Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. We have identified a substrate recognition domain in the C-terminal half of the remodeling ATPase ISWI and determined its structure by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how ISWI interacts with the nucleosomal substrate.

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.

Summary Unlike nuclear multisubunit RNA polymerases I, II and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA Polymerases IV and V are non-essential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their twelve subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits, but differ from each other only in their largest subunits. Use of alternative catalytic second-subunits, which are non-redundant for development and paramutation, yields at least two subtypes of Pol IV, and three subtypes of Pol V in maize. Pol IV/V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

Deficient in DNA Methylation 1 (DDM1) protein is required to maintain the DNA methylation status of Arabidopsis thaliana. DDM1 is a member of the broad SWI2/ SNF2 protein family. Because of its phylogenetic position, DDM1 has been speculated to act as a chromatin-remodeling factor. Here we used a purified recombinant DDM1 protein to investigate whether it can remodel chromatin in vitro. We show that DDM1 is an ATPase stimulated by both naked and nucleosomal DNA. DDM1 binds to the nucleosome and promotes chromatin remodeling in an ATP-dependent manner. Specifically, it induces nucleosome repositioning on a short DNA fragment. The enzymatic activity of DDM1 is not affected by DNA methylation. The relevance of these findings to the in vivo role of DDM1 is discussed.The compaction of eukaryotic genomes into chromatin structures has profound implications for nuclear processes such as replication (1, 2), transcription (3, 4), DNA repair (5), and recombination (6, 7). Evolution has created a diverse repertoire of regulatory mechanisms affecting the dynamics of chromatin structure. One broad group of mechanisms involves a battery of the enzymes that covalently modify histones and DNA (8 -10). Specific regulatory factors recognize the modification status of chromatin fibers and render the structure active or inactive (8).To ensure sufficient structural flexibility of the chromatin, the cell employs a specialized class of multiprotein complexes, which utilize the energy of ATP hydrolysis to change chromatin folding in the poorly understood process of "chromatin remodeling" (11,12). Central to this activity are SWI2/SNF2-type ATPases that form the catalytic core of these remodeling complexes. The SWI2/SNF2 protein family falls within the large superfamily of DEXD/H-ATPases (13). The distinctive signature of the SWI2/SNF2 family is the SNF2_N domain, a variant of the typical DEXD/H domain that contains a well conserved C-terminal extension of ϳ100 amino acids.1 Phylogenetic analysis divides the SWI2/SNF2 family into a number of subfamilies (13), which appear to be present in all major taxa. Outside the SNF2_N and HelicC domains, which form the catalytic module, these subfamilies are not well conserved. Members of three closely related subfamilies (ISWI, Mi2/CHD, and SWI2/ SNF2) have been shown to serve as the catalytic subunits of chromatin-remodeling complexes (15-18). Recently, remodeling activity has also been demonstrated for a complex built around the more distantly related INO80 ATPase (19). All catalytic subunits of the chromatin-remodeling complexes that have been analyzed can act outside the complex context, although they require associated proteins to achieve their full activity (16 -18, 20). CSB/ERCC6 defines a fifth group of remodelers, although it has been analyzed only as the isolated, recombinant protein (21). During recent years it has become clear that energy-dependent remodelers act in concert with histone modifying enzymes (22-27).So far only very few family members have been analyzed in biochemical assay...

The multiprotein complexes involved in active dis-ruption of chromatin structure, homologous to yeast SWI/SNF complex, have been described for human and Drosophila cells. In all SWI/SNF-class complexes characterised so far, one of the key components is the SNF5-type protein. Here we describe the isolation of a plant (Arabidopsis thaliana ) cDNA encoding a 27 kDa protein which we named BSH, with high homology to yeast SNF5p and its human (INI1) and Drosophila (SNR1) counterparts as well as to other putative SNF5-type proteins from Caenorhabditis elegans, fish and yeast. With 240 amino acids, the Arabidopsis BSH is the smallest SNF5-type protein so far identified. When expressed in Saccharomyces cerevisiae, the gene for BSH partially complements the snf5 mutation. BSH is, however, unable to activate transcription in yeast when tethered to DNA. The gene for BSH occurs in single copy in the Arabidopsis genome and is ubiquitously expressed in the plant. Analysis of the whole cell and nuclear protein extracts with antibodies against recombinant BSH indicates that the protein is localised in nuclei. Transgenic Arabidopsis plants with markedly decreased physiological level of the BSH mRNA, resulting from the expression of antisense messenger, are viable but exhibit a distinctive phenotype characterised by bushy growth and flowers that are unable to produce seeds.