SUMMARY Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys 19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys 514 was crucial for enzymatic activity and the ability of yeast cells to grow on non-fermentable carbon sources. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production was dependent on TIP60, the human homolog of ESA1. Our results demonstrate a novel regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.
DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription.DOI: http://dx.doi.org/10.7554/eLife.00726.001
Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-moleculeonly approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxiainducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over onehalf of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.retinal pigment epithelium | pluripotent stem cells | high-throughput screening | differentiation | age-related macular degeneration A ge-related macular degeneration (AMD) is the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries. Dysfunction of the retinal pigment epithelium (RPE) is an early event associated with AMD. The RPE, a monolayer of pigmented cells directly abutting the photoreceptor cell layer, plays many important roles in vision and in maintaining the health and integrity of the retina (1). As the RPE deteriorates, there is progressive degeneration of photoreceptor cells.Successful antiangiogenesis treatments have been developed for the neovascular, or "wet," form of AMD. However, there are no Food and Drug Administration-approved treatment options available for the majority of AMD patients, who suffer from the more common nonneovascular, or "dry," form of the disease. In the past few years, however, transplantation of human pluripotent stem cellderived RPE (hPSC-RPE) has emerged as a promising new therapy for dry AMD. A Phase I clinical trial of human embryonic stem (hES)-derived RPE cells recently reported some preliminary encouraging results (2). Additionally, a Phase I trial that will use RPE cells generated from human induced pluripotent stem cells (hiPSCs) reprogrammed from the patients' own skin cells recently injected their first patient (3).If the promise of hiPSC-based approaches for AMD is to be translated into the clinic, each patient would require individualized generation of RPE cells from his or her stem cells, thereby necessitating the development of simple, efficient, safe, and affordable protocols for RPE generation. Although highly efficient protocols have been established, they rely upon mixtures of growth factors (4-6) with the use of complex biologics derived from animal cells or bacteria, presenting potential clinical challenges. As an alternative approach, use...
Purpose. MicroRNAs (miRNAs) are short, noncoding transcripts that negatively regulate gene expression. They are implicated in diverse cellular processes. The purpose of this study was to obtain a global expression profile of miRNAs in the developing retina and identify differences in miRNA expression between adult rod and cone photoreceptors. Methods. Locked nucleic acid (LNA) microarrays were used to investigate the miRNA transcriptome of the developing mouse retina and brain. Real-time PCR was used to validate the array findings. Laser capture microdissection was used to determine the miRNA spatial pattern of expression. Results. One hundred thirty-eight miRNAs were expressed at at least one of the investigated time points. Several miRNAs showed significant changes in expression between embryonic day 15 and adult age in both retina and brain. Cluster analysis identified subgroups of miRNAs showing defined expression profiles. Globally, correlation of expression was higher, with increasing sequence similarity of the mature miRNAs. The miRNAs with identical seed sequences exhibited highly correlated expression profiles. The co-expression of selected host gene and intronic miRNA pairs was confirmed in adult retina. In some cases, expression profiles of miRNAs showed weak correlation with those of their host transcripts, suggesting posttranscriptional regulation of miRNAs during development. In addition, the miRNA transcriptome of rod- and cone-dominant retinas showed only minor differences, and no miRNAs specific for either cell-type were identified. Conclusions. Global expression profiling revealed dozens of miRNAs with significant expression changes in the developing retina. Precise patterns of expression of miRNAs suggest their specific roles in development.
We present experimental observations of exact dynamic localization of an optical beam in a periodically curved AlGaAs waveguide array. The dynamic localization of the beam is "exact" in that it is observed even when the photonic band of the array is not well described in the nearest-neighbor tight-binding approximation. We present the spatial evolution of the beam around the two-period plane in the structure, explicitly demonstrating the delocalization and subsequent relocalization of the beam. We also emonstrate the strong wavelength dependence of the beam relocalization for a four period structure.
Altered DNA methylation status is associated with human diseases and cancer; however, the underlying molecular mechanisms remain elusive. We previously identified many human transcription factors, including Krüppel-like factor 4 (KLF4), as sequence-specific DNA methylation readers that preferentially recognize methylated CpG (mCpG), here we report the biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. We show that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. In-depth mechanistic studies reveal that recruitment of KLF4 to the methylated cis-regulatory elements of these genes result in chromatin remodeling and transcription activation. Our study demonstrates a new paradigm of DNA methylation-mediated gene activation and chromatin remodeling, and provides a general framework to dissect the biological functions of DNA methylation readers and effectors.DOI: http://dx.doi.org/10.7554/eLife.20068.001
Properties of omnidirectional gap and defect mode of one-dimensional photonic crystal containing indefinite metamaterials with a hyperbolic dispersion
The COVID-19 outbreak has become a global emergency since December 2019. Analysis of SARS-CoV-2 sequences can uncover single nucleotide variants (SNVs) and corresponding evolution patterns. The Global Evaluation of SARS-CoV-2/hCoV-19 Sequences (GESS, https://wan-bioinfo.shinyapps.io/GESS/) is a resource to provide comprehensive analysis results based on tens of thousands of high-coverage and high-quality SARS-CoV-2 complete genomes. The database allows user to browse, search and download SNVs at any individual or multiple SARS-CoV-2 genomic positions, or within a chosen genomic region or protein, or in certain country/area of interest. GESS reveals geographical distributions of SNVs around the world and across the states of USA, while exhibiting time-dependent patterns for SNV occurrences which reflect development of SARS-CoV-2 genomes. For each month, the top 100 SNVs that were firstly identified world-widely can be retrieved. GESS also explores SNVs occurring simultaneously with specific SNVs of user's interests. Furthermore, the database can be of great help to calibrate mutation rates and identify conserved genome regions. Taken together, GESS is a powerful resource and tool to monitor SARS-CoV-2 migration and evolution according to featured genomic variations. It provides potential directive information for prevalence prediction, related public health policy making, and vaccine designs.
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