The splicing regulator SLU7 is required to preserve DNMT1 protein stability and DNA methylation.

Gene expression is finely and dynamically controlled through the tightly coordinated and interconnected activity of epigenetic modulators, transcription and splicing factors and post-translational modifiers. We have recently identified the splicing factor SLU7 as essential for maintaining liver cell identity and genome integrity and for securing cell division both trough transcriptional and splicing mechanisms. Now we uncover a new function of SLU7 controlling gene expression at the epigenetic level. We show that SLU7 is required to secure DNMT1 protein stability and a correct DNA methylation. We demonstrate that SLU7 is part in the chromatome of the protein complex implicated in DNA methylation maintenance interacting with and controlling the integrity of DNMT1, its adaptor protein UHRF1 and the histone methyl-transferase G9a at the chromatin level. Mechanistically, we found that SLU7 assures DNMT1 stability preventing its acetylation and degradation by facilitating its interaction with HDAC1 and the desubiquitinase USP7. Importantly, we demonstrate that this DNMT1 dependency on SLU7 occurs in a large panel of proliferating cell lines of different origins and in in vivo models of liver proliferation. Overall, our results uncover a novel and non-redundant role of SLU7 in DNA methylation and present SLU7 as a holistic regulator of gene expression.

Splicing Factor SLU7 Prevents Oxidative Stress-Mediated Hepatocyte Nuclear Factor 4α Degradation, Preserving Hepatic Differentiation and Protecting From Liver Damage.

BACKGROUND AND AIMS: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. APPROACH AND RESULTS: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7+/- ) mice undergoing chronic (CCl4 ) and acute (acetaminophen) injury. SLU7 expression was restored in CCl4 -injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7+/- mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell's antioxidant machinery. CONCLUSIONS: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.

SLU7: A New Hub of Gene Expression Regulation-From Epigenetics to Protein Stability in Health and Disease.

SLU7 (Splicing factor synergistic lethal with U5 snRNA 7) was first identified as a splicing factor necessary for the correct selection of 3' splice sites, strongly impacting on the diversity of gene transcripts in a cell. More recent studies have uncovered new and non-redundant roles of SLU7 as an integrative hub of different levels of gene expression regulation, including epigenetic DNA remodeling, modulation of transcription and protein stability. Here we review those findings, the multiple factors and mechanisms implicated as well as the cellular functions affected. For instance, SLU7 is essential to secure liver differentiation, genome integrity acting at different levels and a correct cell cycle progression. Accordingly, the aberrant expression of SLU7 could be associated with human diseases including cancer, although strikingly, it is an essential survival factor for cancer cells. Finally, we discuss the implications of SLU7 in pathophysiology, with particular emphasis on the progression of liver disease and its possible role as a therapeutic target in human cancer.

Epigenetic mechanisms and metabolic reprogramming in fibrogenesis: dual targeting of G9a and DNMT1 for the inhibition of liver fibrosis.

OBJECTIVE: Hepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis. DESIGN: G9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-ß1 (TGFß1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology. RESULTS: G9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFß1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFß1 fibrogenic responses in hHSC. TGFß1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity. CONCLUSIONS: Dual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.

DNA Methylation Regulates a Set of Long Non-Coding RNAs Compromising Hepatic Identity during Hepatocarcinogenesis.

BACKGROUND: Long noncoding RNAs (lncRNAs) are emerging as key players in cancer, including hepatocellular carcinoma (HCC). Here we identify the mechanism implicated in the HCC inhibition of a set of lncRNAs, and their contribution to the process of hepatocarcinogenesis. METHODS AND RESULTS: The top-ranked 35 lncRNAs downregulated in HCC (Top35 LNDH) were validated in several human HCC cohorts. We demonstrate that their inhibition is associated with promoter hypermethylation in HCC compared to control tissue, and in HCC human cell lines compared to primary hepatocytes. Moreover, demethylating treatment of HCC human cell lines induced the expression of these lncRNAs. The Top35 LNDH were preferentially expressed in the adult healthy liver compared to other tissues and fetal liver and were induced in well-differentiated HepaRG cells. Remarkably, their knockdown compromised the expression of other hepato-specific genes. Finally, the expression of the Top35 LNDH positively correlates with the grade of tumor differentiation and, more importantly, with a better patient prognosis. CONCLUSIONS: Our results demonstrate that the selected Top35 LNDH are not only part of the genes that compose the hepatic differentiated signature but participate in its establishment. Moreover, their downregulation through DNA methylation occurs during the process of hepatocarcinogenesis compromising hepatocellular differentiation and HCC patients' prognosis.

Epigenetic Biomarkers for the Diagnosis and Treatment of Liver Disease.

Research in the last decades has demonstrated the relevance of epigenetics in controlling gene expression to maintain cell homeostasis, and the important role played by epigenome alterations in disease development. Moreover, the reversibility of epigenetic marks can be harnessed as a therapeutic strategy, and epigenetic marks can be used as diagnosis biomarkers. Epigenetic alterations in DNA methylation, histone post-translational modifications (PTMs), and non-coding RNA (ncRNA) expression have been associated with the process of hepatocarcinogenesis. Here, we summarize epigenetic alterations involved in the pathogenesis of chronic liver disease (CLD), particularly focusing on DNA methylation. We also discuss their utility as epigenetic biomarkers in liquid biopsy for the diagnosis and prognosis of hepatocellular carcinoma (HCC). Finally, we discuss the potential of epigenetic therapeutic strategies for HCC treatment.

Deletion or Inhibition of NOD1 Favors Plaque Stability and Attenuates Atherothrombosis in Advanced Atherogenesis †.

Atherothrombosis, the main cause of acute coronary syndromes (ACS), is characterized by the rupture of the atherosclerotic plaque followed by the formation of thrombi. Fatal plaque rupture sites show large necrotic cores combined with high levels of inflammation and thin layers of collagen. Plaque necrosis due to the death of macrophages and smooth muscle cells (SMCs) remains critical in the process. To determine the contribution of the innate immunity receptor NOD1 to the stability of atherosclerotic plaque, Apoe-/- and Apoe-/- Nod1-/- atherosclerosis prone mice were placed on a high-fat diet for 16 weeks to assess post-mortem advanced atherosclerosis in the aortic sinus. The proliferation and apoptosis activity were analyzed, as well as the foam cell formation capacity in these lesions and in primary cultures of macrophages and vascular SMCs obtained from both groups of mice. Our results reinforce the preeminent role for NOD1 in human atherosclerosis. Advanced plaque analysis in the Apoe-/- atherosclerosis model suggests that NOD1 deficiency may decrease the risk of atherothrombosis by decreasing leukocyte infiltration and reducing macrophage apoptosis. Furthermore, Nod1-/- SMCs exhibit higher proliferation rates and decreased apoptotic activity, contributing to thicker fibrous caps with reduced content of pro-thrombotic collagen. These findings demonstrate a direct link between NOD1 and plaque vulnerability through effects on both macrophages and SMCs, suggesting promising insights for early detection of biomarkers for treating patients before ACS occurs.