Methyl-CpG binding domain (MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a ß-sheet in the MBD to affect embryonic development in Bombyx mori.

Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in ß1-ß6 or α1 in the MBD showed that ß2-ß3-turns in the ß-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, ß4-ß6 and an α-helix, play a role in stabilizing the ß-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without ß4-ß6 and α-helix does not alter embryonic development. These results suggest that MBD2/3-L recognizes and binds to mCpG through the intact ß-sheet structure in its MBD, thus ensuring silkworm embryonic development.

Homeobox protein A1-like and DNA methylation regulate embryo-specific Zinc finger protein 615 gene expression and embryonic development in the silkworm Bombyx mori.

DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the -223 and -190 nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the -223 and -190 nt region of its promoter. Simultaneous RNAi-mediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.

The TIAR-mediated Nrf2 response to oxidative stress is mediated through the Nrf2 noncoding 3'untranslated region in Spodoptera litura.

Nrf2 is a key regulator in the maintenance of cellular redox balance by regulating the expression of genes related to antioxidative responses and detoxification. Nrf2 protein levels are increased in response to oxidative stress. However, the regulation of the Nrf2 3'UTR on Nrf2 translation is unclear. Here, we report that the translational activity of the 3'UTR is required for Spodoptera litura Nrf2 protein expression. Experiments showed that the 3'UTR translation activity of S. litura Nrf2 was much higher than that of the 5'UTR. RNA interference (RNAi) of the expression of T cell internal antigen-related protein (TIAR), an RNA-binding protein that interacts with the 3'UTR of S. litura Nrf2, resulted in Nrf2 mRNA movement out of translationally active polysomes and a decrease in cellular Nrf2 protein levels. TIAR interacted with poly(A)-binding protein (PABP) and translation initiation factors eIF2-2 and eIF2-3 to enhance Nrf2 translation, indicating that the 3'UTR regulates Nrf2 translation. Diethyl maleate (DEM) treatment increased reactive oxygen species (ROS) in cells and enhanced Nrf2 levels, which had been reduced by cycloheximide (CHX), an inhibitor of de novo protein synthesis; Tiar RNAi increased ROS levels in DEM-treated cells, suggesting TIAR-mediated 3'UTR involvement in Nrf2 translation in response to DEM treatment. Thus, we reveal a posttranscriptional regulation mechanism of Nrf2, in which TIAR binds with the Nrf2 mRNA 3'UTR to enhance Nrf2 translation, facilitating the increase in Nrf2 protein levels in response to oxidative stress.

Enhanced mixing state of black carbon with nitrate in single particles during haze periods in Zhengzhou, China.

Black carbon (BC) plays an important role in air quality and climate change, which is closely associated with its mixing state and chemical compositions. In this work the mixing state of BC-containing single particles was investigated to explore the evolution process of ambient BC particles using a single particle aerosol mass spectrometer (SPAMS) in March 2018 in Zhengzhou, China. The BC-containing particles accounted for 61.4% of total detected ambient single particles and were classified into five types including BC-nitrate (BC-N, 52.3%) as the most abundant species, followed by BC-nitrate-sulfate (BC-NS, 22.4%), BCOC (16.8%), BC-fresh (BC-F, 4.5%) and BC-sulfate particles (BC-S, 4.0%). With enhancement of the ambient nitrate concentration, the relative peak area (RPA) of nitrate in BC-N and BCNS particles both increased, yet only the number fraction (Nf) of BCN particles increased while the Nf of BC-NS particles decreased, suggesting that the enhanced mixing state of BC with nitrate was mainly due to the increase in the ambient nitrate mass concentration. In addition, the Nf of BC-N decreased from 65.3% to 28.4% as the absorbing Ångström exponents (AAE) of eBC increased from 0.75 to 1.45, which indicated the reduction of light absorption ability of aged BC particles with the enhanced formation of BC-N particles. The results of this work indicated a change in the mixing state of BC particles due to the dominance of nitrate in PM2.5, which also influenced the optical properties of aged BC particles.