Role of PU.1 and C/EBPα in Remodelling the Interleukin (IL)-1β Enhancer-Promoter Interaction

Background: IL-1b is a potent inflammatory cytokine promptly expressed in activated myeloid immune cells. Among various transcription factors, PU.1 and CCAAT/enhancer-binding protein alpha (C/EBPa) play a key role in the lineage commitment of myeloid cells. To date, however, the exact mechanisms by which these lineage-determining transcription factors employ to regulate the expression of myeloid-specific genes remains elusive; thus, this study explores the role of PU.1 and C/EBPa in remodelling the chromatin conformation that allows ample production of IL-1b. Methods: To examine the mechanism of these lineage-determining transcription factors, production of IL-1b and enhancer-promoter interactions were analyzed in non-myeloid B16-BL6 cells that were ectopically expressed with PU.1 and C/EBPa. Results: Overexpression of PU.1 and C/EBPa rendered B16-BL6 cells response to the bacterial component lipopolysaccharide (LPS) and expressed IL-1b. These cells also expressed a putative enhancer RNA, located ~10 kbs upstream of the IL-1b transcription start site, in response to LPS. Knocking out the enhancer region reduced IL-1b mRNA expression, suggesting that the genomic region is an enhancer. Based on the chromatin conformation capture-qPCR analysis, IL-1b enhancer-promoter interactions were established upon overexpression of PU.1 and C/EBPa, which was further enhanced by LPS. Discussion & Conclusion: These results suggest that PU.1 and C/EBPa are pioneering transcription factors that establish chromatin looping between IL-1b regulatory elements and induce the generation of enhancer RNA, resulting in the production of IL-1b in non-myeloid cells. Interdisciplinary Reflection: Our system that investigates how transcription factors can remodel the chromatin landscape will further expand our understanding of gene regulation

Hdac8 activates akt through upregulating plcb1 and suppressing desc1 expression in mek1/2 inhibition-resistant cells

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phos-pholipase C-β1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition

The transcription factor PU.1 mediates enhancer-promoter looping that is required for IL-1 eRNA and mRNA transcription in mouse melanoma and macrophage cell lines

The DNA-binding protein PU.1 is a myeloid lineage– determining and pioneering transcription factor due to its ability to bind “closed” genomic sites and maintain “open” chromatin state for myeloid lineage–specific genes. The precise mechanism of PU.1 in cell type–specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-B upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1 mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located 10 kbp upstream of the IL-1 transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-B recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1 mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1 transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-B

Histone deacetylase 8 protects human proximal tubular epithelial cells from hypoxia-mimetic cobalt- and hypoxia/reoxygenation-induced mitochondrial fission and cytotoxicity

Cell death by hypoxia followed by reoxygenation (H/R) is responsible for tissue injury in multiple pathological conditions. Recent studies found that epigenetic reprogramming mediated by histone deacetylases (HDACs) is implicated in H/R-induced cell death. However, among 18 different isoforms comprising 4 classes (I-IV), the role of each HDAC in cell death is largely unknown. This study examined the role of HDAC8, which is the most distinct isoform of class I, in the hypoxia mimetic cobalt- and H/R-induced cytotoxicity of human proximal tubular HK-2 cells. Using the HDAC8-specific activator TM-2-51 (TM) and inhibitor PCI34051, we found that HDAC8 played a protective role in cytotoxicity. TM or overexpression of wild-type HDAC8, but not a deacetylase-defective HDAC8 mutant, prevented mitochondrial fission, loss of mitochondrial transmembrane potential and release of cytochrome C into the cytoplasm. TM suppressed expression of dynamin-related protein 1 (DRP1) which is a key factor required for mitochondrial fission. Suppression of DRP1 by HDAC8 was likely mediated by decreasing the level of acetylated histone H3 lysine 27 (a hallmark of active promoters) at the DRP1 promoter. Collectively, this study shows that HDAC8 inhibits cytotoxicity induced by cobalt and H/R, in part, through suppressing DRP1 expression and mitochondrial fission

Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content

<p>Abstract</p> <p>Background</p> <p>Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether <it>Bifidobacteria </it>isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content.</p> <p>Methods</p> <p><it>In vitro </it>culture experiments were performed to evaluate the ability of <it>Bifidobacterium </it>spp. isolated from healthy Koreans (20~30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (10⁸~10⁹CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks.</p> <p>Results</p> <p><it>B. longum </it>SPM1207 reduced serum total cholesterol and LDL levels significantly (<it>p </it>< 0.05), and slightly increased serum HDL. <it>B. longum </it>SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities.</p> <p>Conclusion</p> <p>Daily consumption of <it>B. longum </it>SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.</p

Biochemistry and Molecular Biology Effect of Nerve Growth Factor on the In Vitro Induction of Apoptosis of Human Conjunctival Epithelial Cells by Hyperosmolar Stress

Citation: Kang S-S, Ha S-J, Kim E-S, Shin J-A, Kim JY, Tchah H. Effect of nerve growth factor on the in vitro induction of apoptosis of human conjunctival epithelial cells by hyperosmolar stress. Invest Ophthalmol Vis Sci. 2014;55:535-541

The lipoxygenase gene family: a genomic fossil of shared polyploidy between Glycine max and Medicago truncatula

<p>Abstract</p> <p>Background</p> <p>Soybean lipoxygenases (<it>Lxs</it>) play important roles in plant resistance and in conferring the distinct bean flavor. <it>Lxs </it>comprise a multi-gene family that includes <it>GmLx1</it>, <it>GmLx2 </it>and <it>GmLx3</it>, and many of these genes have been characterized. We were interested in investigating the relationship between the soybean lipoxygenase isozymes from an evolutionary perspective, since soybean has undergone two rounds of polyploidy. Here we report the tetrad genome structure of soybean <it>Lx </it>regions produced by ancient and recent polyploidy. Also, comparative genomics with <it>Medicago truncatula </it>was performed to estimate <it>Lxs </it>in the common ancestor of soybean and <it>Medicago</it>.</p> <p>Results</p> <p>Two <it>Lx </it>regions in <it>Medicago truncatula </it>showing synteny with soybean were analyzed. Differential evolutionary rates between soybean and <it>Medicago </it>were observed and the median Ks values of Mt-Mt, Gm-Mt, and Gm-Gm paralogs were determined to be 0.75, 0.62, and 0.46, respectively. Thus the comparison of Gm-Mt paralogs (Ks = 0.62) and Gm-Mt orthologs (Ks = 0.45) supports the ancient duplication of <it>Lx </it>regions in the common ancestor prior to the <it>Medicago</it>-<it>Glycine </it>split. After speciation, no <it>Lx </it>regions generated by another polyploidy were identified in <it>Medicago</it>. Instead tandem duplication of <it>Lx </it>genes was observed. On the other hand, a lineage-specific duplication occurred in soybean resulting in two pairs of <it>Lx </it>regions. Each pair of soybean regions was co-orthologous to one <it>Lx </it>region in <it>Medicago</it>. A total of 34 <it>Lx </it>genes (15 <it>MtLxs </it>and 19 <it>GmLxs) </it>were divided into two groups by phylogenetic analysis. Our study shows that the <it>Lx </it>gene family evolved from two distinct <it>Lx </it>genes in the most recent common ancestor.</p> <p>Conclusion</p> <p>This study analyzed two pairs of <it>Lx </it>regions generated by two rounds of polyploidy in soybean. Each pair of soybean homeologous regions is co-orthologous to one region of <it>Medicago</it>, demonstrating the quartet structure of the soybean genome. Differential evolutionary rates between soybean and <it>Medicago </it>were observed; thus optimized rates of Ks per year should be applied for accurate estimation of coalescence times to each case of comparison: soybean-soybean, soybean-<it>Medicago</it>, or <it>Medicago</it>-<it>Medicago</it>. In conclusion, the soybean <it>Lx </it>gene family expanded by ancient polyploidy prior to taxon divergence, followed by a soybean- specific duplication and tandem duplications, respectively.</p

Specific Radius Change of Quantum Dot inside the Lipid Bilayer by Charge Effect of Lipid Head-Group

We studied the quantum dot-liposome complex (QLC), which is the giant unilamellar vesicle with quantum dots (QDs) incorporated in its lipid bilayer. A spin coating method in conjunction with the electroformation technique yielded vesicles with highly homogeneous unilamellar structure. We observed QD size dependence of the QLC formation: QLCs form with blue, green and yellow-emission QD (core radius ~1.05 nm, 1.25 nm and 1.65 nm) but not with red-emission QD (core radius ~2.5 nm). In order to explain this size dependence, we made a simple model explaining the QD size effect on QLC formation in terms of the molecular packing parameter and the lipid conformational change. This model predicts that QDs below a certain critical size (radius &amp;#8776; 1.8 nm) can stably reside in a lipid bilayer of 4 - 5 nm in thickness for Egg-PC lipids. This is consistent with our previous experimental results. In the case of red-emission QD, QD-aggregations are only observed on the fluorescent microscopy instead of QLC. We expected that the reduction of packing parameter (P) would lead to the change of specific QD radius. This prediction could be verified by our experimental observation of the shift of the specific QD size by mixing DOPG