DNA Hypomethylation and Histone Variant macroH2A1 Synergistically Attenuate Chemotherapy-Induced Senescence to Promote Hepatocellular Carcinoma Progression

Abstract

Aging is a major risk factor for progression of liver diseases to hepatocellular carcinoma (HCC). Cellular senescence contributes to age-related tissue dysfunction, but the epigenetic basis underlying drug-induced senescence remains unclear.macroH2A1, a variant of histone H2A, is a marker of senescence-associated heterochromatic foci that synergizes with DNA methylation to silence tumor-suppressor genes in human fibroblasts. In this study, we investigated the relationship between macroH2A1 splice variants, macroH2A1.1 and macroH2A1.2, and liver carcinogenesis. We found that protein levels of both macroH2A1 isoforms were increased in the livers of very elderly rodents and humans, and were robust immunohistochemical markers of human cirrhosis and HCC. In response to the chemotherapeutic and DNA-demethylating agent 5-aza-deoxycytidine (5-aza-dC), transgenic expression of macroH2A1 isoforms in HCC cell lines prevented the emergence of a senescent-like phenotype and induced synergistic global DNA hypomethylation. Conversely, macroH2A1 depletion amplified the antiproliferative effects of 5-aza-dC in HCC cells, but failed to enhance senescence. Senescence-associated secretory phenotype and whole-transcriptome analyses implicated the p38 MAPK/IL8 pathway in mediating macroH2A1-dependent escape of HCC cells from chemotherapy-induced senescence. Furthermore, chromatin immunoprecipitation sequencing revealed that this hepatic antisenescence state also required active transcription that could not be attributed to genomic occupancy of these histones. Collectively, our findings reveal a new mechanism by which drug-induced senescence is epigenetically regulated by macroH2A1 and DNA methylation and suggest macroH2A1 as a novel biomarker of hepatic senescence that could potentially predict prognosis and disease progression.

Histone macroH2A1.2 promotes metabolic health and leanness by inhibiting adipogenesis

“…To understand whether macroH2A1.2 could affect the differentiation of pre-adipocytes into mature adipocytes, we used the well-established murine 3T3-L1 cell model. Stable expression of GFP, macroH2A1.2, and its sister splicing variant macroH2A1.1, in 3T3-L1 pre-adipocytes was achieved by lentiviral transduction as previously described [12], and differentiation into mature adipocytes was obtained through a 15 days long protocol based on the sequential addition of dexamethasone, IBMX and insulin [28] (Fig. 7a).…”

“…It is has been shown that 3T3-L1 cell differentiation associates with genome-wide epigenetic dynamic changes in the DNA demethylation/methylation ratio in a time- and stage-dependent manner [31]; DNA hypomethylating agent decitabine blocked the 3T3-L1 adipogenic process [31]. In the context of cancer cells, macroH2A1 incorporation antagonizes the anti-proliferative effects of decitabine [12, 13]. We postulate that macroH2A1.2 might interfere with DNA methylation events during the adipogenic gene expression program.…”

“…Variations in macroH2A1 transcriptional activities to a large extent independent of genome occupancy in response to nutritional and DNA methylation status have been reported in cancer cells [9, 11, 12]. Consistent with these studies, our ChIP-Seq analyses comparing macroH2A1.2 Tg genome binding between 3T3-L1 pre-adipocytes and 3T3 post-differentiated adipocytes revealed high degree of similarity in genome binding patterns, including within the bodies of the subset of genes involved in the adipogenic process that we analyzed.…”

“…In cellular senescence, proliferation becomes arrested and cells acquire a pro-inflammatory senescent secretory phenotype (SASP), with release of chemokines and cytokines [35]. We and others have shown that macroH2A1.1 ectopic expression sustains SASP in fibroblasts and hepatoma cells [12, 36]; a similar signaling loop might take place in the adipose tissue. How to boost macroH2A1.2 expression/activity, to the detriment of macroH2A1.1, with an anti-obesity therapeutic purpose?…”

“…Total and cytoplasmic/nuclear/histone proteins extraction and immunoblotting analyses were performed as previously described [12]. Primary antibodies were as follows: anti-GFP (Abcam, ab13970) anti-macroH2A1.1 (Cell Signaling, Cat 12455), anti-macroH2A1.2 (Cell Signaling, Cat.…”