Maintaining oxidized H3 in heterochromatin is required for the oncogenic capacity of triple-negative breast cancer cells

Abstract

The histone modification of H3 oxidized at lysine 4 (H3K4ox) is catalyzed by lysyl oxidase-like 2 (LOXL2) and is enriched in heterochromatin in triple-negative breast cancer (TNBC) cells. Although H3K4ox has been linked to the maintenance of compacted chromatin, the molecular mechanism underlying this maintenance is unknown. Here we show that H3K4ox is read by the CRL4B complex, leading to the ubiquitination of histone H2A through the E3 ligase RBX1. Finally, interactions between RUVBL1/2 and LOXL2 are involved in the incorporation of the histone variant H2A.Z, which plays an essential role in the mechanism controlling the dynamics of oxidized H3. Maintenance of H3K4ox in chromatin is essential for heterochromatin properties, and disruption of any of the members involved in this pathway blocks the oncogenic properties of TNBC cells. 2018). H3K9me2/3 are recognized by heterochromatin protein 1 (HP1), which has a major role in ensuring heterochromatin compaction, spreading, and inheritance (Bannister et al., 2001).Heterochromatin is not only essential for the maintenance of genome stability but can also directly affect genome integrity, through changes in its highly dynamic structure (Janssen et al., 2018).Therefore, there is a pressing need to improve our understanding of the role of heterochromatin, and the mechanisms by which it is maintained at a mechanistic level. To address this, we employed, unbiased proteomic assays to identify LOXL2 partners, as well as H3K4ox readers. We found that LOXL2 interacts with members of the SRCAP and TIP60 complexes, and that H3K4ox is read by the CRL4B complex. Both of these complexes are involved in loading the histone H2A variant, H2A.Z, into chromatin (Buschbeck and Hake, 2017; Morrison and Shen, 2009). We also discovered that the CRL4B complex is involved in the ubiquitination of H2A by RBX1, that is required for the exchange of the H2A variant, H2A.Z. Notably, both in vitro and in vivo, these series of events are necessary to maintain condensed chromatin in triple-negative breast cancer (TNBC) cells. Knocking down any component of this pathway resulted in inhibition of oncogenic and metastatic traits in TNBC cells, including proliferation, migration, invasion, and in vivo tumor formation capacity. Thus, we propose the existence of a direct link between oxidation of H3, chromatin condensation, and tumorigenic capacities, and argue that any of the components described in the H3K4ox pathway could potentially be targetable factors for treating TNBC. RESULTS The Roles of LOXL2, RUVBL2, and H2A.Z in Chromatin CondensationWe revisited a tandem-affinity purification approach previously published by our lab to identify putative LOXL2 interactors that might be involved in the maintenance or generation of compacted chromatin induced by H3K4ox-LOXL2 (Cebria-Costa et al., 2019;Herranz et al., 2016). Analysis of the new list of interactors showed the presence of two AAA+ ATPases (RUVBL1 and RUVBL2), DMAP1 and BAF53, all of which are members of the SRCAP and TIP60 complexes ( Figure 1A a...