The Many Functions Of Atpdependent Chromatin Remodelling Factors

Given that around 80% of nuclear DNA is packaged by nucleosomes, it is not surprising that ATP-dependent chromatin remodelling factors function in all processes involving DNA such as transcription, replication, recombination and repair (reviewed in Corona and Tamkun, 2004). They affect transcription by changing the accessibility of transcription factors to genes leading either to activation or repression. A compelling link with transcriptional repression has emerged with the discovery of the NURD complexes (containing the CHD-type ATPase Mi-2) which have both nucleosome remodelling and histone deacetylation activities (reviewed in Bowen et al., 2004). NoRC (for Nucleolar chromatin Remodeling Complex) containing the ISWI-homologue SNF2H is involved in the repression of Poll transcription through the recruitment of the SIN3/HDAC co-repressor to the ribosomal DNA promoter (Santoro and Grummt, 2005 and references therein). The role of remodellers in gene regulation impacts development. An illustration of this are the roles of two distinct ATP-dependent chromatin remodelling factors, ISWI and DOM, in controlling specific stem cell self renewal (Xi and Xie, 2005). They do this by regulating responses to peptide factor signalling in the stem cell microenvironment ('niche'). In Drosophila, ISWI was found to control Germline Stem Cell self-renewal and DOM was shown to be essential for Somatic Stem Cell self renewal. These findings suggest that different stem cell types depend on different chromatin remodelling factors to control their self-renewal, at least in part by regulating their gene expression response to the niche signals. Because these chromatin remodelling complexes are highly conserved, it is possible they may play a role in stem cell self-renewal in other organisms, including humans.

Remodelling factors play important regulatory roles to facilitate the many steps of eukaryotic DNA replication. (reviewed in Falbo and Shen, 2006). For example, the mammalian ISWI isoform SNF2H has been shown to be required for efficient DNA replication from a viral origin of replication and through heterochromatin (Collins et al., 2002; Zhou et al., 2005). SNF2H may have also a role in chromatin maturation and the maintenance of epigenetic patterns through replication by being targeted to replication sites in a complex with the Williams Syndrome Transcription Factor (WSTF) that in turn binds directly to replication factor PCNA (Poot et al., 2004). The remodelling event might serve to keep an open state of chromatin after the replication fork passes, thereby creating an opportunity for the epigenetic marks to be copied and transmitted to the next generation (Poot et al., 2005). Roles of various ATP-dependent nucleosome remodelling factors in DNA repair and recombination have also been identified (reviewed in Huang et al., 2005; Shaked et al., 2006). Finally, remodelling factors may also play an important regulatory and architectural role in the maintenance of higher order structure of chromatin (reviewed in Varga-Weisz and Becker, 2006; see also MacCallum et al., 2002).

Several links have emerged between remodelling factors and oncogenesis (reviewed in Cairns, 2001). Subunits of the mammalian SWI/SNF complex possess intrinsic tumour suppressor function or are required for the activity of other tumour suppressor genes. SNF5 (INI1), a core subunit of SWI/SNF is inactivated in malignant rhabdoid tumours, a highly aggressive cancer of early childhood (reviewed in Roberts and Orkin, 2004). Specific mutations in the mammalian SWI2/SNF2 homologue BRG1 have been identified in pancreatic, breast, lung and prostrate cancer cell lines (Wong et al., 2000). SWI/SNF also directly interacts with tumour suppressors and proto-oncogenes such as RB, BRCA1, c-Myc and MLL (Bochar et al., 2000; Cheng et al., 1999; Dunaief et al., 1994; Nie et al., 2003). However, the mechanisms by which the remodelling complex contributes to tumour suppression are yet to be fully understood. Mutations in other ATP-dependent chromatin remodelling factors have been linked to disease, such as in ATR-X, causing X-linked mental retardation syndromes. The phenotypes include facial dysmorphism, urogenital defects, and alpha-thalassaemia (resulting from reduced alpha-globin expression (Xue et al., 2003 and references therein). The SWI/SNF-related SMARCAL1 is mutated in Schimke immuno-osseous dysplasia, a pleiotropic disorder with the diagnostic features of T-cell immunodeficiency, spondyloepi-physeal dysplasia, renal failure, hypothyroidism, episodic cerebral ischemia, and bone-marrow failure (Boerkoel et al., 2002).

5. INSIGHTS FROM STRUCTURES OF SWI2/SNF2-FAMILY MEMBERS

The ATPase domains of SWI/SNF-family members exhibit a conserved architecture with an N-terminal domain containing conserved motifs I, Ia, II and III, required for ATP-hydrolysis, and a C-terminal domain (motifs IV to VI) (Eisen et al., 1995). Functional analysis of these conserved sequence motifs have shown that whereas many of them play key roles in ATP binding and hydrolysis, certain residues within the conserved motif V are specifically required to couple ATP hydrolysis to chromatin remodelling activity: Deletion of eight amino acids in Motif V cripples the chromatin remodelling activity of SWI/SNF without altering its DNA-stimulated ATPase activity (Smith and Peterson, 2005). Therefore, this particular motif seems to be required specifically for coupling the energy from ATP-hydrolysis to the actual biomechanical force required for nucleosome remodelling. Remarkably, Motif V of the human SWI2/SNF2 homolog Brg1 has been shown to be a possible hot spot for mutational alterations associated with cancers (Medina et al., 2004).

Helicases catalyze the processive separation of duplex DNA into single strands. Despite sharing similarity to helicases, none of the chromatin remodelling factors, with the exception of the INO80 complex, have been shown to catalyze the separation of DNA strands (Shen et al., 2000). Instead, they can translocate on double-stranded (ds) DNA in an ATP-hydrolysis dependent manner and are characterized by their ability to generate superhelical torsional strain in DNA (Havas et al., 2000; Saha et al., 2002; Whitehouse et al., 2003). The crystal structure of Rad54, a member of the SWI/SNF family has been solved for both S. solfataricus and zebrafish which helps to understand the mechanism of the SWI/SNF ATPase domain in remodelling processes (Durr et al., 2005; Thoma et al., 2005). It reveals a striking similarity to the SF2 helicases suggesting that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on DNA. Moreover, the motifs for ATP and Mg2+ binding and interlobe-interaction are related to those found in other helicases. However, they contain two SWI2/SNF2-family specific insertions where other helicases have accessory domains and these are likely to play a central role in the remodelling mechanism. Studies on RecG and PcrA helicases have shown that translocation on DNA and strand separation are separable activities (reviewed in Caruthers and McKay, 2002; Singleton and Wigley, 2002). The translocation activity resides within the bi-lobal ATPase core whereas strand separation requires the wedge-like DNA binding accessory domains specific for the particular helicases. The lack of an equivalent wedge-like structure may explain the absence of helicase activity in SWI/SNF ATPases.

The similarities of DNA recognition by the Rad54 ATPase domain and helicases explain how the biochemical activities of SWI2/SNF2 enzymes are generated by a mechanism that is related to that of Dexx-box helicases (Durr et al., 2005). The Rad54 catalytic core consists of two domains, Domain 1 and 2, separated by a deep cleft. Prior to translocation, DNA is bound at Domain 1 at the high affinity DNA binding site. In the presence of ATP, Domain 2 would undergo a conformational change that would push on the upstream minor groove of DNA advancing the ds DNA in the active site of SWI2/SNF2 enzymes. Following advancement of DNA, ATP hydrolysis might relax the structure, allowing rebinding of Domain 1 to DNA, ADP to ATP exchange and rebinding of Domain 2 at a new translocated upstream DNA binding site. It is important to note that even a relatively moderate translocation of DNA by sliding along the minor groove would include a substantial rotation of DNA along the helical axis. Both translocation and rotation could be used by remodelling factors for force generation. Remodelling factors might bind to substrate DNA-protein complexes both by the catalytic domain and by additional substrate binding domains. The screw motion of the DNA at the catalytic domain could not only transport DNA but also generate torque that would lead to disruption of DNA-protein interfaces. The precise mechanism of remodelling is yet not understood completely. However, some models have been proposed recently which will be discussed below.

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