The CHD family of chromatin remodeling enzymes

1.8.1 CHD structure and function

The fourth family of ATP-dependent chromatin remodeling enzymes is evolutionary the least conserved and only exists in mammals, with the exception of a CHD1/2 homolog in yeast (Murawska and Brehm 2011). There are nine members, which share, in addition to the SNF2-like helicase/ATPase members of this family, an N-terminal double chromodomain and in some cases a DNA binding domain, as visualized in figure 1.9 (Sims and Wade 2011). This DNA binding domain is established only for CHD1 while the other family members do not have a canonical but putative DNA binding domain. The tandem chromodomain was shown to interact with acetylated and methylated histones as was especially shown for H3K4me and is a unique feature to CHD proteins among other chromatin remodelers (Flanagan et al. 2005). CHD3 and CHD4 have two PHD zink finger domains which provide an additional DNA binding site, while CHD5-9 harbor additional

domains which have been interpreted as interaction domains though without a yet clarified mechanism (Hall and Georgel 2007).

Each of the domains that bind to chromatin is indispensable and CHD remodelers obviously attach to chromatin through various sites as the mutation of single domains consequently lead to a dissociation of the proteins from chromatin (Marfella and Imbalzano 2007).

CHD remodelers can either act as transcriptional activators or repressors with ascribed functions in multisubunit complexes. They affect each step of transcription, and the decision between transcriptional activation or repression depends on which promoters or transcription factors they bind. The only remodelers with clear repressive functions are CHD3/4, which are part of the NuRD-complex (nucleosome remodeling deacetylase) that also contains histone deacetylases (HDACs) and is involved in gene repression (Hall and Georgel 2007).

1.8.2 CHD in DNA damage response and disease

A role in disease and response to damaged DNA is established for some of the CHD chromatin remodelers. Recently, genetic and transcriptomic analysis of all CHD genes strengthened the significance of CHD in cancer, of which some have mechanistically been

Figure 1.9 Structure and functional domains of CHD8.

The structure of CHD family members is highly heterogenic. Minimal structure similarities are the two N-terminal tandem chromodomains involved in nucleosome binding, the SWF/SNF2-like ATPase/helicase domain as core functional domain which hydrolyses ATP to move histones and nucleosomes, and a helicase C-terminal domain. Only CHD1/2 contain a defined DNA-binding domain, which is not present in any other family member. Those potentially bind chromatin through all other domains. For CHD8, deletion of each domain abrogates its tethering to chromatin. CHD8 possesses two BRK domains of unknown specificity which otherwise exist in CHD7 and CHD9 only, so that these form a subfamily. Figure modified from Hall and Georgel, 2007 with permission from Biochemistry and Cell Biology.

involved in the DDR (Chu et al. 2017). A CHD1-like protein, referred to as ALC1 (amplified in liver cancer) localizes to sites of DNA damage where it promotes DNA repair (Lans et al.

2012). CHD1 as such is required for the conservation of stemness during embryonic development through the prevention of heterochromatin formation (Gaspar-Maia et al.

2009). A tumor-suppressive role is established for CHD5. It is required for the transcription of the tumor suppressor CDKN2A which controls progression through the cell cycle. In contrast to other chromatin remodeling functions, this role seems to be universal throughout cell types. Mutations in CHD5 frequently occur in human gliomas (Kolla et al. 2014). CHD3 and CHD4 are part of the NuRD complex which is involved in DNA repair through the creation of a permissive chromatin environment (O'Shaughnessy and Hendrich 2013).

Upon ionizing radiation, CHD4, a target of ATM, is transiently recruited to DSBs within minutes in a PARP- but not γH2AX-dependent manner and shows enhanced accumulation after ATM depletion. CHD4 controls p300-dependent p53 acetylation, which is enhanced after CHD4 depletion leading to accumulation of p21 and therefore arrest in G1 of the cell cycle (Polo et al. 2010).

Haploinsufficiency of CHD7 leads to the CHARGE syndrome in humans. This genetic disease with distinct organ anomalities is reproducible in mice experiments showing a misregulated gene expression during development and hyperactivation of p53 (Bosman et al. 2005; Van Nostrand et al. 2014; Vissers et al. 2004). Furthermore, CHD7 was recently suggested as biological marker for the outcome of gemcitabine-treated pancreatic cancer.

In vitro, depletion of CHD7 lowers DNA damage signaling as indicated by reduced Chk1 phosphorylation, but leads to a higher percentage of γH2AX-positive cells and reduced survival after treatment with gemcitabine. Patients receiving neoadjuvant treatment with gemcitabine showed a significantly better survival when CHD7 expression was low (Colbert et al. 2014).

1.8.3 The chromatin remodeling factor CHD8

CHD8 is one of the less characterized ATP-dependent chromatin remodelers. Mutations in the CHD8 gene are associated with autism spectrum disorders, and CHD8-heterozygous mice display a haploinsufficient phenotype with large abnormalities in brain development due to a lack of differentiation and perturbed gene expression patterns. CHD8 knockout mice die during early embryogenesis with a phenotype of widespread apoptosis and cardiovascular and brain anomalies. CHD8 is highly expressed in embryonic tissues and stem cells in mice with emphasis on neuronal tissue, but expression levels seems to decrease with differentiation (Nishiyama et al. 2009).

1.8.3.1 Structure and function of CHD8

CHD8 was described to exist in two isoforms which are produced by alternative splicing:

the large isoform harboring the domains as depicted above and the small isoform lacking the C-terminal domains (Nishiyama et al. 2009). However, only the large isoform was found to be expressed in human and mouse tissues (Shingleton and Hemann 2015). It is localized to the nucleus and expression of CHD8 seems to be uniform throughout the cell cycle (Mjelle et al. 2015; Nishiyama et al. 2009). To date, the three-dimensional structure of CHD8 has not been resolved and there are no proteomic analyses for the identification of CHD8 binding partners. Some single experimental results have described CHD8 as being part of multisubunit complexes (see below). Mechanistically, CHD8 is able to slide and bind nucleosomes at DNA. This process requires hydrolysis of ATP, which is stimulated by chromatin 10-fold more than by DNA alone. Compared to other CHD chromatin remodelers, CHD8 requires comparably large DNA fragments for this activity and has low affinity to nucleosomal substrates, which restricts CHD8 remodeling function to large linker DNA or spaced nucleosomes of highly transcribed DNA regions. CHD8 can thus condense DNA with highly spaced nucleosomes as well as decondense compacted chromatin establishing a fixed DNA-nucleosome spacing (Manning and Yusufzai 2017).

1.8.3.2 CHD8-regulated processes

Originally, CHD8 was identified as a negative regulator of Wnt signaling, a pathway which is often constitutively activated in human cancers. Characterization of CHD8 functions comprises the involvement of CHD8 in positive or negative regulation of transcription (Logan and Nusse 2004).

While CHD8 directly binds to ß-catenin upon induction of Wnt signaling, the binding and recruitment of histone H1 via an H1-binding domain on CHD8 is necessary for the transcriptional repression of Wnt-dependent gene transcription. In the same way, the authors suggested CHD8 to be a negative regulator of p53 and detected increased transcription of p53-dependent genes after depletion of CHD8 (Nishiyama et al. 2012;

Nishiyama et al. 2009).

Another mechanism through which CHD8 might regulate transcriptional repression is the maintenance of insulator functions. Chromatin insulators form borders between differentially regulated gene transcription and between eu- and heterochromatin. CHD8 binds to the chromatin insulator CTCF (CCCTC-binding factor), which serves as boundary between differentially regulated chromatin domains. Like CHD8, CTCF can activate and suppress transcription. Two ways for transcriptional repression are the prevention of enhancers from

binding to promoters and a barrier formation of CTCF which prevents the spreading of heterochromatin to euchromatic regions, where CHD8 is essential for both (Ishihara et al.

2006). The dysregulation of insulator activities is associated with an epigenetic suppression of tumor suppressor genes, and CHD8 together with CTCF was found to be lowered during prostate carcinogenesis (Damaschke et al. 2014).

The activation of transcription by CHD8 might function through the association with RNA polymerase II, which was shown to promote the transcription of CHD8-dependent genes of which some are S-phase specific (Rodriguez-Paredes et al. 2009; Subtil-Rodriguez et al.

2014). Another, yet therapeutic involvement of CHD8 in malignancies is through the binding of the chromatin reader BRD4 (bromodomain-containing protein 4), which drives oncogenic gene transcription in myeloic leukemia and, as has been shown recently, in a subset of gynecological cancers. CHD8 is bound to BRD4 through the histone methyltransferase NSD3 and depletion of either factor leads to growth delay and induces differentiation. Thus, inhibition of BRD4 is subject to clinical trials (Jones and Lin 2017; Shen et al. 2015).

Last, CHD8 is part of histone methyl transferase complexes containing the MLL1-protein (mixed-lineage leukemia-1). It promotes malignancy through the methylation of hox genes promotors and induces a CpG methylator phenotype in colorectal cancer cells (Fang et al.

2014; Yates et al. 2010).

Taken together, CHD8 has established roles during embryogenesis and differentiation, mainly by coordinating the transcriptional network. There are contradictory findings as to whether this chromatin remodeler has proto-oncogenic properties as seem to dominate for hematologic malignancies, or whether it executes a tumor suppressor function as seen in many solid tumors.

So far, CHD8 has no established role in the DDR.