CEBPA Research Articles

Expressional Evaluation of C/EBP Family, SREBP1, and Steroid Hormone Receptors in the Epididiymal Fat of Postnatally Developing Mouse.

The differentiation and development of preadipocyte into mature adipocyte are regulated by transcription factors, such as CCAAT enhancer binding protein (Cebp) gene family and sterol regulatory element binding transcription factor 1 (Srebp1). Steroid hormones give influences on the development and function of adipocyte. The present research examined expression patterns of CCAAT enhancer binding protein alpha (Cebpa), CCAAT enhancer binding protein beta (Cebpb), CCAAT enhancer binding protein gamma (Cebpg), sterol regulatory element binding transcription factor 1 (Srebp1), androgen receptor (Ar), and estrogen receptors (Esr) among different epididymal fat parts during postnatal period by quantitative real-time polymerase chain reaction. In the distal epididymal fat, expression of Cebpa, Cebpb, Cebpg, Srebp1, Ar, and Esr2 was increased until 12 months of age, while expression of Esr1 was decreased at 5 months of age and was not detectable after 8 months of age. In the proximal epididymal fat, transcript levels of Cebps and Srebp1 were increased at 8 months of age, followed by decreases of Cebpb and Cebpg transcript levels at 12 months of age. An additional increase of Srebp1 expression was observed at 12 months of age. Expression of Ar and Esr2 were increased until 8 months of age, followed by a drop of Ar expression level at 12 months of age. Expression pattern of Esr1 was similar to that in the distal epididymal fat. In the tail epididymal fat, expression of Cebpa, Cebpg, Srebp1, Ar, and Esr2 was increased with age. Esr1 was not detectable at all. The highest level of Cebpb was observed at 8 months of age. These data suggest the possibility of developmental and functional differentiation among the epididymal fat parts.

Low-density cell culture enhances hepatic function through tight junction formation in HepG2 cells.

An in vitro evaluation system using cultured hepatocytes is the most useful method in preclinical research, such as drug metabolism and toxicity test. Human hepatocytes should be used in an in vitro evaluation system because the expression of drug-metabolizing enzymes varies among animal species. HepG2 cells, a liver cancer-derived cell line, are widely used as a human hepatocyte model; however, their hepatic functions are generally weak. In this study, we showed that low-density HepG2 cell culture induces hepatic function. The morphology of HepG2 cells was altered depending on the cell density at the time of seeding. Low-density cultured HepG2 cells proliferated as tightly packed colonies. The HepG2 cell colonies in low-density culture demonstrated enhanced tight junction formation. Tight junction protein gene expression levels, such as those of zonula occludens-1 (ZO-1), junctional adhesion molecule 1 (JAM), claudin, occludin, and tricellulin, increased in low-density cultured HepG2 cells. Phases I and II metabolic enzymes, phase III transporter gene expression, and CYP3A4 activity also increased in low-density cultured HepG2 cells. Occludin and tricellulin knockdown inhibited the increased hepatic function in low-density cultures. Tricellulin knockdown reduced the expression of hepatocyte nuclear factor 6 (HNF6), CCAAT/enhancer-binding protein alpha (CEBPA), and aryl hydrocarbon receptor (AHR). In addition, the expression of nuclear receptor subfamily 1 group h member 2 (NR1H2) increased in low-density cultures, canceled by occludin and tricellulin knockdown. Our results suggest that low-density HepG2 cell cultures enhance hepatic function by promoting tight junction formation and demonstrate the importance of cell density in drug evaluation using hepatocyte cell lines.

Functional roles for AGPAT6 in milk fat synthesis of buffalo mammary epithelial cells

AGPAT6 plays a crucial role in the triglyceride (TG) synthesis pathway in mammals. However, its roles in buffalo lactation remain unknown. Therefore, we investigated the functional roles of AGPAT6 in milk fat synthesis by transfecting overexpression and lentivirus interference vectors in buffalo mammary epithelial cells (BuMECs) in vitro. AGPAT6 overexpression in BuMECs significantly enhanced the mRNA expression of FABP4, SLC27A6, ACSL1, DGAT1, DGAT2, LPIN1, INSIG1, CEBPA and SREBF1 genes, and significantly reduced that of XDH, CPT1A, LIPE, INSIG2 and PPARGC1A, but has no significant influence to the mRNA abundance of FABP3, GPAM, PPARG and SREBF2. However, the interference with AGPAT6, the mRNA expression of FABP4, SLC27A6, ACSL1, DGAT1, DGAT2, INSIG1, CEBPA, SREBF1, XDH, CPT1A, LIPE, INSIG2 and PPARGC1A genes in BuMECs changed contrary to the overexpression experiment, and that of GPAM, PPARG and SREBF2 also did not change significantly, but the expression of FABP3 was significantly decreased. In addition, the overexpression/interference of AGPAT6 gene significantly increased/decreased TG content in BuMECs. The results here indicate that AGPAT6 gene is involved in TG synthesis in BuMECs, and affects the expression of major genes associated with FA transport and activation, TG synthesis and transcription regulation, FA oxidation and TG degradation during the lipogenesis of milk.

Effects of magnolol and honokiol blend on performance, egg quality, hepatic lipid metabolism, and intestinal morphology of hens at late laying cycle

Magnolol and its isomer honokiol are polyphenols with anti-oxidative and anti-inflammatory activities. We evaluated the effects of magnolol and honokiol supplementation alone or in combination with hen diets during the late laying cycle. A total of 540 Jingfen pink-shell laying hens (50 weeks old) were randomly assigned to six treatments: a control diet and diets supplemented with 300 mg/kg magnolol (M300), honokiol (H300), or 300 mg/kg total phenols with a magnolol/honokiol ratio of 2:1 (M200H100), 1:2 (M100H200), and 1:1 (M150H150). Compared with that of the control, all supplementation groups had higher laying rates and the M300, M100H200, and M150H150 groups showed comparatively lower feed conversion ratios. Magnolol and honokiol supplementation increased the Haugh units of fresh eggs at week 62 and alleviated the decline of the Haugh units of eggs stored for 14 days. Compared with that of the control group, the serum total antioxidant capacity of the M100H200 and M150H150 groups significantly increased, and all supplementation groups had higher total antioxidant capacity and lower malondialdehyde content in the liver. With respect to lipid metabolism, the M200H100 and M150H150 groups had lower total and relative liver weights compared with those of the control and H300 groups. The mRNA expression levels of CCAAT enhancer binding protein alpha, sterol regulatory element binding protein-1, fatty acid synthase and stearyl coenzyme A desaturase 1 involved in lipogenesis; microsomal triglyceride transfer protein and apolipoprotein B involved in fatty acid transport; and the proinflammatory cytokine interleukin-1 beta were lower in all supplementation groups compared with those in the control. With respect to gut health, the heights of the jejunum and ileum villi significantly increased in all supplementation groups compared with those of the control, and the jejunum villus heights of the M300 and M150H150 groups were higher than those of the H300 and M100H200 groups. The H300 and M150H150 groups had higher mRNA expression levels of zonula occludens-1 in the ileum compared with those in the control and M300 groups, whereas all supplementation groups had higher mRNA levels of claudin-1 than that of the control group. In conclusion, magnolol and honokiol improved hen performance and the albumen quality of fresh and stored eggs by improving the antioxidant capacity, liver lipid metabolism, and intestinal health of laying hens. The combination of magnolol and honokiol at a 1:1 ratio may be an optimal choice for hen diet supplementation.

Dysregulated Expression of MiR-19b, MiR-25, MiR-17, WT1, and CEBPA in Patients with Acute Myeloid Leukemia and Association with Graft versus Host Disease after Hematopoietic Stem Cell Transplantation.

Elham JamaliObjectives Acute myeloid leukemia (AML) is a blood malignancy characterized by the proliferation of aberrant cells in the bone marrow and blood that interfere with normal blood cells. We have investigated whether changes in the level of micro-ribonucleic acid (miR)-19b, miR-17, and miR-25, Wilms' tumor (WT1), and CCAAT enhancer-binding protein α (CEBPA) genes expression affect disease prognosis and clinical outcome in AML patients. Materials and Methods The expression level of miR-19-b, miR-17, and miR-25, as well as WT1 and CEBPA genes in a group of patients and controls as well as different risk groups (high, intermediate, and favorite risk), M3 versus non-M3, and graft-versus-host disease (GvHD) versus non-GvHD patients were assessed using a quantitative SYBR Green real-time polymerase chain reaction method. Results When compared with the baseline level at the period of diagnosis before chemotherapy, the expression of miR-19b and miR-17 in AML patients increased significantly after chemotherapy. The level of miR-19b and miR-25 expression in AML patients with M3 and non-M3 French-American-British subgroups differ significantly. MiR-19b and miR-25 expression was elevated in GvHD patients, while miR-19b and miR-25 expression was somewhat decreased in GvHD patients compared with non-GvHD patients, albeit the difference was not statistically significant. Also, patients with different cytogenetic aberrations had similar levels of miR-19-b and miR-25 expression. Conclusion MiR-19b, miR-17, and miR-25 are aberrantly expressed in AML patients' peripheral blood leukocytes, which may play a role in the development of acute GvHD following hematopoietic stem cell transplantation.

LncRNA MIR99AHG enhances adipocyte differentiation by targeting miR-29b-3p to upregulate PPARγ

AimThe aim is to identify new long noncoding RNAs (lncRNAs) involved in adipocyte differentiation. MethodsHigh-throughput RNA sequencing of 3T3-L1 preadipocytes was carried out before and after differentiation to identify the target lncRNAs and miRNAs. The effects of lncRNA, miRNA and the network mechanism on adipocyte differentiation were evaluated in vitro and in vivo. Visceral adipose tissue (VAT) was collected from Chinese subjects with obesity or a normal body mass index (BMI), and the levels of lncRNAs, adipogenic genes and miRNAs were measured. ResultsMIR99AHG, miR-29b-3p were selected as the target lncRNA and miRNA. Short hairpin RNA against MIR99AHG inhibited the differentiation of 3T3-L1 preadipocytes, reduced the expression of the peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT enhancer-binding protein alpha (C/EBPα) and fatty acid binding protein 4 (FABP4) genes, upregulated the expression of miR-29b-3p. Overexpression of MIR99AHG showed the opposite effects. Overexpression of miR-29b-3p inhibited the differentiation of 3T3-L1 preadipocytes and decreased the PPARγ level, while inhibition of miR-29b-3p showed the opposite effects. MIR99AHG and PPARγ competed for binding to miR-29b-3p. In mice with high-fat diet-induced obesity, MIR99AHG and miR-29b-3p mRNA level were increased and decreased, respectively. Tail vein injection of adeno-associated virus 9-MIR99AHG-RNA interference (AAV9-MIR99AHG-RNAi) reduced the body weight, epididymal fat mass, MIR99AHG level and increased the expression of miR-29b-3p. The expression levels of MIR99AHG, PPARγ, C/EBPα and FABP4 in human visceral adipose tissue were higher in the obese group than in the normal weight group. ConclusionsMIR99AHG enhances adipogenesis by regulating miR-29b-3p and PPARγ, providing a new target for therapeutic intervention in obesity.

Detection of TET2 Mutation in Patients with De Novo Acute Myeloid Leukemia: A Mutation Analysis of 51 Iranian Patients.

Acute myeloid leukemia (AML) is a heterogeneous clonal disease that is considered to originate from hematopoietic stem cells, which are characterized by impaired myelopoiesis and blast proliferation. TET oncogene family member 2 (TET2) mutations are frequent in myeloid malignancies and several studies have assessed the clinical importance of TET2 mutations. However, its frequency ratio has not yet been fully clarified. Method:Hence, our study was aimed to analyze TET2mut in patients with de-novo AML and their association with clinical, molecular characteristics and Nucleophosmin 1 (NPM1), Fms-like tyrosine kinase 3 (FLT3), CCAAT Enhancer Binding Protein Alpha (CEBPA) and Wilms’ tumor protein (WT1) gene expression. Fifty-one Iranian patients were screened by polymerase chain reaction (PCR) and direct sequencing to evaluate TET2 mutations frequency. Results:Out of all patients, 10 mutations in 8 patients (15.6%) were detected and closely associated with higher age and higher hemoglobin levels (p-value <0.05). Although FLT3, NPM1 and CEBPA gene expression did not show any significant correlation with TET2mut, cytogenetically normal acute myeloid leukemia (CN-AML) patients appear to bear TET2mut more frequently with lower platelet counts. Monocyte-lineages leukemia has seemed to be more linked with TET2mut in these patients. Conclusion:Our study suggests the frequency of TET2mut in our study (15.6%) is in line with previous studies and reveals the critical role of TET2 in myeloid transformation, especially in leukemia with monocytic subtypes.

α-Glucosidase Inhibitory Activity and Anti-Adipogenic Effect of Compounds from Dendrobium delacourii.

Chemical investigation of Dendrobium delacourii revealed 11 phenolic compounds, and the structures of these compounds were determined by analysis of their NMR and HR-ESI-MS data. All compounds were investigated for their α-glucosidase inhibitory activity and anti-adipogenic properties. Phoyunnanin E (10) and phoyunnanin C (11) showed the most potent α-glucosidase inhibition by comparing with acarbose, which was used as a positive control. Kinetic study revealed the non-competitive inhibitors against the enzyme. For anti-adipogenic activity, densifloral B (3) showed the strongest inhibition when compared with oxyresveratrol (positive control). In addition, densifloral B might be responsible for the inhibition of adipocyte differentiation via downregulating the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer-binding protein alpha (C/EBPα), which are major transcription factors in adipogenesis.

Programming With Varying Dietary Fat Content Alters Cardiac Insulin Receptor, Glut4 and FoxO1 Immunoreactivity in Neonatal Rats, Whereas High Fat Programming Alters Cebpa Gene Expression in Neonatal Female Rats.

Fetal programming refers to an intrauterine stimulus or insult that shapes growth, development and health outcomes. Dependent on the quality and quantity, dietary fats can be beneficial or detrimental for the growth of the fetus and can alter insulin signaling by regulating the expression of key factors. The effects of varying dietary fat content on the expression profiles of factors in the neonatal female and male rat heart were investigated and analyzed in control (10% fat), 20F (20% fat), 30F (30% fat) and 40F (40% fat which was a high fat diet used to induce high fat programming) neonatal rats. The whole neonatal heart was immunostained for insulin receptor, glucose transporter 4 (Glut4) and forkhead box protein 1 (FoxO1), followed by image analysis. The expression of 84 genes, commonly associated with the insulin signaling pathway, were then examined in 40F female and 40F male offspring. Maintenance on diets, varying in fat content during fetal life, altered the expression of cardiac factors, with changes induced from 20% fat in female neonates, but from 30% fat in male neonates. Further, CCAAT/enhancer-binding protein alpha (Cebpa) was upregulated in 40F female neonates. There was, however, differential expression of several insulin signaling genes in 40F (high fat programmed) offspring, with some tending to significance but most differences were in fold changes (≥1.5 fold). The increased immunoreactivity for insulin receptor, Glut4 and FoxO1 in 20F female and 30F male neonatal rats may reflect a compensatory response to programming to maintain cardiac physiology. Cebpa was upregulated in female offspring maintained on a high fat diet, with fold increases in other insulin signaling genes viz. Aebp1, Cfd (adipsin), Adra1d, Prkcg, Igfbp, Retn (resistin) and Ucp1. In female offspring maintained on a high fat diet, increased Cebpa gene expression (concomitant with fold increases in other insulin signaling genes) may reflect cardiac stress and an adaptative response to cardiac inflammation, stress and/or injury, after high fat programming. Diet and the sex are determinants of cardiac physiology and pathophysiology, reflecting divergent mechanisms that are sex-specific.

Oleic acid, independent of insulin, promotes differentiation of goat primary preadipocytes

Context Oleic acid together with insulin is widely used to induce preadipocyte differentiation in humans and mammals, and is also used alone in chicken preadipocytes from abdominal adipose tissue. However, it is not clear whether oleic acid alone promotes goat primary intramuscular preadipocyte differentiation. Aims The aim of the present study was to identify the role of oleic acid in regulating primary preadipocyte differentiation in goats. Methods Three healthy, 7-day-old Jianzhou goats were randomly selected. After slaughter, the longissimus dorsi tissues were collected from each goat under sterile procedures and mixed equally. The primary preadipocytes were then prepared using collagenase type I digestion, and treated with 5 mg/L insulin or different concentrations of oleic acid, including 0 μM, 50 μM, 100 μM, 150 μM and 300 μM. The results were determined using microscopy and Oil Red O staining. The expression of genes related to preadipocyte differentiation were determined by real-time fluorescence quantitative polymerase chain reaction. Results Lower concentrations of oleic acid (50 μM, 100 μM and 150 μM) did not affect the cell morphology and cell growth, whereas 300 μM oleic acid led to severe cytotoxicity compared with the control (0 μM). The treatment of oleic acid (100 μM) enhanced cellular accumulation and lipid droplets deposition significantly, which was not affected by supplementary insulin. In addition, insulin alone treatment did not alter cellular adipogenesis in goat intramuscular preadipocytes. Treatment with oleic acid significantly increased the expression of peroxisome proliferator-activated receptor gamma, CCAAT enhancer-binding protein alpha and fatty acid binding protein 4, and decreased the expression of lipoprotein esterase on Day 2 after cell differentiation, all of which decreased continually on Day 4 and Day 6. Expression of all genes increased significantly on Day 8 after oleic acid treatment in goat intramuscular preadipocytes. Conclusion The results underscore the role of oleic acid independent of insulin in promoting intramuscular preadipocytes in goats, and probably via the control of peroxisome proliferator-activated receptor gamma and CCAAT enhancer-binding protein alpha. Implications These data provide insight into the mechanism underlying preadipocyte differentiation.

Identification of a seven-gene prognostic signature using the gene expression profile of osteosarcoma.

BackgroundOsteosarcoma is a malignant bone tumor that typically occurs in adolescents or children under 20 years of age. Developing efficient clinical prognostic markers is crucial for improving the treatment of osteosarcoma patients.MethodsThree datasets related to osteosarcoma were acquired from the Gene Expression Omnibus (GEO) database. A gene signature model was established using the Limma package in the R software, univariate and multivariate survival analyses, and least absolute shrinkage and selection operator (LASSO) algorithms. The gene signature was then verified using external datasets.ResultsFrom the GEO database, 242 differentially expressed genes were identified. A total of 590 unique genes, including 380 genes from the human protein interaction network, were found to be related to biological processes such as bone development and bone cell development. Univariate Cox survival analyses revealed 43 genes that were associated with the prognosis of osteosarcoma patients. A seven-gene signature [retinitis pigmentosa 2 (RP2), polyhydroxybutyrate (PHB), myosin VI (MYO6), mutL homolog 1 (MLH1), Casein kinase 2 beta (CSNK2B), ribosomal protein L37A (RPL37A), and CCAAT/enhancer binding protein alpha (CEBPA)] was developed using LASSO regression analysis and multivariate regression analysis. This gene signature could stratify the prognostic risk of sample cases in the training set, the test set, and the external verification set (P<0.01). The area under the receiver operating characteristic curve for the 5-year survival was higher than 0.72 in both the training and verification groups.ConclusionsIn this study, a seven-gene signature was developed that is highly efficient at predicting the prognosis of patients with osteosarcoma, and therefore, this signature may be a crucial guide in the treatment of these patients.

Polymorphisms within CEBPA, PRKAG3 and SREBF1 Genes Associated with Fat Deposition in Fat-tail Altay Sheep

Polymorphisms within CEBPA, PRKAG3 and SREBF1 Genes Associated with Fat Deposition in Fat-tail Altay Sheep

Genetic Alteration Profiling of Chinese Lung Adenocarcinoma and Its Effect on Targeted Therapy Efficacy.

BackgroundNon-small cell lung cancer (NSCLC) is the most common type of lung cancer and a highly heterogeneous disease with a diversity of phenotypes and genotypes in different populations. The purpose of this study is to investigate oncogenic alterations of lung adenocarcinoma (LUAD) in eastern China and their significance in targeted therapies.MethodsThis study enrolled 101 LUAD patients and used a customized DNA panel to detect molecular alterations. Comprehensive analysis of mutations and clinical application of genomic profiling was carried out.ResultsThe most commonly mutated genes were epidermal growth factor receptor (EGFR) (53%) and tumor protein p53 (TP53) (32%). The less frequently mutated genes were erb-b2 receptor tyrosine kinase 2 (ERBB2) (25%), ATR serine/threonine kinase (ATR) (20%), CCAAT enhancer binding protein alpha (CEBPA) (16%), RB transcriptional corepressor 1 (RB1) (16%), transcription factor 7 like 2 (TCF7L2) (14%), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) (12%) and spectrin alpha, erythrocytic 1 (SPTA1) (12%). Among them, the frequency of ERBB2, ATR, CEBPA, RB1 and TCF7L2 mutations was much higher than that in the databases. Seventy percent of the patients harbored at least one actionable alteration according to the OncoKB evidence. CEBPA mutations affected the efficacy of EGFR-tyrosine kinase inhibitors. ERBB2, CEBPA and TCF7L2 mutated tumors tend to have higher tumor mutation burden (TMB).ConclusionsLUAD patients from eastern China have a unique profile of mutations. The targeted DNA panel is helpful for personalized treatment decision of LUAD patients, and specific mutations may affect the efficacy of targeted therapies.

Galectin-9/TIM-3 as a Key Regulator of Immune Response in Gliomas With Chromosome 1p/19q Codeletion.

Gliomas with chromosome 1p/19q codeletion were considered a specific tumor entity. This study was designed to reveal the biological function alterations tightly associated with 1p/19q codeletion in gliomas. Clinicopathological and RNA sequencing data from glioma patients were obtained from The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases. Gene set variation analysis was performed to explore the differences in biological functions between glioma subgroups stratified by 1p/19q codeletion status. The abundance of immune cells in each sample was detected using the CIBERSORT analytical tool. Single-cell sequencing data from public databases were analyzed using the t-distributed stochastic neighbor embedding (t-SNE) algorithm, and the findings were verified by in vitro and in vivo experiments and patient samples.We found that the activation of immune and inflammatory responses was tightly associated with 1p/19q codeletion in gliomas. As the most important transcriptional regulator of Galectin-9 in gliomas, the expression level of CCAAT enhancer-binding protein alpha in samples with 1p/19q codeletion was significantly decreased, which led to the downregulation of the immune checkpoints Galectin-9 and TIM-3. These results were validated in three independent datasets. The t-SNE analysis showed that the loss of chromosome 19q was the main reason for the promotion of the antitumor immune response. IHC protein staining, in vitro and in vivo experiments verified the results of bioinformatics analysis. In gliomas, 1p/19q codeletion can promote the antitumor immune response by downregulating the expression levels of the immune checkpoint TIM-3 and its ligand Galectin-9.

Synergetic protective effect of berberine and ginsenoside Rb1 against tumor necrosis factor alpha−induced inflammation in adipocytes

ABSTRACT Obesity significantly impacts living a normal life by increasing morbidity. Additionally, obesity has been shown to be closely associated with severe inflammation in adipocytes. It is widely reported that berberine (BBR) has an anti-inflammatory effect and can reduce glucose and lipid accumulation, whereas ginsenoside Rb1 (Rb1) has been shown to have a significant inhibitory effect on insulin resistance and lipid peroxidation. In this study, we aimed to explore the synergetic effect of BBR and Rb1 on tumor necrosis factor alpha (TNF-α)-treated adipocytes and the mechanisms underlying it. We found that TNF-α reduced cell viability, facilitated the production of inflammatory factors, induced adipogenesis, activated the nuclear factor kappa B (NF-κB) pathway, and increased the expression of peroxisome proliferator-activated receptor gamma, CCAAT enhancer-binding protein alpha, and sterol regulatory element-binding protein-1 c in adipocytes. However, these effects were significantly alleviated by BBR or Rb1. Additionally, a synergetic effect was observed when BBR and Rb1 were used in combination. The effects of BBR in combination with Rb1 on cell proliferation, inflammation, adipogenesis, and the NF-κB pathway in TNF-α-treated adipocytes were significantly abolished by receptor activator of nuclear factor kappa-Β ligand, which is an activator of the NF-κB pathway. Collectively, the results revealed that BBR and Rb1 have a synergetic protective effect against TNF-α-induced inflammation in adipocytes. The mechanism underlying this synergetic effect was found to be inhibition of the NF-κB signaling pathway.

Spatiotemporal dynamics of SETD5-containing NCoR\u2013HDAC3 complex determines enhancer activation for adipogenesis

Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated “primed” state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis.

Induced cell-autonomous neutropenia systemically perturbs hematopoiesis in Cebpa enhancer-null mice

AbstractThe transcription factor C/EBPa initiates the neutrophil gene expression program in the bone marrow (BM). Knockouts of the Cebpa gene or its +37kb enhancer in mice show 2 major findings: (1) neutropenia in BM and blood; (2) decrease in long-term hematopoietic stem cell (LT-HSC) numbers. Whether the latter finding is cell-autonomous (intrinsic) to the LT-HSCs or an extrinsic event exerted on the stem cell compartment remained an open question. Flow cytometric analysis of the Cebpa +37kb enhancer knockout model revealed that the reduction in LT-HSC numbers observed was proportional to the degree of neutropenia. Single-cell transcriptomics of wild-type (WT) mouse BM showed that Cebpa is predominantly expressed in early myeloid-biased progenitors but not in LT-HSCs. These observations suggest that the negative effect on LT-HSCs is an extrinsic event caused by neutropenia. We transplanted whole BMs from +37kb enhancer-deleted mice and found that 40% of the recipient mice acquired full-blown neutropenia with severe dysplasia and a significant reduction in the total LT-HSC population. The other 60% showed initial signs of myeloid differentiation defects and dysplasia when they were sacrificed, suggesting they were in an early stage of the same pathological process. This phenotype was not seen in mice transplanted with WT BM. Altogether, these results indicate that Cebpa enhancer deletion causes cell-autonomous neutropenia, which reprograms and disturbs the quiescence of HSCs, leading to a systemic impairment of the hematopoietic process.

Clinical, Cytogenetic Characteristics and Survival of Acute Leukemia in a National Research Center Database, 10-Year Real-World Data Review

IntroductionMajor resources of our current knowledge on acute leukemia epidemiology and prognosis are based on data from clinical trials. Due to the selective bias of clinical trials, data might differ from the general leukemia population in real-life setting. National Clinical Research Center for Blood Disease established a comprehensive database through the electronic health records (EHR) to facilitate research of the hematologic cancers i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and acute promyelocytic leukemia (APL). The aim of the database is to gain insight into the epidemiology of these cancers, to evaluate treatment responses, to compare results between geographical regions of China. Furthermore, with the privilege of national research center, the database expects to identify prognostic and predictive factors for outcome to improve the quality of treatment and patients care.MethodsThe database development was initiated in 2001. Standard data elements were established to capture the key clinical variables. For individual patients, data from EHRs were extracted, integrated and quality checked. The implement of database facilitated the clinical professions to identify eligible patients, establish research projects, conduct retrospective analysis and follow-up patient outcomes. Continued efforts were made for improving the construction and quality of the database over two decades.We performed a 10-year real-world data review in the database to evaluate the quality of the recorded data and, moreover to describe the clinical, cytogenetic characteristics and survival of acute leukemia patients. The completeness for collected variables was acceptable for statistical analysis. In total, 3,404 patients (1,895 males and 1,509 females) who were diagnosed and treated between Jan. 1, 2010 and Dec. 31, 2020 were enrolled. A substantial proportion (>60%) of patients were residents of the northern and northeast region of China. Demographic and baseline characteristics also included age, age class, baseline blood test, transplantation and research participation. Molecular mutations such as nucleophosmin-1 (NPM1), FMS-related tyrosine kinase 3 (FLT3), and CCAAT/enhancer-binding protein alpha (CEBPA) et al were included in the screening panels. We explored the treatment remission rate and prognosis of different chromosomal karyotype groups among AML patients.ResultsThe patient numbers of the AML, ALL and APL subgroups were 2,345, 769 and 290 respectively. Blood routine results well demonstrated the clinical characteristics of each subgroup (Tbl. 1). In AML group, the frequencies of NPM1, FLT3-ITD, KIT and CEBPA double mutations were 17.9%, 13.2%, 8.7% and 10.1%, respectively (Tbl. 2). In term of ALL, 640 cases (83.2%) were B-ALL and 129 (16.8%) were T-ALL. Among B-ALL, 256 cases (33.3%) were Ph positive. 10-year analysis for overall survival shown that AML patients had better outcomes as compared with ALL group (Fig. 1). In this database, 1,780 AML cases (excluding APL) were enrolled in cytogenetic analysis. The survival rates of different cytogenetic risk groups from our real-world data were separated by the ELN2017 and MRC risk stratification respectively (Fig. 2A-B). Remarkably, we found two rare but recurrent abnormalities, 16 cases with t(7;11) (p15;p15) and 12 cases with t(16;21)(p11;q22/q24;q22). Cases showed high relapse and mortality rate. Compared with the normal karyotype group, the survival of both subentities was worse and transplantation might be recommended in CR1 phase (Fig. 2C), therefore, we recommend that these two subtypes might be regarded as the worse risk group, although neither is mentioned in the current guidelines. The incidence of t(8;21) in our database was 17.9% (Fig. 3). To explore the impact of additional chromosomal abnormalities on the prognosis of t(8;21), we found that the overall survival of patients with additional trisomy 4 was worse than those without trisomy 4 (Fig. 2D), which was rarely mentioned in previous reports.ConclusionThe real-world database is of great importance for defining the comprehensive features of AML, APL and ALL in clinical setting. The results offered a remarkable contribution to our knowledge on acute leukemia and identified the prognosis of rare chromosomal karyotype in AML. [Display omitted] DisclosuresWang: AbbVie: Consultancy; Astellas Pharma, Inc.: Research Funding.

NAD Modulates DNA Methylation and Cell Differentiation.

Nutritional intake impacts the human epigenome by directing epigenetic pathways in normal cell development via as yet unknown molecular mechanisms. Consequently, imbalance in the nutritional intake is able to dysregulate the epigenetic profile and drive cells towards malignant transformation. Here we present a novel epigenetic effect of the essential nutrient, NAD. We demonstrate that impairment of DNMT1 enzymatic activity by NAD-promoted ADP-ribosylation leads to demethylation and transcriptional activation of the CEBPA gene, suggesting the existence of an unknown NAD-controlled region within the locus. In addition to the molecular events, NAD- treated cells exhibit significant morphological and phenotypical changes that correspond to myeloid differentiation. Collectively, these results delineate a novel role for NAD in cell differentiation, and indicate novel nutri-epigenetic strategies to regulate and control gene expression in human cells.

Germline mutations among Polish patients with acute myeloid leukemia

BackgroundA small but important proportion of patients (4–10 %) with AML have germline mutations. They can cause the development of AML at an earlier age, confer a higher risk of relapse or predispose to secondary leukemias, including therapy-related leukemias. The analysis of germline mutations in a patient and his/her family is also critical for the selection of suitable family donors if the patient is a candidate for hematopoietic stem cell transplantation (HSCT).Methods103 unrelated consecutive patients with de novo AML were enrolled in the study. Control group consisted of 103 persons from the general population. We performed NGS sequencing of bone marrow cells and buccal swabs DNA of six genes: CEBPA, DDX41, ETV6, TERT, GATA2, and IDH2 to detect germline pathogenic mutations.ResultsIn the investigated group, 49 variants were detected in six genes. 26 of them were somatic and 23 germline. Germline variants were detected in all six tested genes. Eight pathogenic germline mutations were detected in 7 AML patients, in three genes: CEBPA, ETV6, and IDH2. One patient had two pathogenic germinal mutations, one in ETV6 and one in CEBPA gene. We identified one novel pathogenic germline mutation in CEBPA gene. The difference in frequency of all pathogenic germline mutations between the tested (7.77 %) and control groups (0.97 %) was statistically significant (p = 0.046). In the tested group, the median age at AML diagnosis was 11 years lower in patients with pathogenic germline mutations than in patients without them (p = 0.028).ConclusionsWe showed higher frequency of CEBPA, ETV6, and IDH2 germline mutations in AML patients than in control group, which confirms the role of these mutations in the development of AML. We also showed that the median age at the onset of AML in patients with pathogenic germline mutations is significantly lower than in patients without them.