuclear genome and eventually results in lethality of the progeny. We demonstrate that excision and elimination of micronuclear-specific DNA occurs independently of endoreplication of the new macronuclear genome that takes place during the same period of time. Thus, our data indicate that parental expression of Pdd2p is required for successful DNA elimination and development of somatic nuclei.

A nonessential HP1-like protein affects starvation-induced assembly of condensed chromatin and gene expression in macronuclei of Tetrahymena thermophila.

Heterochromatin represents a specialized chromatin environment vital to both the repression and expression of certain eukaryotic genes. One of the best-studied heterochromatin-associated proteins is Drosophila HP1. In this report, we have disrupted all somatic copies of the Tetrahymena HHP1 gene, which encodes an HP1-like protein, Hhp1p, in macronuclei (H. Huang, E. A. Wiley, R. C. Lending, and C. D. Allis, Proc. Natl. Acad. Sci. USA 95:13624-13629, 1998). Unlike the Drosophila HP1 gene, HHP1 is not essential in Tetrahymena spp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p (DeltaHHP1). However, during a shift to nongrowth conditions, the survival rate of DeltaHHP1 cells is reduced compared to that of wild-type cells. Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p. Activation of two starvation-induced genes (ngoA and CyP) is significantly reduced in DeltaHHP1 cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells. These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response.

The conjusome: a novel structure in Tetrahymena found only during sexual reorganization.

A unique structure, the conjusome, has been identified and initially characterized in Tetrahymena thermophila. The conjusome appears only during a specific phase of conjugation. Immunofluorescence microscopy reveals that the conjusome is strongly labeled by antibodies to the protein Pdd1p. Pdd1p is a chromodomain protein and participates in the formation of chromatin-containing structures in developing macronuclear anlagen. Recent studies suggest that Pdd1p is physically associated with the elimination of specific germ-line sequences from developing macronuclei (anlagen) and may play a role in heterochromatin assembly. The conjusome contains Pdd1p, but it is devoid of any detectable DNA. The conjusome appears before DNA elimination begins in the developing anlagen and after Pdd1p is found in the parental macronucleus. Transmission electron microscopic observations reveal that the conjusome is not a membrane-bounded structure. The conjusome ranges in size from about 1 microm to sizes approaching 7 microm, depending on its maturity. It is composed of a coarse reticulum of a fibrous, electron dense material, interspersed with apparent background cytoplasm. Our initial characterization does suggest a number of possible functions for what may be a new, transient organelle.

Parental expression of the chromodomain protein Pdd1p is required for completion of programmed DNA elimination and nuclear differentiation.

Thousands of DNA elimination events occur during somatic differentiation of many ciliated protozoa. In Tetrahymena, the eliminated DNA aggregates into submacronuclear structures containing the protein Pdd1p, a member of the chromodomain family. We disrupted somatic copies of PDD1, eliminating parental expression of the gene early in the sexual phase of the life cycle. Even though zygotic expression, from the undisrupted germline PDD1 copy, is activated before DNA elimination normally occurs, the somatic knockout cells suffer defects in DNA elimination, genome endoduplication, and nuclear resorption, and eventually die, demonstrating that PDD1 is essential and suggesting Pdd1p is directly involved in establishing a chromatin structure required for DNA elimination.

Pdd1p associates with germline-restricted chromatin and a second novel anlagen-enriched protein in developmentally programmed DNA elimination structures.

Programmed DNA rearrangements, including DNA diminution, characterize the differentiation of somatic from germline nuclei in several developmental systems. Pdd1p (Programmed DNA degradation protein 1), a development-restricted polypeptide, has been implicated in heterochromatin assembly and DNA degradation during ciliate macronuclear development. Here, cross-linking and co-immunoprecipitation were used to verify that Pdd1p-associated chromatin is enriched in germline-restricted DNA. Pdd1p-associated proteins include general core histones and a second anlagen-enriched polypeptide (Pdd2p, formerly known as p43). Immunoblotting analyses demonstrate that, like Pdd1p, Pdd2p is developmentally regulated and present in conjugating cells during the time of germline DNA rearrangements and degradation. Pdd2p is post-translationally modified by phosphorylation at a time in development corresponding to dephosphorylation of Pdd1p and the formation of heterochromatic DNA elimination structures. Following gene cloning, the derived amino acid sequence of the PDD2 gene predicts a novel polypeptide containing multiple putative phosphorylation sites. In situ analyses, using both light and electron microscopy, demonstrate that Pdd1p and Pdd2p co-localize in DNA elimination structures within developing macronuclei. However, unlike Pdd1p, which also localizes to apoptotic macronuclei, Pdd2p appears to be restricted to a higher degree to germline DNA elimination structures. Taken together, the data presented here demonstrate a physical link between Pdd1p and germline-restricted chromatin and establish Pdd2p as the second member of a small group of developmentally restricted polypeptides implicated in programmed DNA elimination.

Programmed DNA degradation and nucleolar biogenesis occur in distinct organelles during macronuclear development in Tetrahymena.

Programmed DNA rearrangements, including DNA degradation, characterize the development of the soma from the germline in a number of developmental systems. Pdd1p (programmed DNA degradation 1 protein), a development-specific polypeptide in Tetrahymena, is enriched in developing macronuclei (anlagen) and has been implicated in DNA elimination and nucleolar biogenesis. Here, immunocytochemistry and fluorescent in situ hybridization (FISH) were employed to follow Pdd1p and two nucleolar markers (Nopp52 and rDNA) during macronuclear development. Both Pdd1p and Nopp52 localize to subnuclear structures, each of which resemble nucleoli. However, while true nucleoli form and persist during development, Pdd1p-positive structures are only present for a brief period of macronuclear differentiation. Accordingly, two distinct organelles can be recognized in anlagen: (1) Pdd1p-positive structures, which lack Nopp52 and rDNA, and (2) developing nucleoli which contain rDNA and Nopp52 but lack Pdd1p. Taken together with recent data corroborating Pdd1p's role in DNA elimination, we favor the hypothesis that Pdd1p structures are unique, short-lived organelles, likely to function in programmed DNA degradation and not in nucleolar biogenesis.

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

Pdd1p, a novel chromodomain-containing protein, links heterochromatin assembly and DNA elimination in Tetrahymena.

During Tetrahymena conjugation, programmed DNA degradation occurs in two separate nuclei. Thousands of germline-specific deletion elements are removed from the genome of the developing somatic macronucleus, and the old parental macronucleus is degraded by an apoptotic mechanism. An abundant polypeptide, Pdd1p (formerly p65), localizes to both of these nuclei at the time of DNA degradation. Here we report that, in developing macronuclei, Pdd1p localizes to electron-dense, heterochromatic structures that contain germline-specific deletion elements. Pdd1p also associates with parental macronuclei during terminal stages of apoptosis. Sequencing of the PDD1 gene reveals it to be a member of the chromodomain family, suggesting a molecular link between heterochromatin assembly and programmed DNA degradation.

Molecular evidence that the myxozoan protists are metazoans.

The evolutionary origins of the protistan phylum, Myxozoa, have long been questioned. Although these obligate parasites are like protozoans in many features, several aspects of their ontogeny and morphology have implied a closer relationship to metazoan lineages. Phylogenetic analyses of 18S ribosomal RNA sequences from myxozoans and other eukaryotes, with the use of parsimony, distance, and maximum-likelihood methods, support the hypothesis that myxozoans are closely related to the bilateral animals. These results suggest that the Myxozoa, long considered an assemblage of protozoans, should be considered a metazoan phylum.