The approach to annotating a genome critically affects the number and accuracy of genes identified in the genome sequence. Genome annotation based on stringent gene identification is prone to ...underestimate the complement of genes encoded in a genome. In contrast, over-prediction of putative genes followed by exhaustive computational sequence, motif and structural homology search will find rarely expressed, possibly unique, new genes at the risk of including non-functional genes. We developed a two-stage approach that combines the merits of stringent genome annotation with the benefits of over-prediction. First we identify plausible genes regardless of matches with EST, cDNA or protein sequences from the organism (stage 1). In the second stage, proteins predicted from the plausible genes are compared at the protein level with EST, cDNA and protein sequences, and protein structures from other organisms (stage 2). Remote but biologically meaningful protein sequence or structure homologies provide supporting evidence for genuine genes. The method, applied to the Drosophila melanogaster genome, validated 1,042 novel candidate genes after filtering 19,410 plausible genes, of which 12,124 matched the original 13,601 annotated genes. This annotation strategy is applicable to genomes of all organisms, including human.
Glioblastoma (GBM) harbors subpopulations of therapy-resistant tumor-initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an ...image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4, a 397 kDa transcription factor. ZFHX4 is required to maintain TIC-associated and normal human neural precursor cell phenotypes in vitro, suggesting that ZFHX4 regulates differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we found that ZFHX4 significantly affects CHD4-mediated gene expression perturbations, which defines ZFHX4 as a master regulator of CHD4. These observations define ZFHX4 as a regulatory factor that links the chromatin-remodeling NuRD complex and the GBM TIC state.
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•An image-based screen identifies regulators of glioblastoma tumor-initiating cells•ZFHX4 is required for the glioblastoma tumor-initiating cell state•ZFHX4 interacts with and regulates CHD4, a core member of the NuRD complex•ZFHX4 and CHD4 colocalize throughout the genome and coregulate gene expression
Glioblastoma (GBM), the most common and aggressive primary brain tumor, contains a subpopulation of stem cell-like, therapy-resistant tumor-initiating cells (TICs). Sabatini, Hahn, and colleagues performed an image-based RNAi screen in order to identify candidate regulators of GBM TIC functions. ZFHX4, identified in this screen, is essential for the stem cell-like state and tumorigenicity of TICs. Additionally, ZFHX4 interacts with CHD4, a core member of the chromatin regulatory NuRD complex, and drives CHD4-dependent gene expression programs.
Summary
A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene‐specific barcoded ...deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non‐essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out‐compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose‐ or sucrose‐containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co‐cultures of deletion strains were grown either in vitro in a chemostat to steady‐state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.
A new technique for the selection of newly synthesized DNA has enabled the selection and amplification of putative origins of replication on the human X chromosome.
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