Dominant CIZ1 fragments drive epigenetic instability and are expressed in early stage cancers

Abstract

CIZ1 is a nuclear matrix protein that is part of the large RNA-dependent supramolecular assembly complexes (SMACs) that form at the inactive X chromosome (Xi) in female cells, and smaller assemblies throughout the nucleus in males and females. It plays a role in maintenance of epigenetic state and gene expression in differentiated cells, via stabilisation of histone post-translational modifications H2AK119ub1 and H3K27me3, added by polycomb repressive complexes (PRC) 1 and 2. Here, we show that expression of the N-terminal replication domain (RD) and C-terminal anchor domain (AD) of human CIZ1 transcript is uncoupled, with consistently elevated AD in early stage breast cancers, and sporadically elevated AD in other common solid tumours. At the protein level CIZ1-Xi SMACs are corrupted in female breast cancers cells, and this is accompanied by elevated AD-encoding transcripts. We modelled the effect of AD fragments in primary murine embryonic fibroblasts and observed dominant-negative interference with CIZ1 SMACs during their assembly in early G1 phase. Mutagenesis identified the matrin 3 homology domain as essential for self-interaction to form stable homodimers in vitro, and as a determinant of its dominant-negative effect in cells, implicating the dimerization interface in CIZ1 SMAC integrity. SMAC disruption was coincident with depletion of PRC1-dependent H2AK119ub1 from Xi chromatin, in a manner abrogated by the PR-deubiquitinase inhibitor PR619, suggesting that CIZ1 SMACs normally stabilise H2AK119ub1 by shielding Xi chromatin from attack by deubiquitinases. Moreover, SMAC disruption was accompanied by changes in gene expression within days. Together, the data suggest that inappropriate expression of CIZ1 AD fragments could drive epigenetic instability in early stage breast cancers by destabilizing the CIZ1 SMACs that normally protect repressed chromatin.

CIZ1 in Xist seeded assemblies at the inactive X chromosome

“…Surprisingly, the number of Xist foci increased in differentiating embryonic stem cells (ESCs) when repeat E was deleted, which implicates repeat E in the integrity or stability of Xist pairs ( Pandya-Jones et al, 2020 ). In experiments where CIZ1 is depleted, Xist pairs remain largely intact arguing against a role for CIZ1 ( Rodermund et al, 2021 ), even though it is itself a homodimeric entity ( Turvey et al, 2023 ). In fact, the bridging entity remains enigmatic and could depend on RNA-RNA interactions ( Van Treeck et al, 2018 ) as on its own, repeat E forms micrometer sized droplets ( Pandya-Jones et al, 2020 ; Ma et al, 2021 ).…”

“…Conversely, limited data suggest that CIZ1 may form a protective ‘shell’ or molecular shield ( Mészáros et al, 2019 ) around the Xi that excludes or includes soluble factors. When CIZ1-Xi assemblies are destabilized by the over-expression of interfering fragments ( Turvey et al, 2023 ), or absent as in CIZ1-null cells ( Stewart et al, 2019 ), underlying chromatin becomes depleted of PRC1-dependent ubiquitination of H2AK119. This might be driven by inappropriate exposure of chromatin to deubiquitylating enzymes (DUBs) is suggested because DUB inhibition abrogates the loss of H2AK119ub in both experimental contexts ( Turvey et al, 2023 ).…”

“…When CIZ1-Xi assemblies are destabilized by the over-expression of interfering fragments ( Turvey et al, 2023 ), or absent as in CIZ1-null cells ( Stewart et al, 2019 ), underlying chromatin becomes depleted of PRC1-dependent ubiquitination of H2AK119. This might be driven by inappropriate exposure of chromatin to deubiquitylating enzymes (DUBs) is suggested because DUB inhibition abrogates the loss of H2AK119ub in both experimental contexts ( Turvey et al, 2023 ). These data are beginning to argue that the CIZ1 in Xist- nucleated assemblies becomes essential only in the later stages of XCI because its primary role is to protect the status of chromatin that was established earlier.…”