A recent discovery by us has helped identify a common denominator the defines the structural basis for nucleosomal repositioning by the ISWI clade of SWI2/SNF2 ATPases.
One feature that sets eukaryotes apart from other forms of life are the multiple essential SWI2/SNF2 ATPases that are at the center of several functionally distinct chromatin remodeling complexes. Our earlier studies had suggested that the SWI2/SNF2 ATPases were probably introduced to eukaryotes from a restriction-modification system of bacterial provenance, wherein it probably facilitated the access of target sites by restriction/modification enzymes (click here to read). We also established that a spectacular radiation of SWI2/SNF2 ATPases, which happened in the period between the first eukaryotic common ancestor and the last eukaryotic common ancestor, spawned the clades of most major chromatin remodeling SWI2/SNF2 ATPases (click here to read).
In functional terms the characterized chromatin remodeling SWI2/SNF2 ATPases can be divided into three broad classes: 1) Those utilizing actin-like proteins. This class might be further divided into those which associate with the Reptin/pontin AAA+ ATPases, i.e. the INO80-like class and those which associated with SWIRM domain containing subunits, i.e. the Brahma-like class. 2) The CHD/MI-2 like remodelers. 3) ISWI remodelers. All these classes can be traced to the last eukaryotic common ancestor. Of course beyond these there are the Rad54-like, Rad5-like and Strawberry notch like versions which are much less understood (see this for a detailed classification of the SWI2/SNF2 ATPases). Of these the Brahma-like remodelers may slide or eject nucleosomes from chromatin. The Ino80-like remodelers include versions that facilitate exchange of canonical nucleosomes with those containing H2A.Z, promoting transcriptional activation by facilitating transcription start site exposure. The CHD/MI-2 like remodelers also tend to slide or eject nucleosomes in both repressive and activating contexts. The ISWI-like remodelers are unique in regulating nucleosome spacing – they might either optimize (e.g. the ACF and CHRAC) it to facilitate repression or randomize it to facilitate transcriptional activation and are the focus of this post.
Several effects have been attributed to the ISWI-like complexes: In Drosophila the loss of dACF1 reduces nucleosome spacing periodicity and shortens the length of DNA per nucleosome. Loss of ISWI in Drosophila results in major decondensation of the male X chromosome and to some degree also the polytene chromosomes. The WICH complex, which combines an ISWI ATPase with the WAC domain tyrosine kinase containing WSTF protein, phosphorylates tyrosine 142 of H2A.X in course of nucleosome repositioning during DNA repair. In vertebrates several distinct ISWI-like complexes have been identified: 1) ACF; 2) CHRAC; 3) WICH; 4) NoRC; 5) WCRF; 6) CECR2-embryonic stem cell/germline; 7) CECR2-somatic cell; 8) NURF. Of these the first six have SNF2H and CECR2-stem cell/germline as the ISWI ATPase, whereas NURF and CECR2-somatic cell have SNF2L as their ATPase subunit. These complexes have been shown to have biological roles by mediating different nucleosomal repositioning events. Prior experiments have demonstrated that their accessory subunits have a role in sensing linker DNA and thereby possibly regulating nucleosomal spacing (Click here to read). However, it remained unknown as to how exactly this was achieved.
It was in this context that we were able to use sequence analysis and comparisons with known structures to develop a unified mechanism (Click here to access the paper). First, using sequence profile searches we were able unify all the large accessory subunits of ISWI ATPases across eukaryotes, such as hACF1, WSTF, RSF1, TIP5, WCRF180, BPTF, yeast Itc1, Ioc3 and Esc8, and the plant HB1 and MBD9 as having a common conserved module. This module is largely alpha helical and is characterized four conserved motifs. The first of these motifs maps to the previously identified DDT motif (however, previously not known from Ioc3); the remaining three motifs are termed the WHIM motifs 1 to 3. Recently, a remarkable structural study by the Richmond group revealed that Ioc3 interacts with the C-terminus the ISWI ATPases, which are characterized by a HAND, SANT and SLIDE domain. These interact with nucleosomal linker DNA and Ioc3. Ioc3 in turn also interacts with nucleosomal linker DNA and together with the C-terminal region of the ISWI protein constitutes a protein ruler that measure out the spacing between two adjacent nucleosomes in a dinucleosome (Click here to read). What our sequence, and structure based unification did was to generalize the findings developed from Ioc3 across all large accessory subunits of ISWI ATPases. As a result we were able show that the DDT and the WHIM1 and WHIM2 motifs tightly pack with each other to form a binding pocket for the trihelical tip of the SLIDE domain in the ISWI ATPase. Based on this mapping, the highly conserved basic residue in WHIM1 is identified as a key feature involved in packing with the DDT motif, and the acidic residue from the GxD signature of WHIM2 emerges as a major determinant of the interaction between the ISWI and its WHIM motif partners. WHIM3 on the other hand, along with the N-terminal portion of WHIM2, constitutes the inter-nucleosomal linker DNA binding site which contacts it in the major groove. This is the major recognition unit for the outer or the external linker DNA element of the dinucleosome. The helix-turn-helix SANT domain from the ISWI ATPase makes a similar DNA contact with the inner linker DNA element in the dinucleosome. Thus, the principle of the protein ruler is a common feature of all ISWI large accessory subunits that is determined by the DDT and WHIM motifs.
Second, most of these proteins have multiple domains for the recognition of histone H3 N-terminal peptides (PHD finger), acetylated histone peptides (bromodomains), monoubiquitinated peptides (the “little finger” type Ub-binding Zn-ribbon), phosphorylated peptides (SJA/FYR) and methylated peptides (AGENET, BMB/PWWP and AUX-RF, a novel Chromo-like domain). Additionally, others like HB1 and MBD9 in plants, BPTF, BAZ2A/B, CECR2 in animals, and previously uncharacterized proteins in chlorophytes and stramenopiles contain DNA-binding domains such as the HARE-HTH, histone H1, CENB-HTH, TAM(MBD), homeo, HMG, BRIGHT, CXXC and AT-hooks. Of these the TAM(MBD) domain in the plant MBD9 proteins is predicted to specifically bind methylated CpG dinucleotides, whereas that in the animal BAZ2 proteins is unlikely to have specific methylated CpG recognition capabilities. The CXXC domain also recognizes the CpG sequence, though most versions prefer unmethylated targets. We have also proposed that the HARE-HTH has a possible role for in discriminating modified DNA. Thus, it appears that a common theme in the WHIM motif proteins is their coupling of measuring out of inter-nucleosomal distant with diverse domains involved in discriminating or catalyzing epigenetic modifications of histones or recognition of specific DNA features such as inter-nucleosomal linker regions and distorted DNA (e.g., histone H1, HMG, BRIGHT domains and AT-hooks) or discrimination of modified DNA marks (CXXC, TAM/MBD and HARE-HTH). One group of WHIM motif proteins from certain chlorophyte, rhodophyte and stramenopile algae combine the WHIM motifs with a RFD module, which is found at the N-termini of the DNMT1 methyltransferase. The RFD module consists of a circularly permuted version of the Sm domain fused to a HTH domain and has been demonstrated to be a key player in heterochromatinization by recruiting repressive proteins such as HDAC2.This suggests that these WHIM motif proteins might couple ISWI-dependent nucleosomal positioning with heterochromatin formation. Another interesting architecture seen in oomycetes combines the WHIM motifs with a Werner’s syndrome type DNA repair nuclease with 3'-5' exonuclease and HRDC domains, suggesting that in these organisms the ISWI-catalyzed chromatin repositioning might be directly combined with DNA repair.
In evolutionary terms the DDT-WHIM proteins and ISWI ATPases can be considered a synapomorphy of eukaryotes suggesting that guided nucleosome positioning was a phenomenon that was already present in the last eukaryotic common ancestor. On the whole, the independent diversity of the domain architectures of paralogous ISWI accessory large subunits in several distinct eukaryotic lineages points to an important role for distinct nucleosome position patterns in facilitating different sets of biological processes. In particular, it would be of great interest to investigate the role of the lineage-specific expansion of the DDT-WHIM motif proteins in ciliates. These unicellular eukaryotes do not have differentiated tissues like animals or plants that also show a multiplicity of DDT-WHIM motif proteins. But they show two functionally distinct types of nuclei – the transcriptionally active macronucleus being derived from the micronucleus following their sexual cycle. The macronucleus is characterized by drastic genomic rearrangements and lack of mitotic chromosome condensation and segregation. We suspect that the lineage-specific expansion of WHIM-DDT proteins in ciliates directly relates with the need for ISWI-dependent maintenance of particular nucleosomal positions in the macronucleus (Click here to access the paper). Our extensive supplement can also be accessed here.