Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP) Gene Transcription Is Regulated by Sp1

Background: Mitochondria-localized glutamic acid-rich protein (MGARP) is a novel mitochondrial transmembrane protein expressed mainly in steroidogenic tissues and in the visual system. Previous studies showed that MGARP functions in hormone biosynthesis and its expression is modulated by the HPG axis. Methodology/principal findings: By bioinformatics, we identified two characteristic GC-rich motifs that are located proximal to the transcription start site (TSS) of MGARP, and each contains two Specificity protein 1 (Sp1) binding elements. We then determined that the −3 kb proximal MGARP promoter is activated in a Sp1-dependent manner using reporter assays and knockdown of Sp1 led to decreased expression of endogenous MGARP messages. We also demonstrated that one of the two GC-rich motifs, GC-Box1, harbors prominent promoter activity mediated by Sp1, and that it requires both GC boxes for full transcriptional activation. These findings suggest a dominant role for these GC boxes and Sp1 in activating the MGARP promoter through a synergistic mechanism. Consistently, the results of an Electrophoretic Mobility Gel Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) confirmed that Sp1 specifically interacts with the GC-rich region. We further found that estrogen receptor α (ERα), a known Sp1 co-activator, could potentiate GC-boxes containing MGARP promoter activity and this effect is mediated by Sp1. Knockdown of Sp1 significantly diminished the MGARP promoter transactivation and the expression of endogenous MGARP mediated by both Sp1 and ERα. Conclusions/significance: The present study identified a proximal core sequence in the MGARP promoter that is composed of two enriched Sp1 binding motifs and established Sp1 as one major MGARP transactivator whose functions are synergistic with ERα, providing a novel understanding of the mechanisms of MGARP gene transcriptional regulation

Fire history and human activity revealed through poly cyclic aromatic hydrocarbon (PAH) records at archaeological sites in the middle reaches of the Yellow River drainage basin, China

Disentangling the role of natural and anthropogenic factors is a major challenge in paleofire studies. Here, we introduce the molecular biomarkers of polycyclic aromatic hydrocarbon (PAHs), combined with charcoal and black carbon in sediments of archaeological sites, to identify anthropogenic signals of environmental change during the middle Holocene. In the prehistoric period, an intensified-fire regime period, inferred on the basis of PAHs, char and macro-charcoal levels, has been attributed to intensified anthropogenic biomass burning from dry crop cultivation between 6500 and 5000 and 3900-3600 yr BP., corresponding to the earliest Chinese settlements during the Miaodigou & Banpo IV Culture and the development of the Erlitou Culture under drier climate conditions. Subsequently, the fire frequency decreased notably between 4500 and 4000 yr BP. as the climate became wetter and there were shifts in the frequency of high-magnitude floods, corresponding to the abandonment of late Neolithic settlements in the floodplain. Changes in the PAH fluxes in sediments are positively correlated with intensified-fire regime periods, consistent with the charcoal and black carbon fluxes during those periods. Principal component analysis of PAH fluxes suggests that these intensified-fire events have a pyrogenic signature, implying that the PAHs were derived from biomass burning of fuelwood and other woody plants. In this study, PAHs in loess sediments have been used as the indicators of anthropogenic biomass burning through time. Meanwhile, PAHs also record the shift of modes of production and the evolution of human settlements as well as the choices made in response to limited natural resources. To comprehensively analyze the paleofire records of PAHs, charcoal and black carbon combined with multiproxy evidence are needed to understand wildfire history as well as human land use and social and cultural development across the study regions in depth

Current Status and Future Potential of Energy Derived from Chinese Agricultural Land: A Review

Energy crisis is receiving attention with regard to the global economy and environmental sustainable development. Developing new energy resources to optimize the energy supply structure has become an important measure to prevent energy shortage as well as achieving energy conservation and emission reduction in China. This study proposed the concept of energy agriculture and constructed an energy agricultural technical support system based on the analysis of energy supply and demand and China’s foreign dependence on energy resources, combined with the function of agriculture in the energy field. Manufacturing technology equipment and agricultural and forestry energy, including crop or forestry plants and animal feces, were used in the system. The current status and future potential of China’s marginal land resources, energy crop germplasm resources, and agricultural and forestry waste energy-oriented resources were analyzed. Developing the function of traditional agriculture in food production may promote China’s social, economic, and environmental sustainable development and achieve energy saving and emission reduction

ERα up-regulates the transcription of the MGARP promoter and acts in synergy with Sp1 to activate MGARP transcriptional activity.

<p>A. pGL3-(−3 kb) reporter and different doses of ERα expression plasmid were co-transfected into HEK-293T cells to determine the dose-dependent manner of ERα in regulating the MGARP promoter by luciferase assay. B. The functional synergy between Sp1 and ERα was determined by cotransfection of the full-length MGARP promoter (−3 kb) or various promoter truncates with or without Sp1 plasmids for Luc assay as indicated. C. The synergystic transactivation activity of ERα and Sp1 under the stimulation of estrogens. The HEK-293T cells were treated with or without 10 nM of estradiol (E2) for 24 hours post transfection of pGL3-(−3 kb) and ERα. Subsequently, the Luc assay was performed at 72 hours post transfection. D. Knockdown of Sp1 diminishes the activation function of ERα on MGARP promoter. HEK-293T cells were co-transfected with pGL3-(−3 kb) reporter and ERα, together with Sp1-specific RNAi (630-RNAi or 1722-RNAi) or control RNAi, in the absence or presence of exogenous Sp1. *** represents p<0.001 and ^# represents p>0.05 (no significant difference). E. RT-PCR shows that down-regulation of Sp1 with Sp1-specific RNAi (630-RNAi or 1722-RNAi) results in a reduction in endogenous MGARP mRNA expression in HEK-293T cells when stimulated by 10 nM E2. The cells were treated with or without 10 nM E2 for 24 hours post transfection and total RNA was harvested at 72 hours post transfection for semiquantitative RT-PCR analysis.</p

ChIP analysis indicates that Sp1 binds to MGARP promoter <i>in vivo</i>.

<p>ChIP was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050053#s2" target="_blank">Materials and Methods</a>. HEK-293T cells and antibodies for RNA polymerase II (Pol II) and Sp1 were used, with IgG as control. The immunoprecipitated chromatin was amplified by PCR with primers specific for the GC-rich region (BOX1&2) of the MGARP promoter (−3 kb), with GAPDH locus as control. M: DNA Marker.</p

GC-box1 plays a major role in MGARP promoter activation and both GC-boxes are required for full transactivation.

<p>The Luc reporters driven by the full-length MGARP promoter (−3 kb) were transfected into HEK-293T cells, as compared to various promoter truncates either missing the GC-Boxes or expressing the GC-Boxes alone, without or with co-transfection of Sp1 plasmids (<b>10 ng</b>) as indicated. Luc activity was examined at 72 hours post transfection. *** represents p<0.001 and ^#represents p>0.05 (no significant difference).</p

Bioinformatics analysis of the MGARP promoter.

<p>A. Promoter analysis comparing the −3 kb upstream region of the MGARP gene between three species (Homo sapiens, Pan troglodytes and Macaca mulatta). B. A summarized display of the −3 kb upstream region in the human MGARP promoter with predicted promoter and transcription factor (TF) binding sites. C. Luciferase (Luc) reporters driven by the 3 Kb MGARP promoter (pGL3-(−3 kb)) and the promoter-less control (pGL3-Basic) were transfected into HEK-293T cells and analysis was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050053#s2" target="_blank">Materials and Methods</a>. <b>*** represents</b> p<0.001 and ^#represents p>0.05 (no significant difference).</p