Modulation of the Fanconi anemia pathway via chemically induced changes in chromatin structure

Abstract

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway.

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 using Proximity Labeling and Affinity Purification

“…These results hint that SLX4 may have a role in chromatin compaction and heterochromatin maintenance. This is not without precedent, as previous studies established links between the Fanconi anemia pathway and chromatin state [88–90].…”

Histone H3K27 methylation modulates the dynamics of FANCD2 on chromatin to facilitate NHEJ and genome stability

“…Histone H3 with hyper-methylation at lysine 27 causes another type of repressive chromatin and is often associated with gene silencing via polycomb group protein (PcG) repression (Pirrotta, 1998(Pirrotta, , 1997Kingston, 2009, 2013). The PRC2 complex has been reported to be related to DSB repair (Campbell et al, 2013;Chou et al, 2010;Kakarougkas et al, 2014;Vierra et al, 2017), however, it is not clear whether the aberrant changes in H3K27me2/3 levels caused by pathological events, such as mutations in EZH2 or presence of H3.1K27M, may directly affect DSB repair efficiency, thus altering genome stability and potentially leading to carcinogenesis. To test this hypothesis, we established stable cell lines ectopically expressing haemagglutinin (HA)-tagged histone H3.1 wild-type (WT) or H3.1K27M mutant by transfecting human primary dermal fibroblasts (HDFs) with lentiviruses ( Fig.…”

“…However, it remained controversial whether the corresponding histone modification, H3K27me2/3, participated in the process (Chou et al, 2010;Campbell et al, 2013). PRC2 has been found to be involved in DSB-induced transcription silencing as well as chromatin-state modulation in FA pathway activation (Kakarougkas et al, 2014;Vierra et al, 2017). However, it remains to be discovered whether PRC2 affects DSB repair by other Fig.…”

“…PRC2 was also required for ionizing-radiation-induced H2AK119ub foci and DSB-induced transcriptional silencing, thus promoting DSB repair (Kakarougkas et al, 2014). Moreover, a recent study has found the PRC2 complex to be involved in the regulation of Fanconi anaemia (FA) pathway activation by modulating chromatin states (Vierra et al, 2017). However, it remains unclear whether H3K27me2/3 also participates in DSB repair or which repair pathway is responsible.…”

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 Using Proximity Labeling and Affinity Purification

“…These results hint that SLX4 may have a role in chromatin compaction and heterochromatin maintenance. This is not without precedent, as previous studies established links between the Fanconi anemia pathway and chromatin state. − …”