Targeted delivery of CEBPA-saRNA for the treatment of pancreatic ductal adenocarcinoma by transferrin receptor aptamer decorated tetrahedral framework nucleic acid.

Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia.

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

DNA methylation patterns of transcription factor binding regions characterize their functional and evolutionary contexts.

BACKGROUND: DNA methylation is an important epigenetic modification which has numerous roles in modulating genome function. Its levels are spatially correlated across the genome, typically high in repressed regions but low in transcription factor (TF) binding sites and active regulatory regions. However, the mechanisms establishing genome-wide and TF binding site methylation patterns are still unclear. RESULTS: Here we use a comparative approach to investigate the association of DNA methylation to TF binding evolution in mammals. Specifically, we experimentally profile DNA methylation and combine this with published occupancy profiles of five distinct TFs (CTCF, CEBPA, HNF4A, ONECUT1, FOXA1) in the liver of five mammalian species (human, macaque, mouse, rat, dog). TF binding sites are lowly methylated, but they often also have intermediate methylation levels. Furthermore, biding sites are influenced by the methylation status of CpGs in their wider binding regions even when CpGs are absent from the core binding motif. Employing a classification and clustering approach, we extract distinct and species-conserved patterns of DNA methylation levels at TF binding regions. CEBPA, HNF4A, ONECUT1, and FOXA1 share the same methylation patterns, while CTCF's differ. These patterns characterize alternative functions and chromatin landscapes of TF-bound regions. Leveraging our phylogenetic framework, we find DNA methylation gain upon evolutionary loss of TF occupancy, indicating coordinated evolution. Furthermore, each methylation pattern has its own evolutionary trajectory reflecting its genomic contexts. CONCLUSIONS: Our epigenomic analyses indicate a role for DNA methylation in TF binding changes across species including that specific DNA methylation profiles characterize TF binding and are associated with their regulatory activity, chromatin contexts, and evolutionary trajectories.

Anti-Obesity Properties of Blackberries Fermented with L. plantarum JBMI F5 via Suppression of Adipogenesis Signaling Mechanisms.

Blackberries (Rubus fruticosus), which are known to include a variety of bioactive substances, have been extensively studied for their antioxidant properties. Blackberries possess multiple health beneficial effects, including anti-inflammation, anti-atherosclerosis, anti-tumor and immunomodulatory activity. However, the potential biological effects and precise molecular mechanisms of the fermented extracts remain largely unexplored. In this research, we demonstrate the effect of blackberries fermented with Lactobacillus for addressing obesity. We investigated the effect of blackberries fermented by Lactobacillus on mice fed a high-fat (60% kcal) diet for 12 weeks. Fermented blackberry administration reduced the body weight and epididymal fat caused by a high-fat diet compared to the obese group. The triglyceride and total cholesterol, which are blood lipid indicators, and the levels of leptin, which is an insulin resistance indicator, were significantly increased in the obese group but were significantly decreased in the fermented blackberries-treated group. Additionally, the expression of adipogenesis marker proteins, such as CEBPα, PPAR-γ and SREBP-1, was significantly increased in the obese group, whereas it was decreased in the fermented blackberries-treated group. These results suggest that fermented blackberries have a protective effect against high-fat-diet-induced obesity by inhibiting adipogenesis and are a potential candidate for the treatment of obesity.

Structure of human DPPA3 bound to the UHRF1 PHD finger reveals its functional and structural differences from mouse DPPA3.

DNA methylation maintenance is essential for cell fate inheritance. In differentiated cells, this involves orchestrated actions of DNMT1 and UHRF1. In mice, the high-affinity binding of DPPA3 to the UHRF1 PHD finger regulates UHRF1 chromatin dissociation and cytosolic localization, which is required for oocyte maturation and early embryo development. However, the human DPPA3 ortholog functions during these stages remain unclear. Here, we report the structural basis for human DPPA3 binding to the UHRF1 PHD finger. The conserved human DPPA3 85VRT87 motif binds to the acidic surface of UHRF1 PHD finger, whereas mouse DPPA3 binding additionally utilizes two unique α-helices. The binding affinity of human DPPA3 for the UHRF1 PHD finger was weaker than that of mouse DPPA3. Consequently, human DPPA3, unlike mouse DPPA3, failed to inhibit UHRF1 chromatin binding and DNA remethylation in Xenopus egg extracts effectively. Our data provide novel insights into the distinct function and structure of human DPPA3.

The expression and clinical significance of UHRF1 in soft tissue sarcomas and its prognostic value.

To explore the expression and prognostic value of UHRF1 gene in soft tissue sarcoma (STS) and its related molecular mechanism. The expression data and clinicopathological parameters of STS were downloaded from the Cancer Genome Atlas (TCGA). The expression level of UHRF1 in STS and adjacent tissues and its relationship with clinicopathological characteristics were analyzed. The expression level of UHRF1 in STS tissues was significantly higher than that in paracancerous tissues (P < .001), and the overall survival (OS) time of patients with high UHRF1 expression was significantly shorter than that of patients with low UHRF1 expression (P = .002). The expression of UHRF1 was correlated with tumor necrosis, histological type and metastasis, and the differences were statistically significant (P = .013; P = .001; P = .002). The area ratio under receiver operating characteristic (ROC) curve between STS tissue and adjacent tissue of UHRF1 expression was 0.994. Number of tumors (HR = 0.416, 95%CI = 0.260-0.666, P < .001), depth of tumor (HR = 2.888, 95%CI = 0.910-9.168, P = .033), metastasis (HR = 2.888, 95% CI = 1.762-4.732, P < .001), residual tumor (HR = 2.637, 95% CI = 1.721-4.038, P < .001) and UHRF1 expression (HR = 1.342, 95% CI = 1.105-1.630, P = .003) were significantly associated with OS, and high expression of UHRF1 (HR = 1.387, 95%CI = 1.008-1.907, P = .044) was an independent risk factor for the prognosis of STS patients. The results of the nomogram exhibited that UHRF1 expression level had a significant effect on the total score value. GSEA enrichment analysis suggested that UHRF1 was involved in 14 signaling pathways regulating mRNA spliceosome, cell cycle, P53 signaling pathway were identified. Single sample gene set enrichment analysis (ssGSEA) exhibited that the expression of UHRF1 in STS was positively correlated with the level of Th2 cell infiltration, and negatively correlated with plasmacytoid dendritic cells (pDC), natural killer cells (NK), Eosinophils, Mast cells, etc. UHRF1 expression is involved in the immune microenvironment of HCC and affects the occurrence and development of HCC. UHRF1 is highly expressed in STS tissues. It is involved in the regulation of multiple tumor-related signaling pathways and immune cell microenvironment, suggesting that UHRF1 may be a potential molecular marker for prognosis prediction and targeted therapy of STS patients.

The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-ß signaling.

The E3 ubiquitin-ligase UHRF1 is an epigenetic regulator coordinating DNA methylation and histone modifications. However, little is known about how it regulates adipogenesis or metabolism. In this study, we discovered that UHRF1 is a key regulatory factor for adipogenesis, and we identified the altered molecular pathways that UHRF1 targets. Using CRISPR/Cas9-based knockout strategies, we discovered the whole transcriptomic changes upon UHRF1 deletion. Bioinformatics analyses revealed that key adipogenesis regulators such PPAR-γ and C/EBP-α were suppressed, whereas TGF-ß signaling and fibrosis markers were upregulated in UHRF1-depleted differentiating adipocytes. Furthermore, UHRF1-depleted cells showed upregulated expression and secretion of TGF-ß1, as well as the glycoprotein GPNMB. Treating differentiating preadipocytes with recombinant GPNMB led to an increase in TGF-ß protein and secretion levels, which was accompanied by an increase in secretion of fibrosis markers such as MMP13 and a reduction in adipogenic conversion potential. Conversely, UHRF1 overexpression studies in human cells demonstrated downregulated levels of GPNMB and TGF-ß, and enhanced adipogenic potential. In conclusion, our data show that UHRF1 positively regulates 3T3-L1 adipogenesis and limits fibrosis by suppressing GPNMB and TGF-ß signaling cascade, highlighting the potential relevance of UHRF1 and its targets to the clinical management of obesity and linked metabolic disorders.

Roles of post-translational modifications of UHRF1 in cancer.

UHRF1 as a member of RING-finger type E3 ubiquitin ligases family, is an epigenetic regulator with five structural domains. It has been involved in the regulation of a series of biological functions, such as DNA replication, DNA methylation, and DNA damage repair. Additionally, aberrant overexpression of UHRF1 has been observed in over ten cancer types, indicating that UHRF1 is a typical oncogene. The overexpression of UHRF1 repressed the transcription of such tumor-suppressor genes as CDKN2A, BRCA1, and CDH1 through DNMT1-mediated DNA methylation. In addition to the upstream transcription factors regulating gene transcription, post-translational modifications (PTMs) also contribute to abnormal overexpression of UHRF1 in cancerous tissues. The types of PTM include phosphorylation, acetylation, methylationand ubiquitination, which regulate protein stability, histone methyltransferase activity, intracellular localization and the interaction with binding partners. Recently, several novel PTM types of UHRF1 have been reported, but the detailed mechanisms remain unclear. This comprehensive review summarized the types of UHRF1 PTMs, as well as their biological functions. A deep understanding of these crucial mechanisms of UHRF1 is pivotal for the development of novel UHRF1-targeted anti-cancer therapeutic strategies in the future.

CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair.

Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

Prevotella copri exhausts intrinsic indole-3-pyruvic acid in the host to promote breast cancer progression: inactivation of AMPK via UHRF1-mediated negative regulation.

Emerging evidence has revealed the novel role of gut microbiota in the development of cancer. The characteristics of function and composition in the gut microbiota of patients with breast cancer patients has been reported, however the detailed causation between gut microbiota and breast cancer remains uncertain. In the present study, 16S rRNA sequencing revealed that Prevotella, particularly the dominant species Prevotella copri, is significantly enriched and prevalent in gut microbiota of breast cancer patients. Prior-oral administration of P. copri could promote breast cancer growth in specific pathogen-free mice and germ-free mice, accompanied with sharp reduction of indole-3-pyruvic acid (IPyA). Mechanistically, the present of excessive P. copri consumed a large amount of tryptophan (Trp), thus hampering the physiological accumulation of IPyA in the host. Our results revealed that IPyA is an intrinsic anti-cancer reagent in the host at physiological level. Briefly, IPyA directly suppressed the transcription of UHRF1, following by the declined UHRF1 and PP2A C in nucleus, thus inhibiting the phosphorylation of AMPK, which is just opposite to the cancer promoting effect of P. copri. Therefore, the exhaustion of IPyA by excessive P. copri strengthens the UHRF1-mediated negative control to inactivated the energy-controlling AMPK signaling pathway to promote tumor growth, which was indicated by the alternation in pattern of protein expression and DNA methylation. Our findings, for the first time, highlighted P. copri as a risk factor for the progression of breast cancer.

Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells.

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

UHRF1P contributes to IL-17A-mediated systemic lupus erythematosus via UHRF1-MAP4K3 axis.

Inflammatory T cells contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Analysis of the T-cell transcriptomics data of two independent SLE patient cohorts by three machine learning models revealed the pseudogene UHRF1P as a novel SLE biomarker. The pseudogene-encoded UHRF1P protein was overexpressed in peripheral blood T cells of SLE patients. The UHRF1P protein lacks the amino-terminus of its parental UHRF1 protein, resulting in missing the proteasome-binding ubiquitin-like (Ubl) domain of UHRF1. T-cell-specific UHRF1P transgenic mice manifested the induction of IL-17A and autoimmune inflammation. Mechanistically, UHFR1P prevented UHRF1-induced Lys48-linked ubiquitination and degradation of MAP4K3 (GLK), which is a kinase known to induce IL-17A. Consistently, IL-17A induction and autoimmune phenotypes of UHRF1P transgenic mice were obliterated by MAP4K3 knockout. Collectively, UHRF1P overexpression in T cells inhibits the E3 ligase function of its parental UHRF1 and induces autoimmune diseases.

[Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with GATA2 Gene Mutation].

OBJECTIVE: To investigate the clinical characteristics, coexisting gene mutations and prognosis of acute myeloid leukemia (AML) patients with GATA2 gene mutation. METHODS: The clinical data of 370 newly diagnosed AML patients treated in our hospital from January 2008 to January 2021 was analyzed retrospectively, the next-generation sequencing technology was used to detect the mutated genes in those patients. The clinical characteristics of AML patients with GATA2 mutations, the co-mutated genes of GATA2 mutations, and the effect of GATA2 mutation on prognosis were analyzed. RESULTS: A total of 23 patients (6.2%) with GATA2 mutation was detected in 370 AML patients. Compared with GATA2 non-mutation group, patients in GATA2 mutation group were mostly normal karyotypes (P =0.037) and in low-risk cytogenetic stratification (P =0.028). The incidence of CEBPAdm and NRAS in GATA2 mutation group was significantly higher than that in GATA2 non-mutation group (P =0.010, P =0.009). There were no statistically significant differences between the two groups in terms of sex, age, white blood cell count (WBC), platelet count, hemoglobin, bone marrow (BM) blast, induction chemotherapy regimen and CR rate (P >0.05). Among the 23 patients with GATA2 mutation, the most common co-mutated genes were CEBPAdm, NRAS (both 39.1%), NPM1, FLT3, TET2, WT1 (all 17.4%), ASXL1 and IDH1 (both 13.0%). Survival analysis showed that there was no statistical difference in 5-year overall survival (OS) and leukemia-free survival (LFS) rates between patients with and without GATA2 mutations in whole cohort (n=370) (P =0.306, P =0.308). Among 306 patients without CEBPAdm, the 5-year OS and LFS rates in GATA2 mutation group showed an increasing trend compared with GATA2 non-mutation group, but the difference was not statistically significant (P =0.092, P =0.056). Among 64 patients with CEBPAdm, there was no statistically significant difference in 5-year OS rate between the GATA2 mutation group and the GATA2 non-mutation group (P =0.104), but the 5-year LFS rate of the GATA2 mutation group was significantly decreased (P =0.047). Among the 23 patients with GATA2 mutation, 16 cases received the "3+7" induction regimen, of which 12 cases received allogeneic hematopoietic stem cell transplantation (allo-HSCT); 7 cases received the "DCAG" induction regimen, of which 3 cases received allo-HSCT. The CR rate was not statistically different between the "3+7" regimen group and the "DCAG" regimen group (P =1.000). The 5-year OS rate and LFS rate in the transplantation group were significantly higher than the chemotherapy group (P =0.021, P =0.020). CONCLUSION: GATA2 mutation is more common in AML patients with normal karyotype and low-risk cytogenetic stratification, and it is significantly associated with CEBPAdm and NRAS co-mutations. The prognostic significance of GATA2 is influenced by CEBPAdm. The choice of "3+7" or "DCAG" induction regimen in patients with GATA2 mutation does not affect their CR rate, while the choice of allo-HSCT can significantly improved the prognosis compared with chemotherapy only.

Inhibition of UHRF1 Improves Motor Function in Mice with Spinal Cord Injury.

Spinal-cord injury (SCI) is a severe condition that can lead to limb paralysis and motor dysfunction, and its pathogenesis is not fully understood. The objective of this study was to characterize the differential gene expression and molecular mechanisms in the spinal cord of mice three days after spinal cord injury. By analyzing RNA sequencing data, we identified differentially expressed genes and discovered that the immune system and various metabolic processes play crucial roles in SCI. Additionally, we identified UHRF1 as a key gene that plays a significant role in SCI and found that SCI can be improved by suppressing UHRF1. These findings provide important insights into the molecular mechanisms of SCI and identify potential therapeutic targets that could greatly contribute to the development of new treatment strategies for SCI.

Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids.

KEY MESSAGE: Two peanut LEC1-type genes exhibit partial functional redundancy. AhNFYB10 could complement almost all the defective phenotypes of lec1-2 in terms of embryonic morphology, while AhNF-YB1 could partially affect these phenotypes. LEAFY COTYLEDON1 (LEC1) is a member of the nuclear factor Y (NF-Y) family of transcription factors and has been identified as a key regulator of embryonic development. In the present study, two LEC1-type genes from Arachis hypogeae were identified and designated as AhNF-YB1 and AhNF-YB10; these genes belong to subgenome A and subgenome B, respectively. The functions of AhNF-YB1 and AhNF-YB10 were investigated by complementation analysis of their defective phenotypes of the Arabidopsis lec1-2 mutant and by ectopic expression in wild-type Arabidopsis. The results indicated that both AhNF-YB1 and AhNF-YB10 participate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. In contrast, AhNF-YB10 complemented almost all the defective phenotypes of lec1-2 in terms of embryonic morphology and hypocotyl length, while AhNF-YB1 had only a partial effect. In addition, 30-40% of the seeds of the AhNF-YB1 transformants exhibited a decreasing germination ratio and longevity. Therefore, appropriate spatiotemporal expression of these genes is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage. On the other hand, overexpression of AhNF-YB1 or AhNF-YB10 at the middle to late stages of Arabidopsis seed development improved the weight, oil content, and fatty acid composition of the transgenic seeds. Moreover, the expression levels of several genes associated with fatty acid synthesis and embryogenesis were significantly greater in developing AhNF-YB10-overexpressing seeds than in control seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil content.

Wnt/ß-catenin signaling regulates amino acid metabolism through the suppression of CEBPA and FOXA1 in liver cancer cells.

Deregulation of the Wnt/ß-catenin pathway is associated with the development of human cancer including colorectal and liver cancer. Although we previously showed that histidine ammonia lyase (HAL) was transcriptionally reduced by the ß-catenin/TCF complex in liver cancer cells, the mechanism(s) of its down-regulation by the complex remain to be clarified. In this study, we search for the transcription factor(s) regulating HAL, and identify CEBPA and FOXA1, two factors whose expression is suppressed by the knockdown of ß-catenin or TCF7L2. In addition, RNA-seq analysis coupled with genome-wide mapping of CEBPA- and FOXA1-binding regions reveals that these two factors also increase the expression of arginase 1 (ARG1) that catalyzes the hydrolysis of arginine. Metabolome analysis discloses that activated Wnt signaling augments intracellular concentrations of histidine and arginine, and that the signal also increases the level of lactic acid suggesting the induction of the Warburg effect in liver cancer cells. Further analysis reveals that the levels of metabolites of the urea cycle and genes coding its related enzymes are also modulated by the Wnt signaling. These findings shed light on the altered cellular metabolism in the liver by the Wnt/ß-catenin pathway through the suppression of liver-enriched transcription factors including CEBPA and FOXA1.

UHRF1 poly-auto-ubiquitination induced by the anti-cancer drug, thymoquinone, is involved in the DNA repair machinery recruitment.

DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.

LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. METHODS: Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). RESULTS: Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. CONCLUSIONS: Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

[Gene Profile and Clinical Significance of Concomitant Mutations in CN-AML Patients with CEBPA Mutation].

OBJECTIVE: To analyze the occurrence of concomitant gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients with CEBPA mutation and its impact on the clinical characteristics and prognosis of the patients. METHODS: 151 newly diagnosed patients with CN-AML in the Second Hospital of Shanxi Medical University from June 2013 to June 2020 were analyzed retrospectively. 34 common genetic mutations associated with hematologic malignancies were detected by next-generation sequencing technology. The occurrence of concomitant gene mutations in patients with CEBPA positive and negative groups was compared, and the correlation between concomitant mutations in different functional groups and the clinical characteristics and prognosis of CN-AML patients with CEBPA mutation was analyzed. RESULTS: In 151 patients with CN-AML, 55 (36.42%) were positive for CEBPA mutation (including 36 cases of CEBPAdm and 19 cases of CEBPAsm), of which 41 (74.55%) had co-mutations with other genes. The main mutated genes were GATA2 (25.45%, 14/55), TET2 (21.82%, 12/55), FLT3 (20.00%, 11/55), NRAS (12.73%, 7/55) and WT1 (9.09%, 9/55), etc. Some cases had two or more concomitant gene mutations. Grouping the mutant genes according to their functions showed that CEBPA+ group had lower mutation rates of histone methylation (P =0.002) and chromatin modification genes (P =0.002, P =0.033), and higher mutation rates of transcription factors (P =0.037) than CEBPA- group. In 55 patients with CEBPA+ CN-AML, the platelet count at diagnosis in signaling pathway gene mutation-positive group was lower than that in the mutation-negative group (P =0.005), the proportion of bone marrow blasts in transcription factor mutation-positive group was higher than that in the mutation-negative group (P =0.003), and the onset age in DNA methylation gene mutation-positive group and chromatin modifier mutation-positive group was older than that in the mutation-negative group, respectively (P =0.002, P =0.008). DFS of CEBPA+ CN-AML patients in signaling pathway gene mutation group was shorter than that in signaling pathway gene mutation-negative group (median DFS: 12 months vs not reached) (P =0.034). Compared with DNA methylation gene mutation-negative group, CEBPA+ CN-AML patients with DNA methylation gene mutation had lower CR rate (P =0.025) significantly shorter OS and DFS (median OS: 20 months vs not reached, P =0.006; median DFS: 15 months vs not reached, P =0.049). OS in patients with histone methylation gene mutation was significantly shorter than that in the histone methylation gene mutation-negative group (median OS: 12 months vs 40 months) (P =0.008). Multivariate analysis of prognostic factors showed that the proportion of bone marrow blasts (P =0.046), concomitant DNA methylation gene mutation (P =0.006) and histone methylation gene mutation (P =0.036) were independent risk factors affecting the prognosis. CONCLUSION: CN-AML patients with CEBPA mutation have specific concomitant gene profile, and the concomitant mutations of different functional genes have a certain impact on the clinical characteristics and prognosis of the patients.