BIOLOGICAL OVERVIEW

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. Both Mi-2 and ISWI are nucleosome-stimulated ATPases and promote nucleosome mobilization. However, Mi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. The complex containing Mi-2 and the RPD3 histone deacetylase shares the nucleosome-stimulated ATPase and nucleosome mobilization properties of recombinant Mi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish Mi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines (Brehm, 2000).

Molecular machines that carry out the fundamental processes of DNA replication, recombination, repair and transcription need to recognize and bind to their DNA substrate. The packaging of DNA into chromatin, however, hinders their access to DNA. Chromatin remodelling factors facilitate DNA binding by creating a more dynamic chromatin structure. Two broad classes of remodelling activities have received much attention in recent years. The first class is comprised of ATP-dependent chromatin remodelling complexes containing ATPases related to SWI2/SNF2 or ISWI. These remodel interactions between histones and DNA in a poorly defined reaction requiring ATP hydrolysis. The second class consists of complexes that acetylate or deacetylate specific lysine residues in the N-terminal tails of histones. Acetylation is believed to lead to a more open chromatin structure by changing the nucleosome-nucleosome interactions involved in the folding of the nucleosomal fiber. Alternatively, acetylated lysine tails could serve as recognition sites for chromatin binding factors, which in turn elicit changes in chromatin structure (Brehm, 2000 and references therein).

Before discussing Drosophila Mi-2 in detail, there will be presented a short review of chromatin remodeling machines and Mi-2 containing complexes in particular. The view that ATP-dependent chromatin remodelling and histone acetylation/deacetylation are carried out by separate entities has recently been challenged. Complexes containing the ATPases Mi-2alpha and/or Mi-2ß as well as histone deacetylases HDAC1 and HDAC2 have been identified in human and frog. These complexes combine ATP-dependent chromatin remodelling and histone deacetylase (HDAC) activities, and have accordingly been named NuRD or NRD [nucleosome remodelling and deacetylation (Tong, 1998; Xue, 1998; Zhang, 1998). The Mi-2 subunits of these complexes belong to the CHD (chromo-helicase/ATPase-DNA binding) family of proteins (Woodage, 1997). Four CHD proteins have been identified in vertebrates: CHD1, CHD2, CHD3 (Mi-2alpha) and CHD4 (Mi-2ß). CHD proteins share two conserved chromo domains and one ATPase domain. In addition, CHD1 and CHD2 have a DNA-binding domain whereas Mi-2alpha and Mi-2ß contain two PHD fingers. The physiological function of CHD proteins is not clear. CHD1 has been localized to decompacted interphase chromosomes in mammalian cells and to regions of high transcriptional activity on Drosophila polytene chromosomes, implicating this family member in transcriptional control (Brehm, 2000 and references therein).

Histone deacetylation of nucleosomal substrates by Mi-2 complexes is stimulated by ATP hydrolysis, arguing that energy-dependent remodelling of the nucleosome is required for the deacetylases to gain access to histone tails. The Mi-2 complexes are believed to repress transcription through their remodelling and deacetylation activities in a targeted manner. Two ways of recruiting Mi-2 complexes to promoter regions have been proposed. (1) Targeting could be mediated via the interaction between the Mi-2 complex and DNA-bound transcriptional repressors. Indeed, the Mi-2 complex copurifies with the Ikaros repressor and functionally interacts with thyroid hormone receptor in vitro (Xue, 1998; Kim, 1999). Furthermore, the Drosophila homolog of Mi-2 physically and genetically interacts with the Hunchback repressor (Kehle, 1998). (2) The Mi-2 complex could be targeted to methylated DNA either directly via its MBD3 subunit or indirectly via association with the MBD2A-methylated DNA-binding protein (Wade, 1999; Zhang, 1999; Brehm, 2000 and references therein).

Several observations suggest that Mi-2 complexes are involved in cell cycle regulation and that their deregulation contributes to cancer. Mi-2 itself was originally identified as an autoimmune antigen in patients suffering from dermatomyositis. These patients suffer an increased risk of developing cancer. Furthermore, Mi-2 is targeted by the E7 oncoprotein of human papilloma virus 16 (Brehm, 1999). Finally, the Mi-2 complex subunit MTA2 is closely related to MTA1, which has been found to be overexpressed in cancer cells with a high potential for metastasis (Brehm, 2000 and references therein).

Mi-2 and other CHD proteins are members of the growing SNF2 superfamily of ATPases. The founding member of this family, yeast SWI2/SNF2, is a well characterized ATP-dependent chromatin remodelling enzyme. However, since the similarity between members of the Snf2 superfamily is restricted to the ATPase domain it is unclear whether all family members are nucleosome remodelling factors. SWI/SNF, Sth1 and ISWI complexes can facilitate factor binding to chromatin and activate transcription from a chromatin template in vitro. Furthermore, both SWI/SNF and ISWI complexes are able to promote movement of a nucleosome along DNA. Both types of ATPase also display a series of distinct biochemical properties. Whereas BRG1 and hBRM ATPase activities are stimulated to the same extent by nucleosomes and naked DNA, the ISWI ATPase is preferentially stimulated by nucleosomes. ATPase activity and nucleosome mobilization by ISWI depends on an intact histone H4 tail. In contrast, remodelling of nucleosomal arrays by SWI/SNF is variably affected by the removal of all four histone tails depending on the precise assay conditions. These findings show that SWI/SNF and ISWI complexes differ in the way they interact with the nucleosome to promote chromatin remodelling (Brehm, 2000 and references therein).

The two unrelated recombinant chromatin remodelling ATPases, Mi-2 and ISWI were compared in tests of nucleosome binding, nucleosome mobilization and ATPase assays. These studies suggest that Mi-2 and ISWI recognize different features of the nucleosome. The activity of the better characterized ATPases ISWI and SWI2/SNF2 depends, to a varying degree, on the presence of histone N-terminal domains. In order to monitor the effect of N-terminal tail sequences, nucleosomes were reconstituted from recombinant Xenopus histones lacking N-termini, and intact and tailless nucleosomes were compared for stimulation of the ATPase activities of recombinant ISWI and Mi-2. The amounts of ISWI and Mi-2 used in this experiment were chosen to give comparable activation by intact nucleosomes. The ATPase activity of ISWI is strongly stimulated by intact nucleosomes reconstituted from recombinant histones in agreement with previous findings (Corona, 1999). Nucleosomes lacking all four histone tails do not stimulate ISWI beyond the levels observed with naked DNA. No significant activation of the ISWI ATPase could be detected by tailless nucleosomes. In sharp contrast to ISWI, Mi-2 is equally well stimulated by intact and tailless nucleosomes, indicating that nucleosomal activation of the Mi-2 ATPase is histone tail independent (Brehm, 2000).

The observations that Mi-2 ATPase activity is stimulated by nucleosomes suggest that Mi-2 can physically associate with nucleosomes. To address this question biotinylated DNA was immobilized on streptavidin-coated paramagnetic beads, and nucleosomes were reconstituted on this DNA. To test for Mi-2 binding, the nucleosomes on beads were incubated with recombinant Mi-2 and then stringently washed to remove loosely bound proteins. Western analysis reveals that Mi-2 binds strongly to nucleosomal arrays assembled from either purified or recombinant histones. No binding to naked DNA is detected under these conditions. It is concluded that Mi-2 binds preferentially to nucleosomal arrays over naked DNA. Mi-2 can also associate with mononucleosomes to form a complex that is sufficiently stable to withstand electrophoresis through polyacrylamide gels (Brehm, 2000).

The abilities of recombinant Mi-2 and ISWI to bind two different mononucleosome species were tested in a bandshift assay. Binding to a mononucleosome (core particle) containing 146 bp of DNA was compared with binding to a nucleosome assembled on the 248 bp fragment. The primary difference between these substrates is that in the 146 bp nucleosome all DNA is entirely in contact with the histone octamer, whereas the 248 bp nucleosome contains ~100 bp of naked DNA extending from the octamer. Incubation with increasing amounts of Mi-2 results in the formation of a Mi-2-core particle complex or a complex of Mi-2 with the 248 bp nucleosome. In contrast, stable ISWI-core particle complexes could not be detected, even if higher amounts of ISWI were used. Incubation of increasing amounts of ISWI with the 248 bp nucleosome produces up to three ISWI-nucleosome complexes, suggesting that more than one ISWI molecule can simultaneously associate with this substrate (Brehm, 2000).

It is concluded that Mi-2 can stably associate with a mononucleosomal substrate lacking free DNA, in agreement with the finding that free DNA does not stimulate the Mi-2 ATPase. In marked contrast, ISWI only binds the nucleosome when free DNA is protruding from the histone octamer. These results strongly suggest that Mi-2 and ISWI recognize different features of the nucleosome (Brehm, 2000).

ATP hydrolysis by Mi-2 is believed to drive remodelling of chromatin by the Mi-2 complex. In order to characterize nucleosome remodelling directly, use was made of a recently established 'nucleosome sliding' assay. The CHRAC complex and recombinant ISWI can mobilize mononucleosomes to change their position on short DNA fragments (Längst, 1999). Nucleosome reconstitution on a 248 bp fragment derived from the mouse rDNA promoter results in the formation of two main species, which differ in the position of the nucleosome relative to the DNA ends and which can be separated by gel electrophoresis under non-denaturing conditions (Längst, 1999). Mononucleosomes positioned near the ends and at the center of the DNA fragment were used as substrates for mobilization by Mi-2 and ISWI. To avoid loss of labelled nucleosomes due to formation of stable enzyme-substrate complexes (as in the bandshift assay) the reaction was stopped by addition of excess unlabelled nucleosomes. Recombinant ISWI moves the centrally positioned nucleosome to the ends of the fragment but not vice versa (Längst, 1999). Remarkably, recombinant Mi-2 behaves in exactly the opposite way: Mi-2 fails to move the central nucleosome but mobilizes the end-positioned nucleosome. Mi-2-promoted nucleosome mobilization is ATP dependent. The effect of removal of individual histone tails on nucleosome mobilization by Mi-2 was also tested. Mi-2 is able to mobilize nucleosomes lacking individual histone N-termini. It is concluded that histone tails are dispensable for stimulation of the Mi-2 ATPase (Brehm, 2000).

The presence of the CHRAC subunits affects the direction of nucleosome sliding by ISWI. Like recombinant Mi-2, the CHRAC complex moves the mononucleosome from the end towards the center of the DNA fragment but fails to mobilize the nucleosome in the opposite direction. In order to investigate whether the presence of Mi-2-associated proteins affects the direction of Mi-2- mediated nucleosome mobilization, immunoprecipitated Mi-2 complexes were tested in the mobilization assay. The alphadMi-2-C antiserum precipitates an activity that mobilizes the end-positioned nucleosome but not the centrally positioned nucleosome. This mobilization activity is ATP dependent. Immunoprecipitated Mi-2 displays the same nucleosome mobilization activity as recombinant Mi-2. Taken together, this analysis of ATPase and nucleosome mobilization activity suggests that the properties of recombinant Mi-2 are fully preserved in a native Mi-2 complex (Brehm, 2000).

Therefore, this comparative analysis has uncovered a number of fundamental differences between these two remodelling enzymes. Both recombinant ATPases can be stimulated by nucleosomes assembled from recombinant histones. Whereas the Mi-2 ATPase does not respond to free DNA, the ISWI ATPase is stimulated by free DNA to some extent. Free DNA also appears to play a role in the interaction of ISWI with the nucleosome: ISWI binds to a nucleosome reconstituted on 248 bp of DNA, which displays free DNA, but not to the core particle consisting entirely of 146 bp of nucleosomal DNA under the stringent conditions of this bandshift assay. In striking contrast, Mi-2 interacts with both nucleosomes equally well, demonstrating that it does not require free DNA for interaction. Taken together, these observations suggest that ISWI, but not Mi-2, recognizes its chromatin substrate in part through an interaction with free DNA (Brehm, 2000).

The ability to promote the movement of a nucleosome along DNA has been demonstrated for the ISWI-containing NURF and CHRAC complexes, recombinant ISWI and the SWI/SNF complex (Hamiche, 1999; Längst, 1999; Whitehouse, 1999). It is conceivable that all remodelling ATPases mobilize nucleosomes in the same way, simply reflecting a common chromatin remodelling activity. Indeed, ISWI and Mi-2 share the intrinsic capacity to promote nucleosome mobilization in an ATP-dependent manner. Surprisingly, however, ISWI and Mi-2 move the nucleosome in opposite directions in these assays. ISWI moves nucleosomes prepositioned at the center to the ends of a DNA fragment but is not able to mobilize end-positioned nucleosomes (Längst, 1999). Mi-2 moves nucleosomes positioned at the end of the DNA fragment to a central position but fails to mobilize the central mononucleosome. Whether this observed directionality of nucleosome movement translates to a regulatory difference in a physiological chromatin context is unclear at present. Nevertheless, the observed difference in the direction of nucleosome movement within the constraints of this assay system suggests that ISWI and Mi-2 interact differently with nucleosomes and that they employ different mechanisms to mobilize them. At present the mechanisms of nucleosome mobilization are not understood. ISWI only moves the central nucleosome, which is flanked on either side by free DNA. Given that free DNA plays a role in substrate recognition by ISWI, it is speculated that ISWI needs to interact with two DNA segments extending from the nucleosome in order to mobilize the nucleosome. This scenario would predict that at least two ISWI molecules simultaneously interact with the central mononucleosome. In agreement with this hypothesis, the formation of multiple ISWI-nucleosome complexes is observed in the bandshift assay, most likely reflecting the binding of multiple ISWI molecules to the 248 bp nucleosome. In contrast, Mi-2 nucleosome binding and ATPase activity is not influenced by free DNA, pointing to a different mechanism of nucleosome mobilization. Interestingly, nucleosome mobilization by Mi-2 in this assay is similar to that of ISWI in the context of the CHRAC complex (Längst, 1999). Identification of the principle that modulates ISWI activity to change the direction of nucleosome mobilization should shed light on the mechanism of nucleosome mobilization by Mi-2 as well (Brehm, 2000).

Guschin (2000) has reported that the highly purified Xenopus Mi-2 complex can redistribute a nucleosome positioned near the end of a fragment derived from the Xenopus thyroid hormone receptor ßA gene towards more central positions in an ATP-dependent manner. It therefore appears that the directionality of Mi2-mediated nucleosome mobilization has been conserved across species (Brehm, 2000).

Nucleosomal stimulation of ISWI ATPase activity and nucleosome mobilization by ISWI are sensitive to the removal of specific histone tails. In contrast, Mi-2 ATPase activity and nucleosome mobilization by Mi-2 are unaffected, suggesting that these are tail-independent processes. The substrate requirements for ATPase and chromatin remodelling activity of Mi-2 reside within the globular domain of the core nucleosome. The differences in dependence on the histone N-termini reinforce the notion that ISWI and Mi-2 approach the nucleosome in fundamentally different ways (Brehm, 2000).

A corresponding comparison between SWI2/SNF2 or Sth1 ATPases and Mi-2 has not been carried out to date. It is not known in which way SWI2/SNF2 and Sth1 ATPases mobilize nucleosomes and whether or not histone tails are required for this activity. It is clear, however, that important differences exist between SWI2/SNF2 or Sth1 and Mi-2 activities. SWI2/SNF2 and Sth1 ATPases are both activated to the same extent by nucleosomes and free DNA. ISWI is preferentially stimulated by nucleosomes but also shows some activation by naked DNA (Corona, 1999). This contrasts sharply with the exclusive stimulation of recombinant Mi-2 and the Mi-2 complex by nucleosomes. In agreement with these results, ATPase activity of the Xenopus Mi-2 complex is stimulated by chicken erythrocyte mononucleosomes but not by salmon sperm DNA (Wade, 1998). In contrast, the ATPase activity of the human NuRD complex is strongly stimulated by naked DNA (Zhang, 1998). It is possible that this discrepancy is due to species-specific differences between different Mi-2 complexes or different experimental setups (Brehm, 2000 and references therein).

Based on this biochemical analysis it is proposed that Mi-2 defines a new class of nucleosome remodelling ATPases. It will be important to determine the molecular basis for the observed differences between Mi-2 and other ATPases in order to gain a better understanding of how chromatin remodelling machines work (Brehm, 2000).

Mi-2 resides in a large complex (~1.0 MDa) that also contains the HDAC RPD3. The estimated size of the complex suggests that it contains further subunits in addition to Mi-2 and RPD3. Most likely some of the additional Mi-2-associated subunits will correspond to proteins identified in vertebrate Mi-2 complexes. Indeed, several of these homologous sequences can be found in Drosophila EST databases (Wade, 1999). The vertebrate Mi-2 complexes have been implicated in mediating transcriptional repression by binding to methylated DNA. In contrast to its vertebrate counterparts, the Drosophila genome is not methylated to any appreciable extent. It will be fascinating to determine whether the Mi-2 complex contains subunits related to the methylated DNA-binding proteins present in the vertebrate complexes and what their role might be in the absence of DNA methylation. A major challenge now is to understand how the different enzymatic subunits of the Mi-2 complex cooperate to regulate chromatin (Brehm, 2000).

GENE STRUCTURE

cDNA clone length - 6475

Bases in 5' UTR - 131

Bases in 3' UTR - 395

PROTEIN STRUCTURE

Amino Acids - 1982

Structural Domains

Mi-2 contains five conserved sequence motifs that are also present in the two human Mi-2 proteins and in two Caenorhabditis elegans ORFs: two chromodomains, a DNA-stimulated adenosine triphosphatase (ATPase) domain, two PHD finger motifs, a truncated helix-turn-helix motif resembling the DNA-binding domain of c-myb, and a motif with similarity to the first two helices of an HMG domain (Kehle, 1998). \

Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. A panel of dMi-2 deletion mutants was tested in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to Drosophila HP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. These results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein-DNA interactions. Furthermore, this study identifies DNA binding as a novel chromodomain-associated activity (Bouazoune, 2002).

Fusion of the chromodomain region and the ATPase domain is sufficient for DNA and nucleosome binding, but not for nucleosome-stimulated ATPase and nucleosome mobilization activities. It follows that additional regions outside these domains make critical contributions to nucleosome remodelling. Conversion of the chromo domain-ATPase domain fusion (dMi-2 484-1271) to an active nucleosome remodeller can be achieved by addition of the remainder of the N-terminal region (NTR) or by addition of the C-terminal region (CTR). This implies that both NTR and CTR provide activities that are redundant in the assay. The CTR binds the repression domains of Hunchback and Tramtrack 69. The C-terminus of mammalian Mi2ß interacts with the KAP-1 co-repressor. The results suggest that CTR function is not restricted to transcription factor binding. Instead, it plays an active role in ATPase regulation: although deletion of the CTR does not affect nucleosome mobilization it makes the ATPase responsive to DNA. In this respect, the dMi-2 1-1271 mutant resembles ATPases of the SWI/SNF subgroup. This observation suggests that the CTR is directly involved in regulation of the ATPase domain: it is required to suppress activity in presence of the 'wrong' effector (DNA), when no remodelling substrate (nucleosome) is available. It is conceivable that the CTR might undergo a change in conformation following nucleosome recognition, which then allows the ATPase domain to function (Bouazoune, 2002).

date revised: 24 October 2000

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