Promoter Characterization and Role of cAMP/PKA/CREB in the Basal Transcription of the Mouse ORMDL3 Gene

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.     

Magnesium is required for specific DNA binding of the CREB B-ZIP domain

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5′-TGACGTCA-3′), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl₂ and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5′-TAACGTTA-3′), C/EBP (5′-TTGCGCAA-3′) and AP-1 (5′-TGAGTCA-3′) sites. The addition of 10 mM MgCl₂ diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using ‘standard’ EMSA conditions (0.25× TBE), CREB bound all the DNA sequences examined. The CREB–CRE complex had an apparent K_d of ~300 pM, PAR of ~1 nM, C/EBP and AP-1 of ~3 nM and SP1 of ~30 nM. The addition of 10 mM MgCl₂ to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl₂. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.     

DNMT3L Modulates Significant and Distinct Flanking Sequence Preference for DNA Methylation by DNMT3A and DNMT3B In Vivo

The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.     

DNA Methylation Modulates Nociceptive Sensitization after Incision

DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT) inhibitor 5-Aza-2′-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2′-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.