Progression into the First Meiotic Division Is Sensitive to Histone H2a-H2b Dimer Concentration in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae contains two genes for histone H2A and two for histone H2B located in two divergently transcribed gene pairs: HTA1-HTB1 and HTA2-HTB2. Diploid strains lacking HTA1-HTB1 (hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2) grow vegetatively, but will not sporulate. This sporulation phenotype results from a partial depletion of H2A-H2B dimers. Since the expression patterns of HTA1-HTB1 and HTA2-HTB2 are similar in mitosis and meiosis, the sporulation pathway is therefore more sensitive than the mitotic cycle to depletion of H2A-H2B dimers. After completing premeiotic DNA replication, commitment to meiotic recombination, and chiasma resolution, the hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2 mutant arrests before the first meiotic division. The arrest is not due to any obvious disruptions in spindle pole bodies or microtubules. The meiotic block is not bypassed in backgrounds homozygous for spo13, rad50Δ, or rad9Δ mutations, but is bypassed in the presence of hydroxyurea, a drug known to inhibit DNA chain elongation. We hypothesize that the deposition of H2A-H2B dimers in the mutant is unable to keep pace with the replication fork, thereby leading to a disruption in chromosome structure that interferes with the meiotic divisions.     

TGF-β Targets the Wnt Pathway Components,APC and β-catenin,as Mv1Lu Cells Undergo Cell Cycle Arrest

Mammalian cells exhibit complex cellular responses to DNA damage, including cell cycle arrest, DNA repair and apoptosis. Defects in any one of these responses can result in carcinogenesis. Absence of the chromatin remodeling complex Swi/Snf is found in many instances of cancer, and we have investigated its role in the UV damage response. The human carcinoma cell line SW13 is deficient in Swi/Snf and is very sensitive to UV radiation. In contrast, SW13 cells with ectopic Brg1 expression regain active Swi/Snf and become significantly more resistant to UV radiation. Sensitivity to UV light correlates well with dramatic UV induced apoptosis in SW13 cells, but not in SW13 cells expressing Brg1. We show that SW13 cells synchronized at the G1/S border progress into S phase after UV irradiation, and this checkpoint deficiency is corrected after Brg1 expression is restored. Interestingly, Brg1 expression in SW13 cells restores expression of two DNA damage responsive genes, Gadd45a and p21. Furthermore, Gadd45a induction and p21 degradation were observed in the Brg1-expressing SW13 cells after UV irradiation. Our findings demonstrate that Swi/Snf protects cells against deleterious consequences of UV induced DNA damage. These results also indicate that Swi/Snf may play a role in replication checkpoint activation after UV damage via regulation of the two PCNA-binding proteins Gadd45a and p21.     

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.     

The Swi2/Snf2 bromodomain is required for the displacement of SAGA and the octamer transfer of SAGA-acetylated nucleosomes

The SWI/SNF and SAGA chromatin-modifying complexes contain bromodomains that help anchor these complexes to acetylated promoter nucleosomes. To study the importance of bromodomains in these complexes, we have compared the chromatin-remodeling and octamer-transfer activity of the SWI/SNF complex to a mutant complex that lacks the Swi2/Snf2 bromodomain. Here we show that the SWI/SNF complex can remodel or transfer SAGA-acetylated nucleosomes more efficiently than the Swi2/Snf2 bromodomain-deleted complex. These results demonstrate that the Swi2/Snf2 bromodomain is important for the remodeling as well as for the octamer-transfer activity of the complex on H3-acetylated nucleosomes. Moreover, we show that, although the wild-type SWI/SNF complex displaces SAGA that is bound to acetylated nucleosomes, the bromodomain mutant SWI/SNF complex is less efficient in SAGA displacement. Thus, the Swi2/Snf2 bromodomain is required for the full functional activity of SWI/SNF on acetylated nucleosomes and is important for the displacement of SAGA from acetylated promoter nucleosomes.     

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.