Recruitment of HNF3β to enhancers identified upstream of CYP2B6 promoter.

<p>As detailed in the <i>Materials and Methods</i>, ChIP assays were used to analyze binding of HNF3β to the HNF3β-a (distal), HNF3β-c (proximal) containing, and the -1.6/-1.4kb regions in cultured HPH (A). After precipitation with HNF3β antibody, de-crosslinked DNA fragments were amplified by PCR. Amplification of the promoter of SULT1E1 was used as negative control as reported previously. In separate experiments, BIACORE SPR affinity assays (B) and (C) were carried out to measure the comparative binding kinetics of CYP2B6 enhancers on HNF3β as described under <i>Materials and Methods</i>. Sensorgrams of the interaction generated by the instrument were analyzed by the software BIAeval 3.2.</p

Transactivation of CYP2B6 5’-flanking reporter constructs by HNF3β.

<p>A computer-based search for the first 2 kb of CYP2B6 upstream resulted in the identification of three potential HNF3β-responsive elements located between -1893bp and -350bp (A). HepG2 cells were transfected with HNF3β expression vector in the presence of CYP2B6 promoter constructs containing sequential deletion fragments (B) or the CYP2B6-2.0k harboring one of the mutated HNF3β binding sites (C). Forty eight hours post-transfection, luciferase activities were determined and expressed relative to the control (pGL3-Basic). Data represent the mean ± SD. (n = 3). (<i>*</i>, <i>p <0</i>.<i>05; **</i>, <i>p<0</i>.<i>01</i>).</p

Effects of HNF3β on hCAR-mediated CYP2B6 activation in HepG2 cells.

<p>HepG2 cells were co-transfected with expression plasmids of hCAR, HNF3β or C/EBPα in the presence (A) or absence (B) of CYP2B6-2kb reporter construct as detailed in the <i>Materials and Methods</i>. Transfected cells were then treated with PB (1mM) and CITCO (1 μM) for 24 h. Dual luciferase activities (A) and CYP2B6 mRNA expression (B) were detected and expressed relative to vehicle control by reporter assay and real-time PCR analysis.</p

Effects of HNF3β on the expression and activity of CYP2B6 in HepG2 cells.

<p>HepG2 cells were infected with Negative Control Adenovirus (Ad-NC) or various amounts of adenovirus expressing HNF3β (Ad-HNF3β) for 48 h. Expression of HNF3β mRNA (A), CYP2B6 mRNA (B), and their proteins (C) were measured using real-time PCR and Western blotting assays, respectively. CYP2B6 enzymatic activity (D) was detected using P450-Glo^™ CYP2B6 Assay kit (Promega). In separate experiments, HepG2 cells were infected with pGreen Negtive Control Lentivirus (pGreen-NC) or HNF3β-RNAi lentivirus (HNF3β-shRNA) for 96 h before measuring the mRNA expression of HNF3β (E) and CYP2B6 (F) by real-time PCR. Results are expressed as the mean ± S.D. (n = 3). (<i>*P <0</i>.<i>05</i>, <i>**P <0</i>.<i>01</i>).</p

Correlation between CYP2B6 and HNF3β expression in HPH.

<p>Total RNA was extracted from HPHs prepared from 35 human liver donors. Expression levels of CYP2B6, hCAR, C/EBPα, HNF4α, HNF3β were measured using real-time RT-PCR assays as detailed in the <i>Materials and Methods</i>. Relative gene expression levels from all donors were normalized against a randomly selected single donor. Leaner regression between CYP2B6 and one of these hepatic transcriptional factors was analyzed individually using Pearson’s Correlation Coefficient (JMP 7.0; SAS, NC).</p

DataSheet1_Nrf2−/− regulated lung DNA demethylation and CYP2E1 DNA methylation under PM2.5 exposure.docx

Cytochrome P450 (CYP450) can mediate fine particulate matter (PM2.5) exposure leading to lung injury. Nuclear factor E2-related factor 2 (Nrf2) can regulate CYP450 expression; however, the mechanism by which Nrf2−/− (KO) regulates CYP450 expression via methylation of its promoter after PM2.5 exposure remains unclear. Here, Nrf2−/− (KO) mice and wild-type (WT) were placed in a PM2.5 exposure chamber (PM) or a filtered air chamber (FA) for 12 weeks using the real-ambient exposure system. The CYP2E1 expression trends were opposite between the WT and KO mice following PM2.5 exposure. After exposure to PM2.5,CYP2E1 mRNA and protein levels were increased in WT mice but decreased in KO mice, and CYP1A1 expression was increased after exposure to PM2.5 in both WT and KO mice. CYP2S1 expression decreased after exposure to PM2.5 in both the WT and KO groups. We studied the effect of PM2.5 exposure on CYP450 promoter methylation and global methylation levels in WT and KO mice. In WT and KO mice in the PM2.5 exposure chamber, among the methylation sites examined in the CYP2E1 promoter, the CpG2 methylation level showed an opposite trend with CYP2E1 mRNA expression. The same relationship was evident between CpG3 unit methylation in the CYP1A1 promoter and CYP1A1 mRNA expression, and between CpG1 unit methylation in the CYP2S1 promoter and CYP2S1 mRNA expression. This data suggests that methylation of these CpG units regulates the expression of the corresponding gene. After exposure to PM2.5, the expression of the DNA methylation markers ten-eleven translocation 3 (TET3) and 5-hydroxymethylcytosine (5hmC) was decreased in the WT group but significantly increased in the KO group. In summary, the changes in CYP2E1, CYP1A1, and CYP2S1 expression in the PM2.5 exposure chamber of WT and Nrf2−/− mice might be related to the specific methylation patterns in their promoter CpG units. After exposure to PM2.5, Nrf2 might regulate CYP2E1 expression by affecting CpG2 unit methylation and induce DNA demethylation via TET3 expression. Our study revealed the underlying mechanism for Nrf2 to regulate epigenetics after lung exposure to PM2.5.</p

Additional file 2 of Type 1 diabetes and diet-induced obesity predispose C57BL/6J mice to PM2.5-induced lung injury: a comparative study

Supplementary Material

Additional file 1 of Type 1 diabetes and diet-induced obesity predispose C57BL/6J mice to PM2.5-induced lung injury: a comparative study

Supplementary Material

Genomic Characterization Revealed PM_2.5-Associated Mutational Signatures in Lung Cancer Including Activation of APOBEC3B

Fine particulate matter (PM2.5) exposure causes DNA mutations and abnormal gene expression leading to lung cancer, but the detailed mechanisms remain unknown. Here, analysis of genomic and transcriptomic changes upon a PM2.5 exposure-induced human bronchial epithelial cell-based malignant transformed cell model in vitro showed that PM2.5 exposure led to APOBEC mutational signatures and transcriptional activation of APOBEC3B along with other potential oncogenes. Moreover, by analyzing mutational profiles of 1117 non-small cell lung cancers (NSCLCs) from patients across four different geographic regions, we observed a significantly higher prevalence of APOBEC mutational signatures in non-smoking NSCLCs than smoking in the Chinese cohorts, but this difference was not observed in TCGA or Singapore cohorts. We further validated this association by showing that the PM2.5 exposure-induced transcriptional pattern was significantly enriched in Chinese NSCLC patients compared with other geographic regions. Finally, our results showed that PM2.5 exposure activated the DNA damage repair pathway. Overall, here we report a previously uncharacterized association between PM2.5 and APOBEC activation, revealing a potential molecular mechanism of PM2.5 exposure and lung cancer