Abscisic acid and desiccation-dependent expression of a novel putative SNF5-type chromatin-remodeling gene in Pisum sativum.

Snf5-like proteins are components of multiprotein chromatin remodeling complexes involved in the ATP-dependent alteration of DNA-histone contacts. Mostly described in yeast and animals, the only plant SNF5-like gene characterized so far has been BSH from Arabidopsis thaliana (L.) Heynh. We report the cloning and characterization of expression of a SNF5-like gene from pea (Pisum sativum L. cv. Lincoln), which has been designated PsSNF5. Southern analysis showed a single copy of the gene in the pea genome. The cDNA contained a 723bp open reading frame encoding a 240 amino acid protein of 27.4kDa with a potential nuclear localization signal. PsSNF5 protein sequence closely resembled BSH, with which it showed an overall amino acid identity of 78.5%. Two-hybrid experiments showed that PsSNF5 is functionally interchangeable with Arabidopsis BSH in the interactions with other components of the remodeling complex. Phylogenetic analysis demonstrated that PsSNF5 clustered with translated expressed sequence tags from other Leguminosae, hypothetically coding for new Snf5-like proteins. RT-PCR expression analysis demonstrated that the PsSNF5 gene is constitutively expressed in all the tissues examined, with minor differences in expression level in different tissues. Nevertheless, expression analysis revealed that PsSNF5 was up-regulated in the last stages of embryo development, when water content decreases. Moreover, abscisic acid and drought stress induced PsSNF5 accumulation in germinating embryos and vegetative tissues, suggesting that chromatin remodeling induced by PsSNF5-containing complexes might contribute to the response to that phytohormone.     

SWI3 subunits of putative SWI/SNF chromatin-remodeling complexes play distinct roles during Arabidopsis development

SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes mediate ATP-dependent alterations of DNA-histone contacts. The minimal functional core of conserved SWI/SNF complexes consists of a SWI2/SNF2 ATPase, SNF5, SWP73, and a pair of SWI3 subunits. Because of early duplication of the SWI3 gene family in plants, Arabidopsis thaliana encodes four SWI3-like proteins that show remarkable functional diversification. Whereas ATSWI3A and ATSWI3B form homodimers and heterodimers and interact with BSH/SNF5, ATSWI3C, and the flowering regulator FCA, ATSWI3D can only bind ATSWI3B in yeast two-hybrid assays. Mutations of ATSWI3A and ATSWI3B arrest embryo development at the globular stage. By a possible imprinting effect, the atswi3b mutations result in death for approximately half of both macrospores and microspores. Mutations in ATSWI3C cause semidwarf stature, inhibition of root elongation, leaf curling, aberrant stamen development, and reduced fertility. Plants carrying atswi3d mutations display severe dwarfism, alterations in the number and development of flower organs, and complete male and female sterility. These data indicate that, by possible contribution to the combinatorial assembly of different SWI/SNF complexes, the ATSWI3 proteins perform nonredundant regulatory functions that affect embryogenesis and both the vegetative and reproductive phases of plant development.     

CHB2, a member of the SWI3 gene family,is a global regulator in Arabidopsis

The SWI/SNF complex is an ATP-dependent chromatin remodeling complex that plays an important role in the regulation of eukaryotic gene expression. Very little is known about the function of SWI/SNF complex in plants compared with animals and yeast. SWI3 is one of the core components of the SWI/SNF chromatin remodeling complexes in yeast. We have identified a putative SWI3-like cDNA clone, CHB2 (AtSWI3B), from Arabidopsis thaliana by screening the expressed sequence tag database. CHB2 encodes a putative protein of 469 amino acids and shares 23% amino acid sequence identity and 64% similarity with the yeast SWI3. The Arabidopsis genome contains four SWI3-like genes, namely CHB1 (AtSWI3A), CHB2 (AtSWI3B), CHB3 (AtSWI3C) and CHB4 (AtSWI3D). The expression of CHB2, CHB3 and CHB4 mRNA was detected in all tissues analyzed by RT-PCR. The expression of CHB1 mRNA, however, could not be detected in the siliques, suggesting that there is differential expression among CHB genes in different Arabidopsis tissues. To investigate the role of CHB2 in plants, Arabidopsis plants were transformed with a gene construct comprising a CHB2 cDNA in the antisense orientation driven by the CaMV 35S promoter. Repression of CHB2 expression resulted in pleiotropic developmental abnormalities including abnormal seedling and leaf phenotypes, dwarfism, delayed flowering and no apical dominance, suggesting a global role for CHB2 in the regulation of gene expression. Our results indicate that CHB2 plays an essential role in plant growth and development.     

Complementation analyses suggest species-specific functions of the SNF5 homology domain

Inactivation on both alleles of the hSNF5/INI1 tumor suppressor gene which encodes a subunit of the human SWI/SNF chromatin remodelling complex occurs in most malignant rhabdoid tumors. No paralog of hSNF5/INI1 is identified in the human genome. In contrast, it has two homologs in the yeast Saccharomyces cerevisiae, SNF5 and SFH1 which encode core components of the ySWI/SNF and RSC complexes, respectively. The homology mainly concerns an approximately 200 amino acid region termed the SNF5 homology domain. We have tested the ability of the hSNF5/INI1-wild type gene product and of chimerical constructs in which the yeast SNF5 domains were replaced by that of the human protein, to complement yeast snf5 and sfh1 phenotypes. Neither growth deficiencies on different carbon sources of snf5 yeasts nor the lethality of the sfh1 phenotype could be rescued. This strongly suggests that the SNF5 homology domain presents species-specific functions.     

A nucleosome interaction module is required for normal function of Arabidopsis thaliana BRAHMA

The BRAHMA (BRM) gene encodes the SNF2-type ATPase of the putative Arabidopsis thaliana SWI/SNF chromatin remodelling complex. This family of ATPases is characterized by the presence of a conserved catalytic domain and an arrangement of auxiliary domains, whose functions in the remodelling activity remains unclear. Here, we characterize, at the molecular and functional level, the carboxy-terminal part of Arabidopsis BRM. We have found three DNA-binding regions that bind various free DNA and nucleosomal probes with different specificity. One of these regions contains an AT-hook motif. The carboxy terminus also contains a bromodomain able to bind histones H3 and H4. We propose that this array of domains constitute a nucleosome interaction module that helps BRM to interact with its substrate. We also characterize an Arabidopsis mutant that expresses a BRM protein lacking the last 454 amino acid residues (BRM-DeltaC), encompassing the bromodomain and two of the three DNA-binding activities identified. This mutant displays an intermediate phenotype between those of the wild-type and a null allele mutant, suggesting that the nucleosome interaction module is required for the normal function of BRM but it is not essential for the remodelling activity of BRM-containing SWI/SNF complexes.