CCAAT Enhancer Binding Protein Beta Research Articles

HIF1α inhibits LPS-mediated induction of IL-6 synthesis via SOCS3-dependent CEBPβ suppression in human dental pulp cells

Hypoxia-inducible factor 1 alpha (HIF1α) is a transcriptional factor that plays a key role in the regulation of various molecules expressed in hypoxic conditions. Ischemic/hypoxic conditions are regarded as a distinct characteristic of dental pulp inflammation due to the encasement of pulp tissue within the rigid tooth structure. This study was performed to examine the role of HIF1α in the regulation of interleukin (IL)-6, a proinflammatory cytokine expressed in inflamed dental pulp, in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). LPS stimulation promoted the expression of IL-6 in hDPCs, while HIF1α suppressed the expression of IL-6. Moreover, HIF1α induced suppressor of cytokine signaling 3 (SOCS3) expression in LPS-stimulated hDPCs, and SOCS3 activity led to downregulate expression of CCAAT enhancer-binding protein beta (CEBPβ), an inducer of IL-6. LPS stimulation promoted HIF1α expression in hDPCs and mouse pulp tissue explants cultured under hypoxic conditions. These findings suggest that HIF1α negatively regulates IL-6 synthesis in LPS-stimulated hDPCs via upregulation of SOCS3 and subsequent downregulation of CEBPβ.

Berberine Inhibits Adipogenesis in Porcine Adipocytes via AMP-Activated Protein Kinase-Dependent and -Independent Mechanisms.

Excessive adipogenesis in adipocytes results in obesity. Berberine, a natural isoquinoline alkaloid, has antiobesity properties. However, the underlying molecular mechanisms have remained unclear up to now. In this study, porcine adipocytes were cultured and treated with berberine. Cellular lipid content was measured by Oil Red O staining extraction. The role of an adenosine monophosphate-activated protein kinase (AMPK) signaling pathway was evaluated by the phosphorylation detection of AMPKα protein and knockdown of AMPK alpha1 (Ampka1) gene. Gene expressions were analyzed by Western blot and real-time reverse transcription-polymerase chain reaction (RT-PCR). The results showed that berberine reduced lipid accumulation in porcine adipocytes in a dose- and time-dependent manner and increased phosphorylation of AMPKα. Furthermore, berberine significantly downregulated the mRNA expression of related genes to adipogenesis including peroxisome proliferator activated receptor gamma 2 (Pparg2), CCAAT/enhancer-binding protein alpha (Cebpa), Cebp beta (Cebpb), sterol regulatory element binding transcription factor 1 (Srebf1), acetyl-CoA carboxylase-1 (Acc-1), fatty acid synthase (Fas), fatty acid binding protein 4 (Fabp4), and stearoyl-CoA desaturase 1 (Scd1). Knockdown of Ampka1 markedly reversed the inhibitory effect of berberine on lipid accumulation and mRNA expression of the above genes except Cebpb in porcine adipocytes. Meanwhile, the protein expression of these adipogenic genes in response to berberine and Ampka1 knockdown paralleled the alterations of their mRNA level. These results suggest that berberine inhibits adipogenesis in porcine adipocytes via AMPK-dependent and -independent multiple mechanisms, which would provide an important idea for the reduction of porcine body fat, as well as the prevention and treatment of human obesity.

Transcription Factor CEBPB Inhibits the Expression of the Human HTR1A by Binding to 5' Regulatory Region in Vitro.

This study identified a transcription factor that might bind to the 5′ regulatory region of the HTR1A and explored the potential effect on 5-HT1A receptor expression. Based on JASPAR predictions, the binding of the transcription factor was demonstrated using the electrophoretic mobility shift assay (EMSA). Vectors over-expressing the transcription factor were co-transfected into HEK-293 and SK-N-SH cells with the recombinant pGL3 vector, and relative fluorescence intensity was measured to determine regulatory activity. Additionally, the qRT-PCR and Western blot were also used to identify whether the transcription factor modulated the endogenous expression of 5-HT1A receptor. The results suggest that the transcription factor CCAA/T enhancer binding protein beta (CEBPB) likely binds to the −1219 to −1209 bp (ATG+1) region of the HTR1A. Two sequences located in the −722 to −372 bp and −119 to +99 bp were also identified. Although the effect of CEBPB on endogenous 5-HT1A receptor expression was not significant, it exhibited the strong inhibition on the relative fluorescence intensity and the mRNA level of HTR1A. CEBPB inhibited the human HTR1A expression by binding to the sequence −1219–−1209 bp. This is useful and informative for ascertaining the regulation of 5-HT1A receptor and mental diseases.

Menin Coordinates C/EBPβ-Mediated TGF-β Signaling for Epithelial-Mesenchymal Transition and Growth Inhibition in Pancreatic Cancer

Menin displays either tumor suppression or promotion functions in a context-dependent manner. Previously, we proposed that Menin acts as a tumor suppressor by inhibiting cell growth in pancreatic ductal adenocarcinoma (PDAC), whereas the relationship between the Menin expression and overall survival rate of PDAC patients has not been completely elucidated, indicating the complexity of Menin functions in PDAC progression. Here, we identify Menin as a promoter of epithelial-mesenchymal transition (EMT), which is largely associated with cell migration or metastasis, with modest activity in cell growth inhibition. Ectopic expression of Menin suppresses the expression of CCAAT/enhancer-binding protein beta (CEBPB) and epithelial-specific genes by histone deacetylation and further enhances the TGF-β signaling-related EMT process. We also demonstrate that CCAAT/enhancer binding protein (C/EBP) beta (C/EBPβ; encoded by CEBPB) acts downstream of Menin and TGF-β signaling for balancing growth inhibition and EMT, and C/EBPβ overexpression could restore the anti-cancer functions of Menin in pancreatic cancer by cooperatively activating CDKN2A/B genes and antagonizing EMT processes. Taken together, our results suggest that Menin functions as an oncogene for cancer metastasis upon C/EBPβ depletion or acts as a tumor suppressor by cooperation with C/EBPβ to activate CDKN2A transcription.

Identifying crucial genes for prognosis in septic patients: Gene integration study based on PRISMA guidelines.

Background:Sepsis is a serious clinical condition with a poor prognosis, despite improvements in diagnosis and treatment.Therefore, novel biomarkers are necessary that can help with estimating prognosis and improving clinical outcomes of patients with sepsis.Methods:The gene expression profiles GSE54514 and GSE63042 were downloaded from the GEO database. DEGs were screened by t test after logarithmization of raw data; then, the common DEGs between the 2 gene expression profiles were identified by up-regulation and down-regulation intersection. The DEGs were analyzed using bioinformatics, and a protein-protein interaction (PPI) survival network was constructed using STRING. Survival curves were constructed to explore the relationship between core genes and the prognosis of sepsis patients based on GSE54514 data.Results:A total of 688 common DEGs were identified between survivors and non-survivors of sepsis, and 96 genes were involved in survival networks. The crucial genes Signal transducer and activator of transcription 5A (STAT5A), CCAAT/enhancer-binding protein beta (CEBPB), Myc proto-oncogene protein (MYC), and REL-associated protein (RELA) were identified and showed increased expression in sepsis survivors. These crucial genes had a positive correlation with patients’ survival time according to the survival analysis.Conclusions:Our findings indicate that the genes STAT5A, CEBPB, MYC, and RELA may be important in predicting the prognosis of sepsis patients.

Mineralocorticoid receptor negatively regulates angiogenesis through repression of STAT3 activity in endothelial cells.

The mineralocorticoid receptor (MR) plays important roles in cardiovascular pathogenesis. The function of MR in angiogenesis is still controversial. This study aimed to explore the role of endothelial MR in angiogenesis and to delineate the underlying mechanism. Endothelial-hematopoietic MR knockout (EMRKO) mice were generated and subjected to hindlimb ischemia and injection of melanoma cells. Laser Doppler measurements showed that EMRKO mice had improved blood flow recovery and increased vessel density in ischemic limbs. In addition, EMRKO accelerated growth and increased the vessel density of tumors. Matrigel implantation, aortic ring assays, and tube formation assays demonstrated that MRKO endothelial cells (ECs) manifested increased angiogenic potential. MRKO ECs also displayed increased migration ability and proliferation. MRKO and MR knockdown both upregulated gene expression, protein level, and phosphorylation of signal transducer and activator of transcription 3 (STAT3). Stattic, a selective STAT3 inhibitor, attenuated the effects of MRKO on tube formation, migration, and proliferation of ECs. At the molecular level, MR interacted with CCAAT enhancer-binding protein beta (C/EBPβ) to suppress the transcription of STAT3. Furthermore, interactions between MR and STAT3 blocked the phosphorylation of STAT3. Finally, stattic abolished the pro-angiogenic phenotype of EMRKO mice. Taken together, endothelial MR is a negative regulator of angiogenesis, likely in a ligand-independent manner. Mechanistically, MR downregulates STAT3 that mediates the impacts of MR deficiency on the angiogenic activity of ECs and angiogenesis. Targeting endothelial MR may be a potential pro-angiogenic strategy for ischemic diseases. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bioinformatics Analysis and Competitive Regulation by Transcription Factors of SIRT5 at the Core Promoter Region Using Bovine Adipocytes.

Sirtuin 5 (SIRT5) belongs to the mitochondrial sirtuin family, which constitutes a highly conserved family of nicotinamide adenine dinucleotide NAD+-dependent deacetylases and ADP-ribosyltransferases that play an important regulatory role in stress resistance and metabolic homeostasis. SIRT5, a member of the mitochondrial sirtuins, has been confirmed to exhibit deacetylase, desuccinylase, and demalonylase enzymatic activities. First, we showed that SIRT5 was expressed at the highest level in the bovine testis, followed by longissimus thoracis and subcutaneous adipose tissue, using real-time quantitative PCR and mRNA levels of SIRT5 during adipocyte differentiation, which increased before the first day and then decreased rapidly. To explore the molecular regulation of bovine SIRT5 expression, we cloned a 2-kb fragment of the 5'-regulatory region and the functional proximal minimal promoter of bovine SIRT5. Electrophoretic mobility shift assays and luciferase reporter assays identified Kruppel-like factor 2 (KLF2), CCAAT enhancer binding protein beta (CEBPβ), peroxisome proliferator-activated receptor alpha (PPARα), myogenic differentiation 1 (MYOD), and nuclear respiratory factor 1 (NRF1) binding sites as transcriptional activators or repressors in the core promoter region of SIRT5. In brief, our study focused on the mechanism underlying the transcriptional regulation of SIRT5 expression in bovine adipocytes.

Knockdown of LXRα Inhibits Goat Intramuscular Preadipocyte Differentiation.

Goat intramuscular fat (IMF) content is mainly determined by the processes of intramuscular preadipocytes adipogenic differentiation and mature adipocyte lipid accumulation. However, the underlying regulators of these biological processes remain largely unknown. Here, we report that the expression of Liver X receptor alpha (LXRα) reaches a peak at early stage and then gradually decreases during goat intramuscular adipogenesis. Knockdown of LXRα mediated by two independent siRNAs significantly inhibits intramuscular adipocytes lipid accumulation and upregulates preadipocytes marker- preadipocyte factor 1 (pref1) expression. Consistently, siRNA treatments robustly decrease mRNA level of adipogenic related genes, including CCAAT enhancer binding protein alpha (Cebpα), Peroxisome proliferator activated receptor gamma (Pparg), Sterol regulatory element binding protein isoform 1c (Srebp1c), Fatty acids binding protein (aP2) and Lipoprotein lipase (Lpl). Next, adenovirus overexpression of LXRα does not affect intramuscular adipocytes adipogenesis manifested by Oil Red O signal measurement and adipogenic specific genes detection. Mechanically, we found that both CCAAT enhancer binding protein beta (Cebpβ) and Kruppel like factor 8 (Klf8) are potential targets of LXRα, indicated by having putative binding sites of LXRα at the promoter of these genes and similar expression pattern during adipogenesis comparing to LXRα. Importantly, mRNA levels of Cebpβ and Klf8 are downregulated significantly in goat LXRα knockdown intramuscular adipocyte. These results demonstrate that loss function of LXRα inhibits intramuscular adipogenesis possibly through down-regulation of Cebpβ and Klf8. Our research will provide new insights into mechanical regulation of goat IMF deposition.

Coordinate regulation by transcription factors and DNA methylation in the core promoter region of SIRT6 in bovine adipocytes

Sirtuin6 (SIRT6) is an ADP-ribosyltransferase and NAD+-dependent deacylase of acetyl groups and long-chain fatty acyl groups, and has been shown as a regulator of insulin secretion, glucose metabolism, lipid metabolism, and cancer. In this study, we determined that the bovine SIRT6 showed higher levels of mRNA expression in the testis, longissimus thoracis, and subcutaneous fat tissue. To elucidate the molecular regulation mechanism of bovine SIRT6 expression, we obtained a 2-kb fragment containing the 5′-regulatory region, and the functional proximal minimal promoter of bovine SIRT6 was identified in the −472/−73 bp region. The CCAAT enhancer binding protein beta (CEBPβ), paired box 6 (PAX6), Kruppel-like factor 2 (KLF2), myb proto-oncogene protein (CMYB), nuclear respiratory factor 1 (NRF1), and E2F transcription factor 1 (E2F1) binding sites, as transcriptional activators or repressors in the core promoter region of SIRT6, were determined by electrophoretic mobility shift assay (EMSA) experiments and luciferase reporter assays. In addition, the results from methylation assay and luciferase report assay showed that the bovine SIRT6 promoter activity was coordinately regulated by methylation and NRF1 or E2F1 during bovine adipocyte differentiation. Taken together, this study illuminated the underlying mechanism of methylation and transcription regulation of SIRT6 expression in bovine adipocytes.

C/EBPβ Deletion Promotes Expansion of Poorly Functional Intestinal Regulatory T Cells

Inflammatory Bowel Diseases [IBDs] are chronic intestinal inflammatory conditions in part mediated by CD4+ T cells. Anti-inflammatory Foxp3+ regulatory T cells [Tregs] maintain immune homeostasis and protect against IBD development via multiple mechanisms, including cytokine secretion and cell-cell interaction. CCAAT enhancer binding protein-beta [C/EBPβ] is a stress-responsive transcription factor linked with IBD susceptibility. Whole-body C/EBPβ deficiency induces CD4+ T cell-predominant hyperproliferation, and we hypothesize that this may be due to impaired Treg function. We used the C/EBPβ-/- mice in the CD45RBHigh adoptive transfer model, to assess C/EBPβ-/- CD4+ T cells for their colitiogenic potential, and C/EBPβ-/- CD4+ Foxp3+ Tregs for their ability to inhibit colitis. We assessed Tregs from the C/EBPβ-/- mice for expression of Treg functional genes and proteins. Naïve C/EBPβ-/- CD4+ T cells are more colitogenic in vivo. The exacerbated colitis does not appear to reflect impaired Treg development, however, as C/EBPβ-/- mice displayed more, rather than fewer intestinal CD4+Foxp3+ Tregs in vivo. Instead, this reflects impaired Treg function as seen by the reduced capacity to suppress T cell proliferation in vitro, along with decreased secretion of the anti-inflammatory cytokine IL-10. These findings were corroborated in vivo by additional adoptive co-transfer studies in which wildtype Tregs prevented colitis but C/EBPβ-/- Tregs did not. C/EBPβ deficiency impairs Treg function and potentiates T cell-mediated colitis. A clearer understanding of the function of this transcription factor may provide a novel therapeutic strategy for IBD.

Aerobic Glycolysis Controls Myeloid-Derived Suppressor Cells and Tumor Immunity via a Specific CEBPB Isoform in Triple-Negative Breast Cancer

Myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immunity. Aerobic glycolysis is a hallmark of cancer. However, the link between MDSCs and glycolysis is unknown in patients with triple-negative breast cancer (TNBC). Here, we detect abundant glycolytic activities in human TNBC. In two TNBC mouse models, 4T1 and Py8119, glycolysis restriction inhibits tumor granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulatingfactor (GM-CSF) expression and reduces MDSCs. These are accompanied with enhanced Tcell immunity, reduced tumor growth and metastasis, and prolonged mouse survival. Mechanistically, glycolysis restriction represses the expression of a specific CCAAT/enhancer-binding protein beta (CEBPB) isoform, liver-enriched activator protein (LAP), via the AMP-activated protein kinase (AMPK)-ULK1 and autophagy pathways, whereas LAP controls G-CSF and GM-CSF expression to support MDSC development. Glycolytic signatures that include lactate dehydrogenase A correlate with high MDSCs and low Tcells, and are associated with poor human TNBC outcome. Collectively, tumor glycolysis orchestrates a molecular network of the AMPK-ULK1, autophagy, and CEBPB pathways to affect MDSCs and maintain tumor immunosuppression.

Receptor/gene/protein-mediated signaling connects methylprednisolone exposure to metabolic and immune-related pharmacodynamic actions in liver.

A multiscale pharmacodynamic model was developed to characterize the receptor-mediated, transcriptomic, and proteomic determinants of corticosteroid (CS) effects on clinically relevant hepatic processes following a single dose of methylprednisolone (MPL) given to adrenalectomized (ADX) rats. The enhancement of tyrosine aminotransferase (TAT) mRNA, protein, and enzyme activity were simultaneously described. Mechanisms related to the effects of MPL on glucose homeostasis, including the regulation of CCAAT-enhancer binding protein-beta (C/EBPβ) and phosphoenolpyruvate carboxykinase (PEPCK) as well as insulin dynamics were evaluated. The MPL-induced suppression of circulating lymphocytes was modeled by coupling its effect on cell trafficking with pharmacogenomic effects on cell apoptosis via the hepatic (STAT3-regulated) acute phase response. Transcriptomic and proteomic time-course profiles measured in steroid-treated rat liver were utilized to model the dynamics of mechanistically relevant gene products, which were linked to associated systemic end-points. While time-courses of TAT mRNA, protein, and activity were well described by transcription-mediated changes, additional post-transcriptional processes were included to explain the lack of correlation between PEPCK mRNA and protein. The immune response model quantitatively discerned the relative roles of cell trafficking versus gene-mediated lymphocyte apoptosis by MPL. This systems pharmacodynamic model provides insights into the contributions of selected molecular events occurring in liver and explores mechanistic hypotheses for the multi-factorial control of clinically relevant pharmacodynamic outcomes.

CCAAT-Enhancer Binding Protein-beta (C/EBPP) Regulates Deltasecretase Expression, Mediating the Pathogenesis in Alzheimer's Disease

Delta-secretase, an age-dependent protease, cleaves both APP and Tau, mediating the formation of amyloid plaques and neurofibrillary tangle in Alzheimer's disease (AD). However, how aging contributes to an increase in delta-secretase expression and AD pathologies remains unclear. Here we show that a CCAAT-enhancer-binding protein (C/EBPβ), an inflammation-regulated transcription factor, acts as a key age-dependent effector elevating both delta-secretase (AEP) and inflammatory cytokines expression, mediating AD pathogenesis. We found that C/EBPβ dictates delta-secretase transcription and protein levels in an age-dependent manner. Notably, overexpression of C/EBPβ in young 3xTg mice strongly increases delta-secretase and accelerates the pathological features of AD, exacerbating cognitive dysfunctions, which is abolished by inactive AEP C189S. Conversely, depletion of C/EBPβ from old 3xTg or 5XFAD mice substantially diminishes delta-secretase and reduces AD pathologies, leading to amelioration of cognitive impairment in these AD mouse models. Thus, our findings support that C/EBPβ plays a pivotal role in AD pathogenesis presumably via increasing both delta-secretase expression and neuroinflammation.

ATF4 overexpression induces early onset of hyperlipidaemia and hepatic steatosis and enhances adipogenesis in zebrafish

Activating transcription factor 4 (ATF4) is constitutively expressed in a variety of tissues, and regulates several pathological features associated with metabolic diseases such as non-alcoholic fatty liver diseases (NAFLD) and obesity. However, the role of ATF4 in animal model systems is poorly understood. To investigate ATF4 functions in zebrafish, we conditionally expressed ATF4 proteins, using a Tet-off transgenic system. We observed early-onset hyperlipidaemia and liver steatosis in ATF4 transgenic zebrafish (ATs) without doxycycline treatment (ATs − Dox). Oil Red O (ORO)-stained signals were predominant in the intravascular blood vessels and liver buds of larval ATs − Dox, indicating that ATF4 functionally promotes lipogenesis. Further, ATF4 overexpression accompanied the stimulation of the unfolded protein response. Therefore, adult ATs − Dox showed increased lipid accumulation, which led, in turn, to liver steatosis. Liver histology and ORO staining of ATs − Dox hepatocytes also indicated oxidative stress and induced NASH-like phenotypes. Moreover, ATF4 overexpression accelerated adipocyte differentiation via CCAAT enhancer binding protein-beta and peroxisome proliferator activated receptor-gamma inducible expression. ATs-Dox zebrafish showed increased weight gain with larger fat pads due to adipocyte hyperplasia. In this study, we report that ATF4 is a potential stimulator of lipid biosynthesis and adipogenesis in zebrafish.

222 Dexamethasone Induces Mesenchymal Trans-differentiation and Promotes Hallmarks of Cancer in Glioblastoma

Abstract INTRODUCTION Dexamethasone is the most commonly used drug in oncologic patients. In brain cancer and specifically glioblastoma, the drug is used in high doses to treat brain tumor associated vasogenic edema. Besides the rather immediate anti-inflammatory properties of dexamethasone, the longer term molecular effects have not been sufficiently studied. This work uncovers possibly deleterious dexamethasone-mediated effects through mesenchymal trans-differentiation and promotion of cellular hallmarks of cancer in glioblastoma patients. METHODS Three independent glioblastoma stem cell (GSC) lines with tumorigenic potential were used for the experiments. RT-PCR, Western Blot, Immunohistochemistry, invasion assays, whole-genome expression profiling, and pathway analyses were done with dexamethasone-exposed versus control cells both in vitro and in vivo orthotopic mouse xenograft models. GSCs were also co-exposed to dexamethasone, temozolomide, and camptothecin; the latter as a potential neutralizer of dexamethasone-induced deleterious effects. A dexamethasone induced gene signature was interrogated for survival prognostication in two large independent clinico-genomic databases comprising more than 500 patients. RESULTS >Dexamethasone induced the mesenchymal activator CCAAT/enhancer binding protein beta (CEBPB) in a dose dependent manner in vitro and in dexamethasone-treated orthotopic tumors. Significantly altered cellular functions included cell movement, cell survival, apoptosis, and angiogenesis. Dexamethasone-treated GSCs were protected against temozolomide-induced apoptosis and had significant increase of invasion both in vitro and in vivo. Furthermore, dexamethasone exposure resulted in significant elevation of cell proliferation and angiogenesis in vivo. A prognostic dexamethasone induced 33 gene signature was derived from orthotopic tumor expression profile analysis and validated in large clinico-genomic databases (p-value: 0.0007). Camptothecin was validated as potential partial neutralizer of dexamethasone-induced oncogenic effects. CONCLUSION Dexamethasone exposure induces a genetic program and CEBPB expression in GSCs that adversely affects key cellular functions and response to therapeutics. High risk scores associated with these genes have negative prognostic value in patients. Our findings further suggest well tolerated low-dose camptothecin as a potential neutralizer of adverse dexamethasone-mediated effects.

Abstract LB-232: Inherited determinants of early recurrent somatic mutations in prostate cancer

Abstract Background: Prostate cancer (PCa) is a clinically heterogeneous hormone regulated disease with a strong genetic component and characterized by distinct molecular subclasses. Since hormone levels and related transcription factors vary during individual’s lifetime, we hypothesized that inherited variants within regulatory elements of the human genome might act as facilitators of specific early somatic events in PCa in age/hormone dependent manner. Methods: We developed a computational approach to nominate heritable facilitators of somatic genomic events in the context of the Androgen Receptor (AR) signaling. Germline variants within functionally active regions of the genome (N=21,364) defined as per ENCODE data were ranked by the so called Trigger Score, which quantifies the fraction of the transcriptome putatively modulated by each variant leveraging individuals’ genotypes and transcript levels from a cohort of human benign prostate tissues. Results: We identified 300 polymorphic regulatory elements that associate with DNA repair and hormone regulated transcript levels. In a cohort of 539 PCa and more than 3,000 controls, a variant on 7p14.3 strongly associated with an early recurrent prostate cancer specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene (OR=5.54, P=1.22e-08). Integrated computational analysis suggested a broad transcriptional regulatory impact of the variant. In vitro luciferase assay in PC-3 cells demonstrated allele-specific enhancer activity of the locus that is concomitantly modulated by AR and by CCAAT/Enhancer Binding Protein beta (CEBPB). ChIP assays experiments showed AR and CEBPB binding to the locus. Upon deletion of the genomic locus via CRISPR-Cas9 followed by transcriptome sequencing, we observed differential transcript levels along chromosome 7 between edited versus control cells, but not in edited versus edited or control versus control, and nominated genes that were also predicted to physically interact with the 7p14.3 locus by previously generated Hi-C data from prostate cells. Conclusions: Our study suggests a relevant role in cancer predisposition of non-coding variants that lead to allele specific transactivation with age dependent and tissue specific effect manifesting in early somatic genomic events. Citation Format: Alessandro Romanel, Sonia Iolanda Garritano, Blerta Stringa, Mirjam Blattner, Davide Dalfovo, Dimple Chakravarty, Kellie Cotter, Priyanka Dhingra, Paola Gasperini, Olivier Elemento, Andrea Sboner, Alberto Inga, Ekta Khurana, MarK Rubin, Francesca Demichelis. Inherited determinants of early recurrent somatic mutations in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-232. doi:10.1158/1538-7445.AM2017-LB-232

Inherited determinants of early recurrent somatic mutations in prostate cancer

Prostate cancer is a highly heritable molecularly and clinically heterogeneous disease. To discover germline events involved in prostate cancer predisposition, we develop a computational approach to nominate heritable facilitators of somatic genomic events in the context of the androgen receptor signaling. Here, we use a ranking score and benign prostate transcriptomes to identify a non-coding polymorphic regulatory element at 7p14.3 that associates with DNA repair and hormone-regulated transcript levels and with an early recurrent prostate cancer-specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene. The locus shows allele-specific activity that is concomitantly modulated by androgen receptor and by CCAAT/enhancer-binding protein (C/EBP) beta (CEBPB). Deletion of this locus via CRISPR-Cas9 leads to deregulation of the genes predicted to interact with the 7p14.3 locus by Hi-C chromosome conformation capture data. This study suggests that a polymorphism at 7p14.3 may predispose to SPOP mutant prostate cancer subclass through a hormone-dependent DNA damage response.

Effect of cyclin-dependent kinase 2 recombinant lentivirus in rats with lipopolysaccharide-induced acute lung injury

Objective To investigate the effect of cyclin-dependent kinase 2 (cdk2) recombinant lentivirus in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods Thirty-six adult SD rats were divided into control group, LPS model group and gene intervention group according to the random number table, with 12 rats per group. Rats with LPS-induced ALI were established by intratracheal injection of LPS. Saline solution (60 μL/kg) was injected in control group at the time point of 0, 24, 48 h respectively. Control-lentivirus (60 μL/kg) and cdk2 recombinant lentivirus (60 μL/kg) were injected respectively in LPS model group and gene intervention group at the time point of 0 h and 24 h. After 48 h, LPS (60 μL/kg) with isotonic saline solution were injected in both LPS model group and gene intervention group. Lung tissue samples from right-lower areas were collected at 24 h postinjury to evaluate the pathological changes with HE staining. Expressions of cdk2, clara cell secretory protein (CCSP), phospholipase A2(PLA2) and p-C/EBP β protein were detected by Western blot. Inflammatory factors of tumor necrosis factor-α(TNF-α), interleukin (IL)-1β, IL-6 and IL-10 in serum were measured with ELISA method. Results Inflammatory infiltration and damage to the alveolar structure were serious in LPS model group than control group, while inflammatory infiltration decreased significantly and alveolar structure tended to be normal in gene- intervention group. Expression of Cdk2 in control group (1.00±0.21) and LPS model group (0.93±0.17) were similar, but both were lower than that in gene intervention group (4.29±0.73) (P<0.05). Expression of CCSP in gene intervention group (3.19±0.38) was significantly higher than that in control group (1.00±0.20) and LPS model group (0.32±0.19) (P<0.05). Expression of PLA2 in LPS model group (4.49±0.51) was higher than that in control group (1.00±0.13) and gene intervention group (1.76±0.26) (P<0.05). Meanwhile, the variation of p-C/EBPβ concentration among the groups was similar to CCSP. Expression of TNF-α in LPS model group[(196.34±30.17)pg/ml] was higher than that in control group [(71.24±5.13)pg/ml] and gene intervention group[(86.32±11.02)pg/ml](P<0.05). Changes in IL-1β, IL-6 and IL-10 among the groups were similar to TNF-α. Conclusions Over-expression of Cdk2 plays a protective role for LPS-induced ALIby up-regulating CCSP and down-regulating inflammatory factors such as PLA2, TNF-α, IL-1β, IL-6 and IL-10, as may relate to the phosphorylation of C/EBPβ. Key words: CCAAT-enhancer binding protein-beta; Phosphorylases; Cyclin-dependent kinase 2; Endotoxins; Acute lung injury

Developmental Control of NRAMP1 (SLC11A1) Expression in Professional Phagocytes.

NRAMP1 (SLC11A1) is a professional phagocyte membrane importer of divalent metals that contributes to iron recycling at homeostasis and to nutritional immunity against infection. Analyses of data generated by several consortia and additional studies were integrated to hypothesize mechanisms restricting NRAMP1 expression to mature phagocytes. Results from various epigenetic and transcriptomic approaches were collected for mesodermal and hematopoietic cell types and compiled for combined analysis with results of genetic studies associating single nucleotide polymorphisms (SNPs) with variations in NRAMP1 expression (eQTLs). Analyses establish that NRAMP1 is part of an autonomous topologically associated domain delimited by ubiquitous CCCTC-binding factor (CTCF) sites. NRAMP1 locus contains five regulatory regions: a predicted super-enhancer (S-E) key to phagocyte-specific expression; the proximal promoter; two intronic areas, including 3′ inhibitory elements that restrict expression during development; and a block of upstream sites possibly extending the S-E domain. Also the downstream region adjacent to the 3′ CTCF locus boundary may regulate expression during hematopoiesis. Mobilization of the locus 14 predicted transcriptional regulatory elements occurs in three steps, beginning with hematopoiesis; at the onset of myelopoiesis and through myelo-monocytic differentiation. Basal expression level in mature phagocytes is further influenced by genetic variation, tissue environment, and in response to infections that induce various epigenetic memories depending on microorganism nature. Constitutively associated transcription factors (TFs) include CCAAT enhancer binding protein beta (C/EBPb), purine rich DNA binding protein (PU.1), early growth response 2 (EGR2) and signal transducer and activator of transcription 1 (STAT1) while hypoxia-inducible factors (HIFs) and interferon regulatory factor 1 (IRF1) may stimulate iron acquisition in pro-inflammatory conditions. Mouse orthologous locus is generally conserved; chromatin patterns typify a de novo myelo-monocytic gene whose expression is tightly controlled by TFs Pu.1, C/ebps and Irf8; Irf3 and nuclear factor NF-kappa-B p 65 subunit (RelA) regulate expression in inflammatory conditions. Functional differences in the determinants identified at these orthologous loci imply that species-specific mechanisms control gene expression.

Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response.

Fasting elicits transcriptional programs in hepatocytes leading to glucose and ketone production. This transcriptional program is regulated by many transcription factors (TFs). To understand how this complex network regulates the metabolic response to fasting, we aimed at isolating the enhancers and TFs dictating it. Measuring chromatin accessibility revealed that fasting massively reorganizes liver chromatin, exposing numerous fasting-induced enhancers. By utilizing computational methods in combination with dissecting enhancer features and TF cistromes, we implicated four key TFs regulating the fasting response: glucocorticoid receptor (GR), cAMP responsive element binding protein 1 (CREB1), peroxisome proliferator activated receptor alpha (PPARA), and CCAAT/enhancer binding protein beta (CEBPB). These TFs regulate fuel production by two distinctly operating modules, each controlling a separate metabolic pathway. The gluconeogenic module operates through assisted loading, whereby GR doubles the number of sites occupied by CREB1 as well as enhances CREB1 binding intensity and increases accessibility of CREB1 binding sites. Importantly, this GR-assisted CREB1 binding was enhancer-selective and did not affect all CREB1-bound enhancers. Single-molecule tracking revealed that GR increases the number and DNA residence time of a portion of chromatin-bound CREB1 molecules. These events collectively result in rapid synergistic gene expression and higher hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARA levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARA target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to restore homeostasis upon fasting.