5.1 Introduction
The chromo-helicase/ATPase- DNA-binding protein 1 is named after three signature motifs and is part of a family of proteins containing these domains called the CHD family (Woodage et a l 1997). The CHD family is part a larger family of SNF2 related helicase/ATPases which contains proteins that have functions in transcriptional regulation, recombination and DNA repair (Eisen et a l 1995). CEQDl was first cloned in mouse, but there are now several CHD related proteins in the databases in organisms ranging from yeast to humans. The D rosophila CH D l was cloned by virtue of its similarity to the mouse protein, but has not been extensively studied (Stokes et a l 1996). The yeast homologue of C H D l, C hdlp, has been found to be an ATP-dependent chromatin modifying factor (Tran et a l 2000) and the closest homologue of CHDl in
Drosophila, dMi-2 (or CHD4) has also been shown to be an ATPase that promotes
nucleosome mobilisation (Brehm et a l 2000). It is therefore likely that Drosophila CHDl is a chromatin remodelling factor which could be involved in regulating transcription. A possible interaction with the transcription factor Mirror is therefore very intriguing. Many transcription factors regulate gene expression by recruiting chromatin modifying proteins
Chapter 5
and it would be very interesting to find out CH D l is a co-factor for Mirror in transcriptional regulation.
5.1.1 Introduction to Chromo domains and ATPase/Helicase domains
Members of the CHD family of proteins are unique in that they contain both a chromo (chromatin organization modifier) domain and an ATPase/helicase domain. Chromo domains were first recognized as a 50 amino acid motif in D ro so p h ila
heterochromatin protein 1 (H Pl) and the Polycomb protein (Paro and Hogness 1991). Chromo domains have since been identified in a variety of proteins from fungi and plants to mammals that all seem to be connected to chromatin structure and/or function (Eissenberg 2001). The structure of the chromo domain fold consists of three P-strands and an a-helix and is related to a DNA binding fold found in the archeal chromatin proteins (Ball et al. 1997). The Chromo domain has been shown to function in protein- protein as well as protein-RNA and protein DNA interactions all of which are related to a general function of targeting chromo domain proteins to specific regions or positions in the nucleus. Protein-protein interactions mediated by chromo domains include interactions with specifically modified histones ie the H Pl chromo domain which interacts with the H3 tail methylated at lysine 9. Chromo domain interactions are also important within protein complexes, such as the Polycomb chromo domains which targets the PcG silencing complex to chromatin (Eissenberg 2001). Protein-RNA interactions are mediated by the MOF and MSL2 chromo domains, two proteins which are part of the dosage compensation complex in Drosophila (Akhtar et al. 2000). The chromo domain of
Drosophila dMi-2, which is most closely related to the CH D l chromo domain (see
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The ATPase domain of CHDl is part of the SNF2 subfamily of ATPase/helicases (Eisen et al. 1995). Proteins containing these domains are usually found in large protein complexes and function to increase the accessibility of nucleosomal DNA in an ATP- dependent manner (Narlikar et al. 2002). The SNF2 type of ATPase/helicase domain is part of the DEAD/H superfamily of helicases (Bork and Koonin 1993), but none of the SNF2 related ATPase proteins have been found to possess helicase activity. The domain and is made up of seven conserved “helicase” motifs (I-VII) which are highlighted in the CHDl sequence in figure 5.3. There are three subclasses of ATP-dependent chromatin remodelling complexes which are classified according to the ATPase subunit they contain. Figure 5.1 gives an overview of the complexes that have been studied in yeast, mammals
and Drosophila.
The SWI/SNF family of complexes contain members of the SWI/SNF subfamily of ATPases which include SWI2/SNF2 and STHl from yeast, BRG l and BRM from mammals and Brahma from Drosophila (Tsukiyama 2002). Proteins in this subclass contain a bromodomain, a motif which interacts with acetylated hi stone tails, in addition to the ATPase domain. The SWI/SNF ATPases can both change the translational position of nucleosomes on DNA and cause conformational changes that affect the DNA-protein contacts within the nucleosome in an ATP dependent manner (Narlikar et al. 2002). The in vivo functions of the different SWI/SNF complexes varies although they are all involved in regulating transcription and are mainly thought to cause activation of transcription. SWI/SNF complexes have been shown to have specific effects on subsets of genes in yeast, mammals and Drosophila, but have also been suggested to have more general roles in maintaining chromatin structure (Tsukiyama 2002).
The ISWI family of complexes contain ISW l and 2 in yeast, hSNF2 in humans and the ISWI ATPase in Drosophila. These ATPase subunits contain a SANT domain which is found in many proteins involved in transcriptional control and is predicted to be a DNA binding motif (Aasland et al. 1996). As opposed to the conformational changes
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observed with SWI/SNF, the ISWI ATPases are thought to use sliding of nucleosomes on DNA as their main remodelling activity (Narlikar et al. 2002). In vivo, the ISWI complexes are mainly thought to be involved in the negative regulation of transcription and creating an inaccessible chromatin structure (Tsukiyama 2002).
The CHD or Mi-2 family of complexes is the least well studied subclass of chromatin remodellers. In yeast, the CHD proteins Chdpl and hrpi have not been isolated as part of a complex to date. In mammals the NuRD complex which contains the Mi- 2/CHD3/4 ATPases is the only well characterised complex in this subclass (Xue et al.
1998), (Tong et al. 1998), although a similar complex is thought to exist in Drosophila
(Brehm et al. 2000). The known in vitro and in vivo functions of the CHD family are discussed below.
Figure 5.1. ATP-dependent Chromatin remodelling factors SWI2/SFN2 family ATPase «tomdoL. domain ISWI family ATPase SANT \M B b S i
Rsc
Yeast j - X - v S N | . f . i ' l ' S THI ISWI LSW2 ( p«-«* - ' _ hSWI/SNF RSF liACF/ WCRF hCHRAC Human Drosophila dSWT/SNF H r m ! B \ I * S . V - I I . M ' W I 2 BAP NLIRF A( RS JPI Chapter 5 CHD/Mi-2 family PHC finqers .. ■ . :b e chromo domain NuKD 'V—- — C'HRAC A t K A C P f A, RFJO, ( A C H _ )Figure 5.1 Taken from Narlikar et al, 2002. The SWI (switchingVSNF (sucrose non- fermenting) family of complexes include yeast SWI/SFN and RSC (remodels structure of chromatin) which contains the STHl (Snf two homologue) ATPase. Drosophila SWI/SFN complexes contain BRM (Brahma) and human complexes contain human BRM or BRGl (Brm/SWI2-related gene 1). The ISWI (Imitation Switch) family of complexes comprises ISWI and 2 in yeast. In Drosophila, the ISWI ATPase is found in the NURF (nucleosome remodelling factor) complex, CHRAC (chromatin accessibility complex) and ACF (ATP- dependent chromatin assembly and remodelling factor). The human ISWI homologue, SNF2h is found in human CHRAC and ACF as well as RSF (remodelling and spacing factor). Some of the proteins in the CHD family of ATPases are found in the NuRD (nucelosome remodelling and histone deacetylation) complex.
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5.1.2 The CHD family o f proteins
The CHD family of proteins can be subdivided into two classes according to the domains they contain addition to the chromo and ATPase domains (Woodage et al. 1997). CH Dl and CHD2 contain a C-terminal DNA binding domain whereas CHD3 and CHD4, also called M i-2a and Mi-2P, contain N-terminal PHD zinc fingers. A comparison between some of the different CHD family members as well as other SNF2-like ATPases from Drosophila is presented in figure 5.2. The CHD3/4 subclass has been much more extensively studied than the CHD 1/2 subclass. The CHDl proteins from yeast, mammals
and Drosophila will be discussed in detail in section 5.1.3.
The Mi-2 proteins were originally identified as autoantigens in the human disease dermatomyositis (Seelig et al. 1996). The first clues to their function came from the purification of large complexes containing histone deacetylases. Hyperacetylation of histone tail lysines is linked to active transcription whereas hypoacetylation is linked to a repressed chromatin state (Narlikar et al. 2002). Like the ATP-dependent chromatin remodellers, histone acetyltransferases (HATs) and histone deacetylases (HDACs), also reside in large multi-subunit complexes. The HAT and HD AC complexes can be recruited to specific chromatin sites by transcription factors to activate or repress transcription. When antibodies to human HDACl and HDAC2 were used to purify a multi-subunit complex it was found to contain both CHD3 and CHD4 (Tong et al. 1998). In parallel, antibodies raised against CHD4 were used to purify a complex containing HDACl and 2 (Xue et al. 1998). One variant of this complex called NuRD or NRD (nucleosome remodelling and deacetylation) can be seen in figure 5.1. CHD3/4 and HDACs have also been found as part of other complexes, such as the PYR complex which share some subunits with the NuRD complex (O'Neill et al. 2000). Amongst the other components of the NuRD complex, the presence of the methylated-DNA-binding protein MDB3 is of special interest. Méthylation of DNA is also linked to transcriptional repression and
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suggests that these complexes may be recruited to methylated DNA to create a repressed chromatin state (Tsukiyama 2002).
The association of CHD3 and CHD4 with a histone deacetylase suggests that they are involved in transcriptional repression. Indeed, antibodies against the NuRD complex partially relieves repression by thyroid hormone receptor in Xenopus oocytes (Xue et al.
1998). In mammalian cells a CHD3/4 complex interacts with the zinc-finger repressor Ikaros and may be involved T cell differentiation (Tsukiyama 2002). A CHD3/4 - Ikaros complex may also be involved in repression of y-globin during y- to (3-globin switching (O'Neill et al. 2000). In addition to specific targeting by transcription factors, CHD3/4 complexes may also exhibit a more constitutive association with chromatin which causes chromatin acétylation in a non-targeted fashion (Li et al. 2002).
The Drosophila Mi-2 protein is the closest homologue of Drosophila CHDl and
was first identified in a yeast two-hybrid screen for proteins that interact with the transcription factor Hunchback (Kehle et al. 1998). The Hunchback protein is involved in the initial repression of HOX genes during embryonic development, but the maintenance of repression requires the Polycomb-group (PcG) proteins. Drosophila Mi-2 (or CHD4) was shown to interact with Hunchback in vitro, and to study a possible in vivo interaction, mutant alleles of dMi-2 were generated. Homozygous dMi-2 mutants survive until larval stages due to maternal contribution of RNA and/or proteins, but germ-line and somatic clones are cell lethal. Mutants in dMi-2 were however shown to interact genetically with
hunchback causing derepression of Ubx. Interestingly, dMi-2 was also shown to interact with PcG mutations suggesting that Mi-2 may serve as a link between the initial repression of Hox genes during embryonic development and the long term maintenance by PcG proteins. Drosophila Mi-2 has also been shown to interact physically and genetically with another transcription factor, Tramtrack69 (Murawsky et al. 2001). Like Hunchback, Tramtrack69 is a zinc-finger transcription factor and has been shown to regulate nervous
Chapter 5
system development. Tramtrack69 and dMi-2 have been shown to associate in vivo by im m unoprécipitation and they also show overlapping localisation on polytene chromosomes.
Drosophila Mi-2, like its vertebrate homologues, is found in a large complex in vivo. Gel filtration experiments indicate that the dMi-2 complex is around 1.2 MDa (Murawsky et al. 2001). Using a dMi-2 antibody, the Drosophila RPD3 HDAC could be immunoprecipitated as part of the dMi-2 complex. The complex was shown to have both ATPase and HDAC activity [Brehm, 2000 #22. A recent report investigated the role of the chromo domains in dMi-2 function and found that deletion of these motifs leads to loss of nucleosome binding and remodelling activities (Bouazoune et a l 2002). It was found that the chromo domains did not interact with histone tails like the H Pl chromo domain, but instead displayed DNA binding activity. This is a novel activity of chromo domains and it remains to be seen if other CHD chromo domains share this function.
In addition to CHDl and dMi-2, there is a third protein in the Drosophila genome with extensive homology to this family. It has been named CHD3, but is as yet only a predicted protein and has not been studied. Another protein. Kismet, also contains two chromo domains and an ATPase domain which is highly homologous to the CHD ATPase domain. However, Kismet does not contain putative DNA binding domain and also contains a motif found only in the Brahma family of ATPases (Daubresse et al. 1999). The functions of Kismet will be discussed with other Drosophila ATPases below.
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Figure 5.2. Comparison of Drosophila C H Dl with other ATPases
DmCHDl (1889aa) ATPase DNA HsCHD2 (1739aa) MmCHDl (171 laa)
71%
"1j 47% r
50% 1 71% 1 I 48% 50% DmMi-2 (1982aa)n
48% 25% DmKismet _ _(5322aa)
UUL
QUI
4 5%
23%
DmBRM
(1638aa) 43% Jà.
DmISWI
(1027)
I
.42%]
Figure 5.2. Schematic representation of various ATPase containing proteins in the CHD family and in Drosophila. C= Chromo domain, ATPase = ATPase/helicase domain, DNA DNA binding domain, P= PHD fingers, B= Bromo domain, S = SANT domain.
Percentages represent the amino acids identify between the domain and the equivalent domain in Drosophila CHDl.