Chromatin profiling using targeted DNA adenine methyltransferase.

Chromatin is the highly complex structure consisting of DNA and hundreds of associated proteins. Most chromatin proteins exert their regulatory and structural functions by binding to specific chromosomal loci. Knowledge of the identity of these in vivo target loci is essential for the understanding of the functions and mechanisms of action of chromatin proteins. We report here large-scale mapping of in vivo binding sites of chromatin proteins, using a novel approach based on a combination of targeted DNA methylation and microarray technology. We show that three distinct chromatin proteins in Drosophila melanogaster cells each associate with specific sets of genes. HP1 binds predominantly to pericentric genes and transposable elements. GAGA factor associates with euchromatic genes that are enriched in (GA)n motifs. A Drosophila homolog of Saccharomyces cerevisiae Sir2p is associated with several active genes and is excluded from heterochromatin. High-resolution, genome-wide maps of target loci of chromatin proteins ('chromatin profiles') provide new insights into chromatin structure and gene regulation.

Nuclear organization and gene expression: homologous pairing and long-range interactions.

Genetic studies have demonstrated that pairing interactions between homologous chromosomes and long-range associations between nonhomologous sites can influence gene expression. Recent work has revealed that such influences are widespread in eukaryotes and that chromosome architecture is likely to be of fundamental importance for nuclear structure and function.

Large-scale chromosomal movements during interphase progression in Drosophila.

We examined the effect of cell cycle progression on various levels of chromosome organization in Drosophila. Using bromodeoxyuridine incorporation and DNA quantitation in combination with fluorescence in situ hybridization, we detected gross chromosomal movements in diploid interphase nuclei of larvae. At the onset of S-phase, an increased separation was seen between proximal and distal positions of a long chromsome arm. Progression through S-phase disrupted heterochromatic associations that have been correlated with gene silencing. Additionally, we have found that large-scale G1 nuclear architecture is continually dynamic. Nuclei display a Rabl configuration for only approximately 2 h after mitosis, and with further progression of G1-phase can establish heterochromatic interactions between distal and proximal parts of the chromosome arm. We also find evidence that somatic pairing of homologous chromosomes is disrupted during S-phase more rapidly for a euchromatic than for a heterochromatic region. Such interphase chromosome movements suggest a possible mechanism that links gene regulation via nuclear positioning to the cell cycle: delayed maturation of heterochromatin during G1-phase delays establishment of a silent chromatin state.

Centromeres are specialized replication domains in heterochromatin.

The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, we examined replication and chromatin assembly at centromeres in Drosophila cells. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones.

Changes in chromosomal localization of heterochromatin-binding proteins during the cell cycle in Drosophila.

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brownDominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.

The centromere paradox: stable inheritance with rapidly evolving DNA.

Every eukaryotic chromosome has a centromere, the locus responsible for poleward movement at mitosis and meiosis. Although conventional loci are specified by their DNA sequences, current evidence favors a chromatin-based inheritance mechanism for centromeres. The chromosome segregation machinery is highly conserved across all eukaryotes, but the DNA and protein components specific to centromeric chromatin are evolving rapidly. Incompatibilities between rapidly evolving centromeric components may be responsible for both the organization of centromeric regions and the reproductive isolation of emerging species.

Gene families: the taxonomy of protein paralogs and chimeras.

Ancient duplications and rearrangements of protein-coding segments have resulted in complex gene family relationships. Duplications can be tandem or dispersed and can involve entire coding regions or modules that correspond to folded protein domains. As a result, gene products may acquire new specificities, altered recognition properties, or modified functions. Extreme proliferation of some families within an organism, perhaps at the expense of other families, may correspond to functional innovations during evolution. The underlying processes are still at work, and the large fraction of human and other genomes consisting of transposable elements may be a manifestation of the evolutionary benefits of genomic flexibility.

Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation.

Epigenetic silenced alleles of the Arabidopsis SUPERMAN locus (the clark kent alleles) are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites, where X = A, T, C, or G). A genetic screen for suppressors of a hypermethylated clark kent mutant identified nine loss-of-function alleles of CHROMOMETHYLASE3 (CMT3), a novel cytosine methyltransferase homolog. These cmt3 mutants display a wild-type morphology but exhibit decreased CpXpG methylation of the SUP gene and of other sequences throughout the genome. They also show reactivated expression of endogenous retrotransposon sequences. These results show that a non-CpG DNA methyltransferase is responsible for maintaining epigenetic gene silencing.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Dual recognition-incision enzymes might be involved in mismatch repair and meiosis.

Mismatch repair in many organisms depends on three proteins: the mismatch-recognition protein MutS, a nicking endonuclease MutH, and MutL, which acts as a scaffold between these. However, many genomes lack MutL but possess MutS. In one of these cases, in a coral mitochondrial genome, a gene is present that encodes a MutS protein fused to an HNH nicking endonuclease, potentially eliminating the requirement for MutL. Likewise, many prokaryotes could operate similarly, independently of MutL by encoding a fused MutS-Smr (MutS2) protein. Smr, which is proposed to be a nicking endonuclease, can also be found separately in many eukaryotes, where it might play a role in mismatch repair or meiotic chromosome crossing-over.

Position effect and related phenomena.

Position-effect variegation in Drosophila, the mosaic expression of genes juxtaposed to heterochromatin, remains an enigmatic long-range phenomenon. While the chromatin-conformation model has been challenged, compelling contrary evidence is lacking. Nevertheless, progress has been made in the genetic and molecular analysis of genes involved in the process of heterochromatin formation and in the characterization of genetic elements normally located in pericentric heterochromatin. In addition, telomeric position effect in yeast provides a new model system for the study of the quasi-stable inheritance of an inactivated state.

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.

Position-effect variegation after 60 years.

Position-effect variegation in Drosophila--the mosaic expression of a gene placed adjacent to a junction between euchromatin and heterochromatin--remains an enigma. However, new insights are being gained from recent studies of genetic modifiers, new model systems, and variegating genes showing exceptional behavior.

Something from nothing: the evolution and utility of satellite repeats.

Large blocks of tandemly repeated sequences, or satellites, surround the centromeres of complex eukaryotes. During mitosis in Drosophila, satellite DNA binds proteins that, during interphase, bind other sites. The requirement for a repeat to borrow a partner protein from those available at mitosis might limit the spectrum of repeat units that can be expanded into large blocks. To account for the ubiquity and pericentric localization of satellites, we propose that they are utilized to maintain regions of late replication, thus ensuring that the centromere is the last region to replicate on a chromosome.

A moveable 5' splice site in adenine phosphoribosyltransferase genes of Drosophila species.

In two distantly related Drosophila species, the use of alternate 5' splice sites to process an intron in pre-mRNA from homologous adenine phosphoribosyltransferase (APRT)-encoding genes led to RNAs encoding nonfunctional peptides in addition to APRT. The production of aberrantly spliced transcripts as a normal feature of gene expression supports a general model of eucaryotic gene evolution through alternative splicing and moveable splice junctions.

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.

Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae.

We have mapped a signal sequence for mRNA 3'-end formation in Saccharomyces cerevisiae by using a Drosophila melanogaster DNA segment that complements a yeast adenine-8 mutation. That the 3' end of the transcript in S. cerevisiae nearly coincides with that in D. melanogaster is consistent with the possibility that mRNA termini are similarly determined in both organisms. Deletion analysis reveals that the complete signal is no more than 21 base pairs long. Part of the signal is the sequence TTTTTATA, which is seen in the termination region of several yeast genes. TTTTTATA appears to be able to act autonomously as a partial termination signal. The efficiency of the complete signal is affected by substitution of sequences downstream from it. This modulation of the effect of a signal is consistent with termination in S. cerevisiae, resembling rho-dependent termination in bacteria.

The brown protein of Drosophila melanogaster is similar to the white protein and to components of active transport complexes.

The brown gene of Drosophila melanogaster is required for deposition of pteridine pigments in the compound eye and other tissues. We isolated a ca. 150-kilobase region including brown by microdissection and chromosome walking using cosmids. Among the cDNAs identified by hybridization to the cosmids, one class hybridized to a genomic region that is interrupted in two brown mutants, bw and In(2LR)CK, and to 2.8- and 3.0-kilobase poly(A)+ RNAs which are altered in the mutants. Nucleotide sequencing of these cDNAs revealed that the two transcripts differ as a consequence of alternative poly(A) addition and that both encode the same predicted protein of 675 amino acids. Searches of available databases for amino acid sequence similarities detected a striking overall similarity of this predicted protein to that of the D. melanogaster white gene. The N-terminal portion aligned with the HisP family of membrane-associated ATP-binding proteins, most of which are subunits of active transport complexes in bacteria, and to two regions of the multidrug resistance P-glycoprotein. The C-terminal portion showed a structural similarity to integral membrane components of the same complexes. Taken together with earlier biochemical evidence that brown and white gene products are necessary for uptake of a pteridine precursor and genetic evidence that brown and white proteins interact, our results are consistent with suggestions that these proteins are subunits of a pteridine precursor permease.