Roxanne Tymon

Abstract

In order to accommodate for the extremely large size of our genome it must be wrapped, coiled, looped, and condensed. As a result, DNA processes require precise mechanisms to de-condense and re-condense its chromosomes, which cause the recruitment of ATP-dependent chromatin remodeling complexes. One specific family of chromatin remodeling complexes is the imitation switch (ISWI) family, which has been shown to function in nucleosome spacing, DNA damage repair, transcription, and replication. The ISWI complex’s unique C-terminal HAND-SANT-SLIDE domain is crucial to its specific function. A big topic of research has been to understand the precise mechanisms of how ISWI targets nucleosomes and determines which type of chromatin regulatory function is needed. This review will touch on what is currently known of ISWI’s structure and function.

 

 

DNA structure is very important to gene regulation. The large size of DNA requires that a packed chromatin structure is needed to fit within the nucleus. Therefore, when a gene is activated or repressed, the chromatin needs to be unwound or rewound, respectively. This tightly regulated process is called chromatin remodeling, and requires the role of chromatin remodeling complexes. During remodeling, histones within the nucleosome can be shifted, removed, or exchanged for another histone variant (1).

Chromatin remodeling complexes use ATP as an energy source to regulate transcription, DNA repair, replication, and recombination (1). There are five main subfamilies of ATP-dependent chromatin remodeling complexes that stem from the Snf2 superfamily. The subfamilies, CHD, INO80, SWI/SNF, ISWI, and ATRX, all share similar ATPase domains, while also containing unique features that specify individualized functions (2). One particular ATP-dependent chromatin remodeling complex family of interest is imitation switch (ISWI). Interestingly, initial research has connected ISWI to cancer mechanisms. For example, duplications in the 17q chromosome containing the NURF gene, a member of the ISWI family, have been shown to be involved in human cancers such as brain, breast, lung, liver, and prostate (3).

The ISWI family can be divided into multiple members that vary throughout species. In yeast, the ISWI members consist of ISW1a, ISW1b, and ISW2. The fly orthologs are NURF (nucleosome remodeling factor), CHRAC (chromatin accessibility complex), and ACF (ATP-utilizing chromatin assembly and remodeling factor) (1). Human orthologs include NURF, CHRAC, ACF, WICH, NoRC, RSF, and CERF (4). Each ISWI complex is composed of 2-4 subunits making up three types of domains (ATPase, non-catalytic, and distinctive) (1). For example, the NURF protein in humans is composed of BPTF, SNF2L, and pRBAP46/48 subunits (3).

All members of the Snf2 superfamily contain a similar ATPase subunit within the center region of their gene, which is broken into two domains (DExx and HELICc) separated by an insertion sequence. Each subfamily of complexes is then specialized by the varying domains flanking the ATPase region. The ISWI family has a unique short insertion sequence dividing the ATPase domains. Ultimately, the key factor that individualizes the ISWI family is the HAND-SANT (ySWI3, yADA2, hNCoR, hTFIIIB)-SLIDE (SANT-like ISWI) domain (HSS) located at its C-terminus (1).

The ISWI complex is very important to many chromatin regulatory functions, but much is yet to still be understood. Crystal structures of the C-terminus of ISWI have begun to aid in further understanding why ISWI specifically is targeted to certain processes over other chromatin remodeling complexes (5). Further, experiments have been conducted to highlight proteins that ISWI interacts with during such processes as replication and DNA repair (6,4). Specific examination of ISWI family members in different species and the specific subunits of these complexes have been and will continue to help narrow down answers to better understand the precise mechanisms of how ISWI targets nucleosomes and determines which type of chromatin regulatory function is needed. This review will focus on the unique structure and function of the ISWI chromatin remodeling complex family.

Function

ISWI Remodels Chromatin The generalized function of ISWI is to promote chromatin remodeling. Chromatin remodeling can take on many forms, but the ISWI complex focuses on nucleosome spacing. ISWI uses ATP to slide the nucleosome position until it is restricted by the length of the linker DNA separating two nucleosomes (2). It is hypothesized that this repositioning opens up the DNA allowing for access to DNA binding proteins (1). Additionally, the histone H4 tail has been shown to be important in nucleosome interactions with ISWI during remodeling. Specifically, experiments mutating the tail of histone H4 had negative effects on these interactions, resulting in disruptions in remodeling (5).

Variation among species appears to be at play in the actual mechanism of how ISWI interacts with nucleosomes. In humans, it has been shown that two ACF complexes bind a nucleosome forming a dimer of ATPases, which then coordinates sliding in two different directions (7). In contrast, yeast experiments have shown that one ISW1a complex can interact with two nucleosomes to adjust the linker DNA length between them (8). Further studies examining other human and Drosophila ISWI complexes would reveal if similar mechanisms of nucleosome interactions are at work.

ISWI as a whole has been shown to function in nucleosome sliding, and further research by Xiao and colleagues (9) helped narrow down specific subunits that are involved in this process. In their experiments, they analyzed nucleosome position preference of the different subunits of the Drosophila ISWI complex, NURF. A nucleosome mobility assay showed that the combination of only the NURF301 (non-catalytic) and ISWI (catalytic) subunits in the presence of ATP resulted in the nucleosomes shifting to the preferred position (N3). The combination of only the ISWI, NURF38 (distinctive), and NURF55 (non-catalytic) subunits plus ATP caused the nucleosomes to remain in position N2 and not move to the preferred N3 position. This indicates that both the NURF301 and ISWI subunits play an important role in nucleosome spacing in Drosophila (9). The NURF301 subunit shows similarities to the human ortholog BPTF, indicating a possibility that a similar mechanism occurs in humans.

The generalized function of ISWI can be further applied to specialized functions including regulating DNA damage, regulating transcription, and participating in replication processes. The question of exactly how ISWI and not other remodeler complexes is specifically targeted for these functions remains unanswered at this time, although the HSS domain is believed to be involved.

ISWI Regulates DNA Damage In order for DNA lesions to be repaired, it is important that the chromatin is accessible to repair proteins. ATP-dependent chromatin remodeling complexes are therefore crucial to regulating the DNA damage response (DDR). Specifically, the ISWI family has been identified as a key player in the DDR through the work of Aydin and colleagues (4). In the presence of double strand breaks, ISWI has been shown to be recruited in multiple different ways to the site of damage. These complex parallel pathways include the ATPase domain of ISWI and other proteins that work together to modify specific histones within the nucleosome. Such modifications include direct phosphorylation and indirect ubiquitination by ISWI at H2AX within the nucleosome adjacent to the break. Through the work of multiple protein complexes, the chromatin is remodeled, which in turn recruits repair proteins that function in homologous recombination and non-homologous end joining repair (4).

Many studies of ISWI complexes must be conducted in vitro because of the complexity of trying to control chromatin remodeling in vivo. The Erdel and Rippe labs (10) were able to examine ISWI subunits in vivo by fluorescently tagging proteins of interest. In this study, they observed the kinetics of specific subunits being recruited to the target site where UV damage was created in U2OS cells. The SNF2L (catalytic), SNF2H (catalytic), and ACF1 (non-catalytic) subunits of human ISWI complexes were recruited to the lesion some seconds after induction and remained for 2-3 minutes. Examination of PCNA recruitment revealed an earlier binding and a longer lasting interaction than ISWI subunits at the site of damage. Erdel and Rippe therefore proposed a “continuous sampling model” where ISWI binds and releases nucleosomes until it encounters damage, in which case it binds. Based on the time-frame of PCNA, it is suggested that PCNA acts faster than ISWI and may stabilize the chromatin when recruited to sites of DNA damage (10).

ISWI Regulates Transcription Chromatin structure is critical to the regulation of transcription depending upon whether transcription factors can access the DNA or not. ISWI has been shown to be involved in regulating transcription. An experiment using a mutant ISWI protein (dominant negative) in metaphase chromosomes of Drosophila larvae neuroblasts revealed an unraveled chromosome structure (11). These results indicate that ISWI is acting in regulating the compaction of the chromosomes throughout the whole genome. Microarray analysis of mutants where ISWI function is lost showed increased expression in 75% of the Drosophila larvae genes tested (11). Therefore based on these two experiments, ISWI is thought to repress transcription by promoting the compaction of chromatin. An analysis of histone levels in the presence of dominant negative ISWI protein revealed a reduction in histone H1 (11). Since histone H1 is important in chromatin assembly to stabilize the 30nm fiber structure, it is suggested that ISWI is involved in chromatin assembly, which therefore represses transcription. The precise mechanism of how ISWI promotes the interactions of histone H1 and the nucleosome is unclear at this time.

Additional experiments have examined ISWI’s involvement in transcription, but the actual mechanism is still unclear. ISWI has been shown to interact with transcription factors and activators through experiments conducted by Xiao and colleagues (9). GST pull-down assays revealed that NURF301 (subunit of the Drosophila ISWI complex NURF) bound to the GAGA transcription factor and the HSF and VP16 transcription activators. Transcription activator binding was specific to the NURF301 subunit only. Interactions with the GAGA transcription factor were also observed with the ISWI subunit, but not as strongly as with NURF301. Based on these findings, it shows that ISWI is recruited during transcription to regulate nucleosome sliding (9).

ISWI Regulates Replication ISWI has been shown to be involved in DNA replication through experiments using SNF2H (ATPase domain of the ACF human ISWI complex) in the Poot and collaborators’ labs (6). Immunostaining and co-immunopreciptiation experiments demonstrated that the Williams syndrome transcription factor (WSTF) recruits SNF2H to the replication foci throughout S phase. WSTF was also shown to interact with the PCNA during S phase, which then leads to SNF2H recruitment. Additionally, western blot analysis revealed increased heterochromatin protein when either WSTF or SNF2H were depleted through RNAi. Therefore, the WSTF-SNF2H complex may be involved in chromatin remodeling during S phase to promote nucleosome mobility associated with euchromatin (6).

In addition to chromatin remodeling, ISWI has been shown to be involved in chromosomal segregation during mitosis through the work of Yokoyama and colleagues (12). A sedimentation assay revealed that ISWI binds microtubules. ISWI is the only chromatin remodeling complex known to bind to microtubules leading to the possibility that the HSS domain may be involved in this process. Interestingly, a mutation in the ATPase domain of ISWI revealed that ATPase activity is not required for ISWI to bind microtubules, unlike in chromatin remodeling. Further experiments depleting ISWI in Xenopus egg extracts or Drosophila cells showed normal spindles in metaphase, but rapidly disappearing spindles in anaphase as well as disruptions in chromosomal segregation. This indicates that ISWI is needed for stabilizing the microtubules and aiding in chromosome segregation during anaphase. Surprisingly for being a chromatin remodeling complex, depletion experiments discovered that ISWI is not involved in de-condensing chromosomes during mitosis (12).

Structure

3D Structure of the C-terminus of the ISWI ATPase Subunit Now that we’ve examined the functions of ISWI, a closer look at its structure will help tie together the relationship between structure and function. Drosophila melanogaster crystal structures of the C-terminus of ISWI helped further understand the aspects that make this complex unique (5). Overall, the protein is composed of 12 α-helices that can be divided into 3 domains and one spacer region, which are all tightly connected (Figure 1). The first 4 helices make up the HAND domain, which got its name from its similar appearance to an open hand structure. Next is the SANT domain, which takes on a compact structure of 3 helices. A cluster of hydrophobic residues keeps the HAND and SANT domain close together. After the SANT domain is a 33 residue spacer, which is involved in hydrophobic interactions and the formation of salt bridges. Lastly, the SLIDE domain contains the final 3 helices, and takes on a large loop structure (5).

HSS and Helicase Domains of the ISWI ATPase Subunit To better understand how ISWI is targeted to nucleosomes, it is important to examine its structure. The combination of the HAND, SANT, and SLIDE domains together at the C-terminus of the ATPase ISWI subunit have been shown to be important in nucleosome interaction. Unmodified histone tails within the nucleosome interact with the SANT domain, while linker nucleosomal DNA interacts with the SLIDE domain (5). Additionally, Grune and colleagues demonstrated through nucleosome assays that both the N-terminus (ATPase domain) and the C-terminus individually interact with nucleosomal DNA (5).

Experiments deleting the SLIDE domain greatly diminished the activation of the ATPase domain, while deleting the SANT domain had no effect (5). Nucleosome sliding and spacing assays revealed that when SLIDE was deleted, nucleosome remodeling was inhibited. Based on these results, it is clear that SLIDE must be present and active for nucleosome remodeling to occur, but it is not essential for SANT (5). One model proposes that the SLIDE and ATPase domains must coordinate together during nucleosome remodeling (Figure 2). This model suggests that a conformational change in the DNA-binding domain (SLIDE) results in a loosening of the DNA wrapped around the histones, thus creating a small loop structure on one side. The DNA is then moved through the ATPase domain towards the other side of the histone core (1). The SLIDE domain pushes the DNA towards the ATPase domain, which then pulls the DNA creating a coordinated balance of forces (2,13). Lastly, conformational changes in the SLIDE domain return the nucleosome to its normal structure ready for another round of remodeling (1).

ISWI Involvement in Disease

Future research to better understand the mechanisms of ISWI and its structural components are crucial to learning about disease. Cancer is of particular interest in connection to ISWI. The NURF protein of the ISWI complex has been reported to possibly play a role in human cancers. A comparative genome hybridization array was used in one specific study performed by Buganim and colleagues (14) that examined duplications in the 17q chromosome, which codes for the BPTF gene (non-catalytic subunit of NURF). To address this question, the authors maintained human embryonic lung cells altered with a translocation of chromosome 17. Increased cell proliferation and BPTF mRNA levels were observed, possibly linking the BPTF gene to human cancers with duplications in chromosome 17 such as lung, colon, and prostate cancer (14).

Another study of human cancers by Ye and collaborators (15) examined the SNF2L (catalytic) subunit, of the human NURF complex within the ISWI complex family. siRNA knockdown experiments of SNF2L comparing normal and highly malignant cancer cell lines resulted in reduced cell growth, increased apoptosis, and increased DNA damage in only the cancer cells. These inhibitory effects on cancer cells show that SNF2L could possibly play a role in cancer therapy (15). Continued research needs to be conducted to further examine this possibility.

Conclusions

There are still unanswered questions about the ISWI ATP-dependent chromatin remodeling complex family, but much has been highlighted to pinpoint ISWI during important regulated chromatin processes. Just like in all biology, structure greatly relates to function. The unique HAND-SANT-SLIDE domain of ISWI clearly plays a role in specializing ISWI function during nucleosome sliding. Figure 3 displays a simplified summary model of how the specialized structure of ISWI leads to nucleosome spacing, which in turn is involved in regulating DNA damage repair, transcription, and replication. Questions still remain. For instance, how is the HSS domain of ISWI specifically targeted to nucleosomes to function in particular DNA processes? Also, how can the mechanisms of ISWI be used to possibly act as a therapy target for certain diseases, like cancer?

 

References

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