The DNA-binding properties of the ARID-containing subunits of yeast and mammalian SWI/SNF complexes

SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that are highly conserved from yeast to human. From yeast to human the complexes contain a subunit with an ARID (A-T-rich interaction domain) DNA-binding domain. In yeast this subunit is SWI1 and in human there are two closely related alternative subunits, p270 and ARID1B. We describe here a comparison of the DNA-binding properties of the yeast and human SWI/SNF ARID-containing subunits. We have determined that SWI1 is an unusual member of the ARID family in both its ARID sequence and in the fact that its DNA-binding affinity is weaker than that of other ARID family members, including its human counterparts, p270 and ARID1B. Sequence analysis and substitution mutagenesis reveals that the weak DNA-binding affinity of the SWI1 ARID is an intrinsic feature of its sequence, arising from specific variations in the major groove interaction site. In addition, this work confirms the finding that p270 binds DNA without regard to sequence specificity, excluding the possibility that the intrinsic role of the ARID is to recruit SWI/SNF complexes to specific promoter sequences. These results emphasize that care must be taken when comparing yeast and higher eukaryotic SWI/SNF complexes in terms of DNA-binding mechanisms.     

ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development.

The ARID family of DNA binding proteins was first recognized approximately 5 years ago. The founding members, murine Bright and Drosophila dead ringer (Dri), were independently cloned on the basis of their ability to bind to AT-rich DNA sequences, although neither cDNA encoded a recognizable DNA binding domain. Mapping of the respective binding activities revealed a shared but previously unrecognized DNA binding domain, the consensus sequence of which extends across approximately 100 amino acids. This novel DNA binding domain was designated AT-rich interactive domain (ARID), based on the behavior of Bright and Dri. The consensus sequence occurs in 13 distinct human proteins and in proteins from all sequenced eukaryotic organisms. The majority of ARID-containing proteins were not cloned in the context of DNA binding activity, however, and their features as DNA binding proteins are only beginning to be investigated. The ARID region itself shows more diversity in structure and function than the highly conserved consensus sequence suggests. The basic structure appears to be a series of six alpha-helices separated by beta-strands, loops, or turns, but the structured region may extend to an additional helix at either or both ends of the basic six. It has also become apparent that the DNA binding activity of ARID-containing proteins is not necessarily sequence specific. What is consistent is the evidence that family members play vital roles in the regulation of development and/or tissue-specific gene expression. Inappropriate expression of ARID proteins is also increasingly implicated in human tumorigenesis. This review summarizes current knowledge about the structure and function of ARID family members, with a particular focus on the human proteins.     

染色质重塑因子ARID1A的肿瘤抑制作用

哺乳动物SWI/SNF复合物是一种ATP依赖的染色质重塑复合物, 在细胞增殖、分化、发育和肿瘤抑制过程中发挥着重要作用。ARID1A是一种SWI/SNF复合物亚基, 此外还是一种ARID家族成员, 具有非序列特异性DNA结合活性。ARID1A发挥着肿瘤抑制作用, 在多种肿瘤如卵巢癌、膀胱癌和胃癌等存在频繁基因突变。ARID1A可通过上调p21和下调E2F-反应基因表达而抑制细胞增殖。ARID1A与肿瘤抑制作用的发现对癌症发生的理解和癌症新治疗有重要裨益。文章介绍了ARID1A的基本特征、肿瘤发生的关联及生物学作用, 以期对ARID1A有一个全面理解。     

Solution structure of SWI1 AT-rich interaction domain from Saccharomyces cerevisiae and its nonspecific binding to DNA

SWI1 is a subunit of the SWI/SNF complex involved in chromatin remodeling. It contains an AT-rich interaction domain (ARID) which has the potential DNA binding activity. In this study, we determined the solution structure of the SWI1 ARID domain from Saccharomyces cerevisiae by nuclear magnetic resonance spectroscopy. Yeast SWI1 ARID domain is composed of seven alpha helices, six of which are conserved among the ARID family. In addition, the DNA-binding activity of the SWI1 ARID domain was confirmed by chemical shift perturbation assay. Similar to its human homolog, the yeast SWI1 ARID domain binds DNA nonspecifically.     

DNA-binding properties of ARID family proteins

The ARID (A–T Rich Interaction Domain) is a helix–turn–helix motif-based DNA-binding domain, conserved in all eukaryotes and diagnostic of a family that includes 15 distinct human proteins with important roles in development, tissue-specific gene expression and proliferation control. The 15 human ARID family proteins can be divided into seven subfamilies based on the degree of sequence identity between individual members. Most ARID family members have not been characterized with respect to their DNA-binding behavior, but it is already apparent that not all ARIDs conform to the pattern of binding AT-rich sequences. To understand better the divergent characteristics of the ARID proteins, we undertook a survey of DNA-binding properties across the entire ARID family. The results indicate that the majority of ARID subfamilies (i.e. five out of seven) bind DNA without obvious sequence preference. DNA-binding affinity also varies somewhat between subfamilies. Site-specific mutagenesis does not support suggestions made from structure analysis that specific amino acids in Loop 2 or Helix 5 are the main determinants of sequence specificity. Most probably, this is determined by multiple interacting differences across the entire ARID structure.     

抑癌基因ARID1A在肿瘤中的研究进展

染色质重塑复合物相关基因在癌症中频繁突变,这种现象逐渐引起研究者的重视。然而,染色质重塑活动如何引起癌症发生,对此机理研究甚少。ARID1A是SWl／SNF（BRG1相关因子）染色质重塑复合物中的一个亚基,具有DNA结合活性,可以与富含AT的DNA序列特异性结合。近来基因组测序发现,ARID1A在卵巢癌、肝癌、胃癌、乳腺癌等肿瘤中频繁发生突变,这些突变导致ARID1A在肿瘤中表达降低,表明ARID1A是个潜在的抑癌基因。该文将针对ARID1A在各种癌症中的缺失及失活机制、ARID1A的生物学功能和潜在抑癌机理以及与,临床预后之间关系等方面做一综述,以期为肿瘤诊断、治疗提供新思路。     

Nomenclature of the ARID family of DNA-binding proteins

The ARID is an ancient DNA-binding domain that is conserved throughout the evolution of higher eukaryotes. The ARID consensus sequence spans about 100 amino acid residues, and structural studies identify the major groove contact site as a modified helix-turn-helix motif. ARID-containing proteins exhibit a range of cellular functions, including participation in chromatin remodeling, and regulation of gene expression during cell growth, differentiation, and development. A subset of ARID family proteins binds DNA specifically at AT-rich sites; the remainder bind DNA nonspecifically. Orthologs to each of the seven distinct subfamilies of mammalian ARID-containing proteins are found in insect genomes, indicating the minimum age for the organization of these higher metazoan subfamilies. Many of these ancestral genes were duplicated and fixed over time to yield the 15 ARID-containing genes that are found in the human, mouse, and dog genomes. This paper describes a nomenclature, recommended by the Mouse Genomic Nomenclature Committee (MGNC) and accepted by the Human Genome Organization (HUGO) Gene Nomenclature Committee, for these mammalian ARID-containing genes that reflects this evolutionary history.     

Dynamics of the Mrf-2 DNA-binding domain free and in complex with DNA

Mrf-2 is a member of a new class of DNA-binding proteins known as the AT-rich interaction domain family or ARID. Chemical shift indices and characteristic NOE values indicate that the three-dimensional structure of the Mrf-2 ARID in complex with DNA is nearly identical to that of the free protein. The backbone dynamics of the Mrf-2 domain free and in complex with DNA have been characterized by (15)N NMR relaxation measurements and model-free analysis. Chemical shift perturbations and dynamic studies suggest that two flexible interhelical loops, the flexible C-terminal tail, and one alpha-helix are involved in DNA recognition, indicating the importance of protein dynamics in DNA binding. Some well-structured regions, in particular the putative DNA-contacting helix, in Mrf-2 show a decrease in the order parameters (S(2)) upon complex formation. The less well-structured loops and the unstructured C-terminus show reduced flexibility upon DNA binding. In addition, the model-free analysis indicates motions on the picosecond to nanosecond and micro- to millisecond time scales at the DNA-binding surface of the bound Mrf-2 ARID, suggesting a model where interactions between the protein and DNA are highly dynamic.     

Characterization of a human SWI2/SNF2 like protein hINO80: demonstration of catalytic and DNA binding activity

The proteins belonging to SWI2/SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. We have identified a human gene with a putative DNA binding domain, which belongs to the INO80 subfamily of SWI2/SNF2 proteins. Here we report the cloning, expression, and functional activity of the domains from hINO80 gene both in terms of the DNA dependent ATPase as well as DNA binding activity. A differential expression of the various domains within this gene is detected in human tissues while a ubiquitous expression is detected in mice. The intranuclear localization is demonstrated using antibodies directed against the DBINO domain of hINO80.     

The Drosophila retained/dead ringer gene and ARID gene family function during development

The recently discovered ARID family of proteins interact with DNA through a phylogenetically conserved sequence termed the A/T Interaction Domain (ARID). The retained/dead ringer (retn/dri) gene of Drosophila melanogaster is a founding member of the ARID gene family, and of the eARID subfamily. This subfamily exhibits an extended region of sequence similarity beyond the core ARID motif and a separate conserved domain termed the REKLES domain. retn/dri is involved in a range of developmental processes, including axis patterning and muscle development. The retn/dri ARID motif has been shown by in vitro studies to exhibit sequence-specific DNA binding activity. Here we demonstrate that the ARID domain is essential for the in vivo function of retn/dri during embryonic development by showing that a mutant form of RETN/DRI, deleted for part of the ARID domain and unable to bind DNA in vitro, cannot rescue the retn/dri mutant phenotype. In the presence of wild-type RETN/DRI this construct acts as a dominant negative, providing additional support for the proposal that RETN/DRI acts in a multiprotein complex. In contrast, we are yet to find an in vivo role for the REKLES domain, despite its clear evolutionary conservation. Finally, we have used germline clone analysis to reveal a requirement for retn/dri in the Drosophila preblastoderm syncytial mitoses.     

Skeletrophin, a novel RING molecule controlled by the chromatin remodeling complex, is downregulated in malignant melanoma

Recent experiments have revealed that aberrant functionality of the chromatin remodeling complex is related to tumorigenicity in various malignant tumors. Skeletrophin is an actin-binding cytoskeleton-related molecule, which is induced by the overexpression of truncated human SWI1 (SMARCF1). Human SWI1 is a sub-unit of the chromatin remodeling complex and binds chromatin through its ARID (AT-rich interactive domain). Truncated SWI1 lacks one of the two glucocorticoid-receptor binding domains and inhibits the intact human SWI1 in a dominant negative manner. Skeletrophin, was therefore identified as a candidate molecule for the indication of change to a malignant phenotype due to the aberrant function of the chromatin remodeling complex. Surprisingly, the skeletrophin gene is located in 1p36.32, where the putative tumor suppressor gene of cutaneous malignant melanoma has long been postulated to be on. Cutaneous malignant melanoma is a highly aggressive tumor. To overcome the clinical problem of malignant melanoma and highly invasive and metastatic activity, it is important to unravel the molecular mechanism responsible for melanoma progression. Recent studies including those from our laboratories have elucidated that skeletrophin is a novel RING-HC type ubiquitin ligase and that the ubiquitin ligase pathway mediated by skeletrophin acts to oppose melanoma cell invasion. Here, we summarize the characterization of skeletrophin, with emphasis on its biological activity, the disruption of which is linked with melanoma progression.