Aging research has been very successful at identifying signaling pathways and evolutionarily conserved genes that extend lifespan with the assumption that an increase in lifespan will also increase ...healthspan. However, it is largely unknown whether we are extending the healthy time of life or simply prolonging a period of frailty with increased incidence of age-associated diseases. Here we use Caenorhabditis elegans, one of the premiere systems for lifespan studies, to determine whether lifespan and healthspan are intrinsically correlated. We conducted multiple cellular and organismal assays on wild type as well as four long-lived mutants (insulin/insulin-like growth factor-1, dietary restriction, protein translation, mitochondrial signaling) in a longitudinal manner to determine the health of the animals as they age. We find that some long-lived mutants performed better than wild type when measured chronologically (number of days). However, all long-lived mutants increased the proportion of time spent in a frail state. Together, these data suggest that lifespan can no longer be the sole parameter of interest and reveal the importance of evaluating multiple healthspan parameters for future studies on antiaging interventions.
Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) or ChIP followed by genome tiling array analysis (ChIP-chip) have become standard technologies for genome-wide ...identification of DNA-binding protein target sites. A number of algorithms have been developed in parallel that allow identification of binding sites from ChIP-seq or ChIP-chip datasets and subsequent visualization in the University of California Santa Cruz (UCSC) Genome Browser as custom annotation tracks. However, summarizing these tracks can be a daunting task, particularly if there are a large number of binding sites or the binding sites are distributed widely across the genome.
We have developed ChIPpeakAnno as a Bioconductor package within the statistical programming environment R to facilitate batch annotation of enriched peaks identified from ChIP-seq, ChIP-chip, cap analysis of gene expression (CAGE) or any experiments resulting in a large number of enriched genomic regions. The binding sites annotated with ChIPpeakAnno can be viewed easily as a table, a pie chart or plotted in histogram form, i.e., the distribution of distances to the nearest genes for each set of peaks. In addition, we have implemented functionalities for determining the significance of overlap between replicates or binding sites among transcription factors within a complex, and for drawing Venn diagrams to visualize the extent of the overlap between replicates. Furthermore, the package includes functionalities to retrieve sequences flanking putative binding sites for PCR amplification, cloning, or motif discovery, and to identify Gene Ontology (GO) terms associated with adjacent genes.
ChIPpeakAnno enables batch annotation of the binding sites identified from ChIP-seq, ChIP-chip, CAGE or any technology that results in a large number of enriched genomic regions within the statistical programming environment R. Allowing users to pass their own annotation data such as a different Chromatin immunoprecipitation (ChIP) preparation and a dataset from literature, or existing annotation packages, such as GenomicFeatures and BSgenome, provides flexibility. Tight integration to the biomaRt package enables up-to-date annotation retrieval from the BioMart database.
We have determined the three-dimensional (3D) architecture of the
Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational ...modeling. Using chromosome conformation capture carbon copy (5C), we derive ∼13 kb resolution 3D models of the
Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The
parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the
parS sites and chromosome segregation.
► Chromatin interaction mapping and modeling elucidate
Caulobacter genome structure ► The genome is ellipsoidal with periodically arranged arms ► The
parS region shapes whole genome structure and affects chromatin compaction ► The
parS region is the only genomic region stably attached to the cell envelope
CRISPR-Cas systems are a diverse family of RNA-protein complexes in bacteria that target foreign DNA sequences for cleavage. Derivatives of these complexes have been engineered to cleave specific ...target sequences depending on the sequence of a CRISPR-derived guide RNA (gRNA) and the source of the Cas9 protein. Important considerations for the design of gRNAs are to maximize aimed activity at the desired target site while minimizing off-target cleavage. Because of the rapid advances in the understanding of existing CRISPR-Cas9-derived RNA-guided nucleases and the development of novel RNA-guided nuclease systems, it is critical to have computational tools that can accommodate a wide range of different parameters for the design of target-specific RNA-guided nuclease systems. We have developed CRISPRseek, a highly flexible, open source software package to identify gRNAs that target a given input sequence while minimizing off-target cleavage at other sites within any selected genome. CRISPRseek will identify potential gRNAs that target a sequence of interest for CRISPR-Cas9 systems from different bacterial species and generate a cleavage score for potential off-target sequences utilizing published or user-supplied weight matrices with position-specific mismatch penalty scores. Identified gRNAs may be further filtered to only include those that occur in paired orientations for increased specificity and/or those that overlap restriction enzyme sites. For applications where gRNAs are desired to discriminate between two related sequences, CRISPRseek can rank gRNAs based on the difference between predicted cleavage scores in each input sequence. CRISPRseek is implemented as a Bioconductor package within the R statistical programming environment, allowing it to be incorporated into computational pipelines to automate the design of gRNAs for target sequences identified in a wide variety of genome-wide analyses. CRISPRseek is available under the GNU General Public Licence v3.0 at http://www.bioconductor.org.
Progression from brown preadipocytes to adipocytes engages two transcriptional programs: the expression of adipogenic genes common to both brown fat (BAT) and white fat (WAT), and the expression of ...BAT-selective genes. However, the dynamics of chromatin states and epigenetic enzymes involved remain poorly understood. Here we show that BAT development is selectively marked and guided by repressive H3K27me3 and is executed by its demethylase Jmjd3. We find that a significant subset of BAT-selective genes, but not common fat genes or WAT-selective genes, are demarcated by H3K27me3 in both brown and white preadipocytes. Jmjd3-catalyzed removal of H3K27me3, in part through Rreb1-mediated recruitment, is required for expression of BAT-selective genes and for development of beige adipocytes both in vitro and in vivo. Moreover, gain- and loss-of-function Jmjd3 transgenic mice show age-dependent body weight reduction and cold intolerance, respectively. Together, we identify an epigenetic mechanism governing BAT fate determination and WAT plasticity.
•H3K27me3 marks BAT genes, but not common fat genes or WAT genes, in preadipocytes•Demethylation by Jmjd3 is required for BAT gene expression and browning of WAT•Rreb1 regulates Ucp1 and Cidea expression by recruiting Jmjd3 to their promoters•Jmjd3 regulates age-dependent body weight gain and cold intolerance in mice
Pan, Huang et al. show that H3K27me3 demarcates brown-fat-selective genes, but spares common-fat- and white-fat-selective genes, in preadipocytes. Jmjd3-catalyzed erasure of H3K27me3 is required for brown fat gene expression and browning of white fat. Gain- and loss-of-function Jmjd3 transgenic mice show body weight reduction and cold intolerance, respectively.
Recent clinical and experimental evidence suggests that endoplasmic reticulum (ER) stress contributes to the life-and-death decisions of β cells during the progression of type 1 and type 2 diabetes. ...Although crosstalk between inflammation and ER stress has been suggested to play a significant role in β cell dysfunction and death, a key molecule connecting ER stress to inflammation has not been identified. Here we report that thioredoxin-interacting protein (TXNIP) is a critical signaling node that links ER stress and inflammation. TXNIP is induced by ER stress through the PERK and IRE1 pathways, induces IL-1β mRNA transcription, activates IL-1β production by the NLRP3 inflammasome, and mediates ER stress-mediated β cell death. Collectively, our results suggest that TXNIP is a potential therapeutic target for diabetes and ER stress-related human diseases such as Wolfram syndrome.
► TXNIP is a critical signaling node that links ER stress and inflammation ► TXNIP is induced by ER stress through the PERK and IRE1 pathways ► TXNIP mediates ER stress-mediated β cell death
The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic ...programs and target genes in cancer cells remain poorly understood. Here, we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD, and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent but engages the SRC1–3 co-activators to promote downstream transcription. Furthermore, we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.
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•Tead and AP1 coordinate downstream transcription in diverse cancer cells•JNK-independent Tead-AP1 interaction engages SRC1–3 co-activators•Tead and AP1 regulate the activity of Dock-Rac/CDC42 module•Tead-AP1 cooperation controls migration and invasion through a core set of target genes
Tead and AP1 are two families of transcription factors involved in tumorigenesis. Here, Liu et al. show Tead-AP1 co-occupancy on active enhancer or promoter regions in diverse cancer cells. This Tead-AP1 cooperation engages SRC1–3 co-activators and drives a core set of downstream target genes to coordinate cancer cell migration and invasion.
The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology ...requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.
Direct genomic manipulation at a specific locus is still not feasible in most vertebrate model organisms. In vertebrate cell lines, genomic lesions at a specific site have been introduced using ...zinc-finger nucleases (ZFNs). Here we adapt this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdr (also known as kdra). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early-stage embryos are easily accessible.
Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor ...(Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented “hyper-angiogenesis” defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.
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► Deep sequencing reveals microRNAs essential for zebrafish vascular development ► miR-221 is required for angiogenesis and acts in the Vegfc/Flt4 signaling pathway ► miR-221 acts autonomously to promote endothelial tip cell migration and proliferation ► miR-221 targets cdkn1b and pik3r1 to induce proliferation and PI3K output
Angiogenesis requires coordination of distinct cell behaviors between sprouting tip cells and the stalk cells connected to the patent circulatory system. Nicoli et al. identify a microRNA, miR-221, that is required for tip cell migration and proliferation through the repression of targets involved in PI3K signaling and cell cycle inhibition.
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