A model of chromatin reorganization induced by CHD8 depletion for efficient

gemcitabine treatment

Higher order chromatin structure profoundly influences genome maintenance and DNA repair. Chromatin structures are non-randomly distributed within the nucleus, one of the best examples being the organization of DNA into euchromatin and heterochromatin.

Euchromatin contains actively transcribed genes, and facultative heterochromatin occurs by gene silencing for example during cellular differentiation (Nair and Kumar 2012).

Constitutive heterochromatin represents around 25 % of the genome and is characterized by low gene density but highly repetitive sequences that increase the risk for recombination and might necessitate an even tighter control of genome maintenance (Goodarzi and Jeggo 2012; Soria et al. 2012). Although chromatin is undoubtedly needed for the regulation of virtually all DNA-associated processes to take place, it primarily forms a barrier to efficient DNA repair. High compaction in heterochromatin hinders the access for repair factors even more, but due to its high abundance, it forms the major environment in which the whole DDR takes place (Bakkenist and Kastan 2015). A cell deals with such a challenge and threat to genomic integrity by tightly regulated mechanisms:

When damaged DNA is encountered, chromatin needs to be opened first to elicit a fully activated DDR and needs to be restored when damage is repaired. One well characterized process is the CHD3-dependent relaxation of heterochromatin: ATM, when activated, phosphorylates the heterochromatin-associated protein KAP-1, which loosens contact to DNA and to CHD3, which in turn diffuses from chromatin to open it. After repair, the restoration of KAP-1 to DNA tethers CHD3 to the NuRD complex, again leading to the reformation of heterochromatin (Klement et al. 2014).

Indeed, spreading of γH2AX is impaired when meeting heterochromatin border and γH2AX foci form slower in heterochromatin (Brunton et al. 2011; Soria et al. 2012). Also, repair of DSBs seems to occur 2-fold slower in heterochromatin than in euchromatin and is predominantly dependent on ATM, whereas in euchromatin some studies found that it is not (Goodarzi et al. 2008).

Heterochromatin is refractory to the spreading of γH2AX and γH2AX foci occur mutually exclusively with heterochromatin marks but cluster in the periphery of those (Goodarzi and Jeggo 2012). In yeast, γH2AX spreading is immediately stopped when encountering a heterochromatic region (Kim et al. 2007).

The generation of global chromatin environments requires boundary elements, to which chromatin modifying enzymes are recruited. One of those boundary elements is CTCF.

Indeed, we observed that the induction of DNA damage signaling upon CHD8 kd is dependent on the presence of CTCF in a preliminary result, but here both factors appear to have antagonistic effects on the DDR (Fig. 5.2).

The relevance of the above-mentioned becomes clear when we take into account that, indeed, an open chromatin structure as found in stem cell like populations confers higher chemoresistance through a higher capacitie to repair DNA. CHD8 can be integrated in a model of chromatin organization to modulate the DDR and chemoresistance as follows: The linker histone H1 has been found to delicately influence chemoresistance as its deletion leads to an open chromatin structure and hyperresistance to a variety of DNA damaging agents (Hargreaves and Crabtree 2011; Murga et al. 2007). CHD8 could do the same by providing an open chromatin environment. It evidently binds histone H1 to chromatin (Nishiyama et al. 2009) and it would be of interest whether depletion of CHD8 leads to a

Figure 5.2 Depletion of CTCF does not increase the cellular DNA damage response but rescues the effect of CHD8 depletion in UV-treated cells.

U2OS and PANC-1 cells were depleted of CHD8 and CTCF as indicated and treated with 20 J/m² UV-C or left untreated. One hour after treatment, cells were harvested and analyzed by immunoblotting.

phenotype comparable to H1 depletion. However, open chromatin structures do not unilaterally promote DNA repair and cell survival. Decompacted DNA becomes hydrolyzed easily activating the DDR (Walerych et al. 2015). Further, facilitating DDR leads to an apoptotic response (Bakkenist and Kastan 2015). As a consequence, decompaction of chromatin may initially lead to increased γH2AX accumulation and cell death, but in the long term increases the fraction of chemoresistant cells. If we apply this model to the phenotype we see after CHD8 knockdown, we should not only observe increased DNA damage response but also chemoresistance. Indeed, as a preliminary result, we do see a slight -- though not significant -- reduction of cell growth in CHD8-depleted, gemcitabine-treated cells initially. This is independent of the presence of p53. As soon as cells start to recover

Figure 5.3 Influence of CHD8 knockdown on cell proliferation after gemcitabine treatment.

U2OS cells were transfected with the siRNAs as indicated. After the replacement of transfection mix and recovery, they were trypsinized, counted and seeded and either treated with gemcitabine or left untreated as indicated. The confluence of a plate was determined every day using bright field microscopy and digital image analysis.

Figure 5.4 A model for CHD8 in the DNA damage response.

This speculative model integrates transcription and chromatin remodeling functions of CHD8. Either the decompaction of chromatin after CHD8 knockdown or the enhanced transcription lead to accumulation of DNA damage and repair factors, resulting in increased DNA damage signaling and apoptosis on the one hand but a higher fraction of cells which can repair their DNA and therefore resume proliferation and acquire chemoresistance on the other hand.

from gemcitabine treatment, CHD8-depleted cells show a slight growth advantage (Fig.

5.3). Furthermore, we observe increased EdU incorporation and slightly faster progression through cell cycle after CHD8 depletion.

Thus, a speculative model emerges bringing together the decompaction of chromatin as potential mechanistic basis for CHD8’s role in the DNA damage response together with the expression of KAT5 and MDC1 following CHD8 knockdown (see Fig. 5.4): CHD8 knockdown could either specifically activate the transcription of KAT5 and MDC1 or both factors could be induced as a secondary effect of chromatin decompaction after CHD8 knockdown. The acetylation of histones by KAT5 supports an open chromatin structure, which facilitates the clustering of highly transcribed MDC1 even more. Both initially leads to increased DNA damage signaling but also the activation of ATM and DNA repair pathways, leading to a low response to DNA damaging agents.