A conserved Swi2/Snf2 ATPase motif couples ATP hydrolysis to chromatin remodeling

Yeast (Saccharomyces cerevisiae) SWI/SNF is a prototype for a large family of ATP-dependent chromatin-remodeling enzymes that facilitate numerous DNA-mediated processes. Swi2/Snf2 is the catalytic subunit of SWI/SNF, and it is the founding member of a novel subfamily of the SF2 superfamily of DNA helicase/ATPases. Here we present a functional analysis of the diagnostic set of helicase/ATPase sequence motifs found within all Swi2p/Snf2p family members. Whereas many of these motifs play key roles in ATP binding and/or hydrolysis, we identify residues within conserved motif V that are specifically required to couple ATP hydrolysis to chromatin-remodeling activity. Interestingly, motif V of the human Swi2p/Snf2p homolog, Brg1p, has been shown to be a possible hot spot for mutational alterations associated with cancers.     

Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.     

The NEF4 complex regulates Rad4 levels and utilizes Snf2/Swi2-related ATPase activity for nucleotide excision repair

Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae. Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits. We report previously unrecognized similarity between Rad7 and F-box proteins. Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase. Mutational analysis provides strong support for this model. The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function. Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4. Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria. Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23. As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4.     

Sth1p,a Saccharomyces cerevisiae Snf2p/Swi2p homolog,is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins.

The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle.     

Characterization of a cDNA clone coding for a sea urchin histone H2A variant related to the H2A.F/Z histone protein in vertebrates.

A cDNA clone coding for a sea urchin histone H2A variant has been isolated. The coding region of the clone has been sequenced and the sequence found to be closely related to the H2A.F sequence in chickens. The nucleotide sequence of the sea urchin H2A.F/Z is 74% conserved when compared to chicken H2A.F and 51% conserved compared to sea urchin H2A early and 60% compared to sea urchin H2A late. The nucleotide-derived amino acid comparisons show that H2A.F/Z is 97% homologous with H2A.F in chickens and 57% and 56% homologous when compared to sea urchin H2A early and late respectively. There are between 3-6 copies of the H2A.F/Z sequence in the S. purpuratus genome. The H2A.F/Z gene sequence codes for the previously identified H2A.Z protein. All embryonic stages and adult tissues tested contain mRNA for H2A.F/Z. The mRNA appears in the poly A+ RNA fraction after chromatography over oligo dT cellulose.     

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.