Structural and functional insights into the epigenetic regulator MRG15.

MORF4-related gene on chromosome 15 (MRG15), a chromatin remodeller, is evolutionally conserved and ubiquitously expressed in mammalian tissues and cells. MRG15 plays vital regulatory roles in DNA damage repair, cell proliferation and division, cellular senescence and apoptosis by regulating both gene activation and gene repression via associations with specific histone acetyltransferase and histone deacetylase complexes. Recently, MRG15 has also been shown to rhythmically regulate hepatic lipid metabolism and suppress carcinoma progression. The unique N-terminal chromodomain and C-terminal MRG domain in MRG15 synergistically regulate its interaction with different cofactors, affecting its functions in various cell types. Thus, how MRG15 elaborately regulates target gene expression and performs diverse functions in different cellular contexts is worth investigating. In this review, we provide an in-depth discussion of how MRG15 controls multiple physiological and pathological processes.

Tumor cell SPTBN1 inhibits M2 polarization of macrophages by suppressing CXCL1 expression.

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment, and the M2-type TAMs can promote tumor growth, invasion and angiogenesis, and suppress antitumor immune responses. It has been reported that spectrin beta, non-erythrocytic 1 (SPTBN1) may inhibit the infiltration of macrophages in Sptbn1+/-  mouse liver, but whether tumor SPTBN1 affects TAMs polarization remains unclear. This study investigated the effect and mechanism of tumor cell SPTBN1 on polarization and migration of TAMs in hepatoma and breast cancer. By analyzing tumor immune databases, we found a negative correlation between SPTBN1 and abundance of macrophages and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. By reverse transcription-quantitative real-time PCR assays and cell migration assays, the migration and M2 polarization of macrophages were enhanced by the culture medium from hepatocellular carcinoma cell line PLC/PRF/5, SNU449, and breast cancer cell line MDA-MB-231 with SPTBN1 suppression, which could be reversed by CXCL1 neutralizing antibody MAB275. Meanwhile, the ability of migration and colony formation of PLC/PRF/5, SNU449, and MDA-MB-231 cells were promoted when coculture with M2 macrophages. We also found that SPTBN1 regulated CXCL1 through p65 by cytoplasmic-nuclear protein isolation experiments and ChIP-qPCR. Our data suggest that tumor cell SPTBN1 inhibits migration and M2-type polarization of TAMs by reducing the expression and secretion of CXCL1 via inhibiting p65 nuclear localization.

Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology.

BACKGROUND: Protein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes. RESULTS: The FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification. CONCLUSION: The TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.

Deletion of BACH1 Attenuates Atherosclerosis by Reducing Endothelial Inflammation.

BACKGROUND: The transcription factor BACH1 (BTB and CNC homology 1) suppressed endothelial cells (ECs) proliferation and migration and impaired angiogenesis in the ischemic hindlimbs of adult mice. However, the role and underlying mechanisms of BACH1 in atherosclerosis remain unclear. METHODS: Mouse models of atherosclerosis in endothelial cell (EC)-specific-Bach1 knockout mice were used to study the role of BACH1 in the regulation of atherogenesis and the underlying mechanisms. RESULTS: Genetic analyses revealed that coronary artery disease-associated risk variant rs2832227 was associated with BACH1 gene expression in carotid plaques from patients. BACH1 was upregulated in ECs of human and mouse atherosclerotic plaques. Endothelial Bach1 deficiency decreased turbulent blood flow- or western diet-induced atherosclerotic lesions, macrophage content in plaques, expression of endothelial adhesion molecules (ICAM1 [intercellular cell adhesion molecule-1] and VCAM1 [vascular cell adhesion molecule-1]), and reduced plasma TNF-α (tumor necrosis factor-α) and IL-1ß levels in atherosclerotic mice. BACH1 deletion or knockdown inhibited monocyte-endothelial adhesion and reduced oscillatory shear stress or TNF-α-mediated induction of endothelial adhesion molecules and/or proinflammatory cytokines in mouse ECs, human umbilical vein ECs, and human aortic ECs. Mechanistic studies showed that upon oscillatory shear stress or TNF-α stimulation, BACH1 and YAP (yes-associated protein) were induced and translocated into the nucleus in ECs. BACH1 upregulated YAP expression by binding to the YAP promoter. BACH1 formed a complex with YAP inducing the transcription of adhesion molecules. YAP overexpression in ECs counteracted the antiatherosclerotic effect mediated by Bach1-deletion in mice. Rosuvastatin inhibited BACH1 expression by upregulating microRNA let-7a in ECs, and decreased Bach1 expression in the vascular endothelium of hyperlipidemic mice. BACH1 was colocalized with YAP, and the expression of BACH1 was positively correlated with YAP and proinflammatory genes, as well as adhesion molecules in human atherosclerotic plaques. CONCLUSIONS: These data identify BACH1 as a mechanosensor of hemodynamic stress and reveal that the BACH1-YAP transcriptional network is essential to vascular inflammation and atherogenesis. BACH1 shows potential as a novel therapeutic target in atherosclerosis.

Loss of SPTBN1 Suppresses Autophagy Via SETD7-mediated YAP Methylation in Hepatocellular Carcinoma Initiation and Development.

BACKGROUND & AIMS: Loss of Spectrin beta, non-erythrocytic 1 (SPTBN1) plays an important role in the carcinogenesis of hepatocellular carcinoma (HCC); however, the mechanisms underlying its involvement remain poorly understood. Defects in autophagy contribute to hepatic tumor formation. Hence, in this study, we explored the role and mechanism of SPTBN1 in the autophagy of hepatic stem cells (HSCs) and HCC cells. METHODS: Expansion, autophagy, and malignant transformation of HSCs were detected in the injured liver of Sptbn1+/- mice induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment. Hippo pathway and Yes-associated protein (YAP) stabilization were examined in isolated HSCs, Huh-7, and PLC/PRF/5 HCC cells and hepatocytes with or without loss of SPTBN1. RESULTS: We found that heterozygous SPTBN1 knockout accelerated liver tumor development with 3,5-diethoxycarbonyl-1,4-dihydrocollidine induction. Rapamycin promoted autophagy in murine HSCs and reversed the increased malignant transformation induced by heterozygous SPTBN1 deletion. Loss of SPTBN1 also decreased autophagy and increased YAP stability and nuclear localization in human HCC cells and tissues, whereas YAP inhibition attenuated the effects of SPTBN1 deficiency on autophagy. Finally, we found that SPTBN1 positively regulated the expression of suppressor of variegation 3-9-enhancer of zeste-trithorax domain containing lysine methyltransferase 7 to promote YAP methylation, which may lead to YAP degradation and inactivation. CONCLUSIONS: Our findings provide the first demonstration that loss of SPTBN1 impairs autophagy of HSCs to promote expansion and malignant transformation during hepatocarcinogenesis. SPTBN1 also cooperates with suppressor of variegation 3-9-enhancer of zeste-trithorax domain containing lysine methyltransferase 7 to inactive YAP, resulting in enhanced autophagy of HCC cells. These results may open new avenues targeting SPTBN1 for the prevention and treatment of HCC.

SPTBN1 inhibits growth and epithelial-mesenchymal transition in breast cancer by downregulating miR-21.

SPTBN1 (spectrin beta, non-erythrocytic 1) has been linked to tumor progression and epithelial-mesenchymal transition (EMT). However, the role of SPTBN1 has yet to be investigated in breast cancer. This study aimed to evaluate the viability, growth, and migration ability of the breast cancer cell line MDA-MB-231 and BT549 using CCK-8 assay, xenograft models, and Transwell assays. The expression of SPTBN1, EMT-related genes, and miRNA21 in breast cancer cells and tissues were assessed by quantitative real-time polymerase chain reaction (qPCR) and Western blot. SPTBN1 staining of breast cancer tissues was analyzed by the Human Protein Atlas databases. Both chromatin immunoprecipitation qPCR and immunofluorescence were performed to detect how SPTBN1 regulates miRNA21. Our results showed that the expression of SPTBN1 in primary breast cancer tumors was dramatically lower than that in normal tissues and that lower levels of SPTBN1 were associated with significantly shorter progression-free survival. We also discovered that the loss of SPTBN1 promotes EMT, the viability of MDA-MB-231 and BT549 in vitro, and the growth of MDA-MB-231 tumor xenografts in vivo by upregulating miR-21 level. Furthermore, loss of SPTBN1-mediated miR-21 upregulation was dependent on the stability and nuclear translocation of NF-κB p65. Therefore, SPTBN1 is a pivotal regulator that inhibits EMT and the growth of breast cancer.

CDK9 inhibitor CDKI-73 is synergetic lethal with PARP inhibitor olaparib in BRCA1 wide-type ovarian cancer.

Poly (adenosine diphosphate ribose) polymerase (PARP) inhibitors benefit a small percentage of ovarian cancer patients with homologous recombination (HR) deficiency (HRD), which greatly limits the applications of PARP inhibitors. Given the function of CDK9 in homologous recombination repair (HRR), here, we show how to extend the utility of PARP inhibitors in BRCA1-proficient ovarian cancer by targeting CDK9. We found that high CDK9 expression is associated with a higher tumor stage in epithelial ovarian cancer patients, and CDK9 is co-expressed with BRCA1 by analyzing a public database. By using a CDK9 inhibitor CDKI-73, we found that its combination with the PARP inhibitor olaparib significantly suppressed cell viability and colony formation and induced apoptosis in BRCA1-proficient ovarian cancer cells. Consistently, the combination treatment remarkably reduced the tumor growth in mouse xenograft models. We demonstrated that CDKI-73 could downregulate BRCA1 expression, resulting in hypersensitivity to olaparib in BRCA1-proficient ovarian cancer. Taken together, our results show a synergetic effect of CDKI-73 combined with olaparib in BRCA1-proficient ovarian cancer, facilitating the clinical use of CDK9 as a predictive biomarker to exploit PARP inhibitors.

Bach1-induced suppression of angiogenesis is dependent on the BTB domain.

The transcription factor Bach1 impairs angiogenesis after ischemic injury by suppressing Wnt/ß-catenin signaling; however, the specific domains responsible for the anti-angiogenic effects of Bach1 remain unclear. This study determined the role of the BTB domain of Bach1 in ischemic angiogenesis. Bach1 is highly expressed in circulating endothelial cells from acute myocardial infarction patients and is the early induction gene after ischemia. Mice were treated with adenoviruses coding for GFP (AdGFP), Bach1 (AdBach1), or a Bach1 mutant lacking the BTB domain (AdBach1-ΔBTB) after surgically induced hind-limb ischemia. Measures of blood-flow recovery, capillary density, and the expression of vascular endothelial growth factor (VEGF) and heme oxygenase-1 (HO-1) were significantly lower and ROS levels were higher in the AdBach1 group, but not in AdBach1-ΔBTB animals. Furthermore, transfection with AdBach1, but not AdBach1-ΔBTB, in human endothelial cells was associated with significant declines in 1) capillary density and hemoglobin content in the Matrigel-plug assay, 2) proliferation, migration, tube formation, and VEGF and HO-1 expression in endothelial cells. Bach1 binds directly with TCF4, and this interaction is mediated by residues 81-89 of the Bach1 BTB domain and the N-terminal domain of TCF4. Bach1, but not Bach1-ΔBTB, also co-precipitated with histone deacetylase 1 (HDAC1), while the full-length HDAC1 proteins, but not HDAC1 mutants lacking the protein-interaction domain, co-precipitated with Bach1. Collectively, these results demonstrate that the anti-angiogenic activity of Bach1 is crucially dependent on molecular interactions that are mediated by the protein's BTB domain, and this domain could be a drug target for angiogenic therapy.

BTB and CNC homology 1 (Bach1) promotes human ovarian cancer cell metastasis by HMGA2-mediated epithelial-mesenchymal transition.

Transcriptional factor BTB and CNC homology 1 (Bach1) has been linked to tumor progression and metastasis, but the mechanisms underlying the effects of Bach1 on tumor growth and metastasis are largely uncharacterized. Here, we report that Bach1 expression was significantly higher in human epithelial ovarian cancer (EOC) tissues than in normal ovarian tissues and that higher levels of Bach1 were associated with tumor stage and poorer overall and progression-free survival. We found that Bach1 enhanced the expression of epithelial-mesenchymal transition (EMT) genes, including Slug and Snail, and promoted cell migration by recruiting HMGA2 in the human EOC cell line A2780. Bach1 overexpression enhanced and Bach1 knockout reduced the expression of Slug and the metastasis of EOC cells in a tumor metastasis mouse model. Bach1 expression was positively correlated with Slug and HMGA2 expression in human ovarian cancer tissues. In addition, Bach1 activated p-AKT and p-p70S6K, increased the expression of cyclin D1, and promoted the growth of ovarian cancer cells in vitro and tumor xenografts in vivo. Together, our findings reveal that Bach1 enhances tumor growth and recruits HMGA2 to promote EMT and ovarian cancer metastasis.

Bach1: Function, Regulation, and Involvement in Disease.

The transcription factor BTB and CNC homology 1 (Bach1) is widely expressed in most mammalian tissues and functions primarily as a transcriptional suppressor by heterodimerizing with small Maf proteins and binding to Maf recognition elements in the promoters of targeted genes. It has a key regulatory role in the production of reactive oxygen species, cell cycle, heme homeostasis, hematopoiesis, and immunity and has been shown to suppress ischemic angiogenesis and promote breast cancer metastasis. This review summarizes how Bach1 controls these and other cellular and physiological and pathological processes. Bach1 expression and function differ between different cell types. Thus, therapies designed to manipulate Bach1 expression will need to be tightly controlled and tailored for each specific disease state or cell type.