Mammalian aging driven by transcription going awry.

The mechanisms that underlie increased cryptic transcription during senescence and aging have been poorly understood. Sen et al. recently identified cryptic transcription start sites (cTSSs) and chromatin state changes that may contribute to cTSS activation in mammals. Their results indicate that enhancer-promoter conversion may drive cryptic transcription in senescence.

Loss of chromatin structural integrity is a source of stress during aging.

Dysfunction and dysregulation at multiple levels, from organismal to molecular, are associated with the biological process of aging. In a eukaryotic nucleus, multiple lines of evidence have shown that the fundamental structure of chromatin is affected by aging. Not only euchromatic and heterochromatic regions shift locations, global changes, such as reduced levels of histones, have been reported for certain aged cell types and tissues. The physiological effects caused by such broad chromatin changes are complex and the cell's responses to it can be profound and in turn influence the aging process. In this review, we summarize recent findings on the interplay between chromatin architecture and aging with an emphasis on the cellular response to chromatin stress and its antagonistic effects on aging.

Paleogenomics of echinoids reveals an ancient origin for the double-negative specification of micromeres in sea urchins.

Establishing a timeline for the evolution of novelties is a common, unifying goal at the intersection of evolutionary and developmental biology. Analyses of gene regulatory networks (GRNs) provide the ability to understand the underlying genetic and developmental mechanisms responsible for the origin of morphological structures both in the development of an individual and across entire evolutionary lineages. Accurately dating GRN novelties, thereby establishing a timeline for GRN evolution, is necessary to answer questions about the rate at which GRNs and their subcircuits evolve, and to tie their evolution to paleoenvironmental and paleoecological changes. Paleogenomics unites the fossil record and all aspects of deep time, with modern genomics and developmental biology to understand the evolution of genomes in evolutionary time. Recent work on the regulatory genomic basis of development in cidaroid echinoids, sand dollars, heart urchins, and other nonmodel echinoderms provides an ideal dataset with which to explore GRN evolution in a comparative framework. Using divergence time estimation and ancestral state reconstructions, we have determined the age of the double-negative gate (DNG), the subcircuit which specifies micromeres and skeletogenic cells in Strongylocentrotus purpuratus We have determined that the DNG has likely been used for euechinoid echinoid micromere specification since at least the Late Triassic. The innovation of the DNG thus predates the burst of post-Paleozoic echinoid morphological diversification that began in the Early Jurassic. Paleogenomics has wide applicability for the integration of deep time and molecular developmental data, and has wide utility in rigorously establishing timelines for GRN evolution.

Dose-dependent nuclear ß-catenin response segregates endomesoderm along the sea star primary axis.

In many invertebrates, the nuclearization of ß-catenin at one pole of the embryo initiates endomesoderm specification. An intriguing possibility is that a gradient of nuclear ß-catenin (nß-catenin), similar to that operating in vertebrate neural tube patterning, functions to distinguish cell fates in invertebrates. To test this hypothesis, we determined the function of nß-catenin during the early development of the sea star, which undergoes a basal deuterostomal mode of embryogenesis. We show that low levels of nß-catenin activity initiate bra, which is expressed in the future posterior endoderm-fated territory; intermediate levels are required for expression of foxa and gata4/5/6, which are later restricted to the endoderm; and activation of ets1 and erg in the mesoderm-fated territory requires the highest nß-catenin activity. Transcription factors acting downstream of high nß-catenin segregate the endoderm/mesoderm boundary, which is further reinforced by Delta/Notch signaling. Significantly, therefore, in sea stars, endomesoderm segregation arises through transcriptional responses to levels of nß-catenin activity. Here, we describe the first empirical evidence of a dose-dependent response to a dynamic spatiotemporal nß-catenin activity that patterns cell fates along the primary axis in an invertebrate.

Histone methylation and aging: lessons learned from model systems.

Aging induces myriad cellular and, ultimately, physiological changes that cause a decline in an organism's functional capabilities. Although the aging process and the pathways that regulate it have been extensively studied, only in the last decade have we begun to appreciate that dynamic histone methylation may contribute to this process. In this review, we discuss recent work implicating histone methylation in aging. Loss of certain histone methyltransferases and demethylases changes lifespan in invertebrates, and alterations in histone methylation in aged organisms regulate lifespan and aging phenotypes, including oxidative stress-induced hormesis in yeast, insulin signaling in Caenorhabiditis elegans and mammals, and the senescence-associated secretory phenotype in mammals. In all cases where histone methylation has been shown to impact aging and aging phenotypes, it does so by regulating transcription, suggesting that this is a major mechanism of its action in this context. Histone methylation additionally regulates or is regulated by other cellular pathways that contribute to or combat aging. Given the numerous processes that regulate aging and histone methylation, and are in turn regulated by them, the role of histone methylation in aging is almost certainly underappreciated.

Probabilistic error correction for RNA sequencing.

Sequencing of RNAs (RNA-Seq) has revolutionized the field of transcriptomics, but the reads obtained often contain errors. Read error correction can have a large impact on our ability to accurately assemble transcripts. This is especially true for de novo transcriptome analysis, where a reference genome is not available. Current read error correction methods, developed for DNA sequence data, cannot handle the overlapping effects of non-uniform abundance, polymorphisms and alternative splicing. Here we present SEquencing Error CorrEction in Rna-seq data (SEECER), a hidden Markov Model (HMM)-based method, which is the first to successfully address these problems. SEECER efficiently learns hundreds of thousands of HMMs and uses these to correct sequencing errors. Using human RNA-Seq data, we show that SEECER greatly improves on previous methods in terms of quality of read alignment to the genome and assembly accuracy. To illustrate the usefulness of SEECER for de novo transcriptome studies, we generated new RNA-Seq data to study the development of the sea cucumber Parastichopus parvimensis. Our corrected assembled transcripts shed new light on two important stages in sea cucumber development. Comparison of the assembled transcripts to known transcripts in other species has also revealed novel transcripts that are unique to sea cucumber, some of which we have experimentally validated. Supporting website: http://sb.cs.cmu.edu/seecer/.

Yeast EndoG prevents genome instability by degrading cytoplasmic DNA.

In metazoans release of mitochondrial DNA or retrotransposon cDNA to cytoplasm can cause sterile inflammation and disease 1. Cytoplasmic nucleases degrade these DNA species to limit inflammation 2,3. It remains unknown whether degradation these DNA also prevents nuclear genome instability. To address this question, we decided to identify the nuclease regulating transfer of these cytoplasmic DNA species to the nucleus. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. Nuclear mtDNA (NUMTs) and retrotransposon cDNA insertions increase dramatically in nondividing stationary phase cells. Yeast EndoG (Nuc1) nuclease limits insertions of cDNA and transfer of very long mtDNA (>10 kb) that forms unstable circles or rarely insert in the genome, but it promotes formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of cytoplasmic DNA to nucleus in aging or during meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs can originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating cytoplasmic DNA play a role in preserving genome stability.

Yeast EndoG prevents genome instability by degrading cytoplasmic DNA.

In metazoans release of mitochondrial DNA or retrotransposon cDNA to cytoplasm can cause sterile inflammation and disease. Cytoplasmic nucleases degrade these DNA species to limit inflammation. It remains unknown whether degradation these DNA also prevents nuclear genome instability. To address this question, we decided to identify the nuclease regulating transfer of these cytoplasmic DNA species to the nucleus. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. Nu clear mt DNA (NUMTs) and retrotransposon cDNA insertions increase dramatically in nondividing stationary phase cells. Yeast EndoG (Nuc1) nuclease limits insertions of cDNA and transfer of very long mtDNA (>10 kb) that forms unstable circles or rarely insert in the genome, but it promotes formation of short NUMTs (∼45-200 bp). Nuc1 also regulates transfer of cytoplasmic DNA to nucleus in aging or during meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs can originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating cytoplasmic DNA play a role in preserving genome stability.

Loosening chromatin and dysregulated transcription: a perspective on cryptic transcription during mammalian aging.

Cryptic transcription, the initiation of transcription from non-promoter regions within a gene body, is a type of transcriptional dysregulation that occurs throughout eukaryotes. In mammals, cryptic transcription is normally repressed at the level of chromatin, and this process is increased upon perturbation of complexes that increase intragenic histone H3 lysine 4 methylation or decrease intragenic H3 lysine 36 methylation, DNA methylation, or nucleosome occupancy. Significantly, similar changes to chromatin structure occur during aging, and, indeed, recent work indicates that cryptic transcription is elevated during aging in mammalian stem cells. Although increased cryptic transcription is known to promote aging in yeast, whether elevated cryptic transcription also contributes to mammalian aging is unclear. There is ample evidence that perturbations known to increase cryptic transcription are deleterious in embryonic and adult stem cells, and in some cases phenocopy certain aging phenotypes. Furthermore, an increase in cryptic transcription requires or impedes pathways that are known to have reduced function during aging, potentially exacerbating other aging phenotypes. Thus, we propose that increased cryptic transcription contributes to mammalian stem cell aging.

A conserved gene regulatory network subcircuit drives different developmental fates in the vegetal pole of highly divergent echinoderm embryos.

Comparisons of orthologous developmental gene regulatory networks (GRNs) from different organisms explain how transcriptional regulation can, or cannot, change over time to cause morphological evolution and stasis. Here, we examine a subset of the GRN connections in the central vegetal pole mesoderm of the late sea star blastula and compare them to the GRN for the same embryonic territory of sea urchins. In modern sea urchins, this territory gives rise to skeletogenic mesoderm; in sea stars, it develops into other mesodermal derivatives. Orthologs of many transcription factors that function in the sea urchin skeletogenic mesoderm are co-expressed in the sea star vegetal pole, although this territory does not form a larval skeleton. Systematic perturbation of erg, hex, tbr, and tgif gene function was used to construct a snapshot of the sea star mesoderm GRN. A comparison of this network to the sea urchin skeletogenic mesoderm GRN revealed a conserved, recursively wired subcircuit operating in both organisms. We propose that, while these territories have evolved different functions in sea urchins and sea stars, this subcircuit is part of an ancestral GRN governing echinoderm vegetal pole mesoderm development. The positive regulatory feedback between these transcription factors may explain the conservation of this subcircuit.

Altered Chromatin States Drive Cryptic Transcription in Aging Mammalian Stem Cells.

A repressive chromatin state featuring trimethylated lysine 36 on histone H3 (H3K36me3) and DNA methylation suppresses cryptic transcription in embryonic stem cells. Cryptic transcription is elevated with age in yeast and nematodes, and reducing it extends yeast lifespan, though whether this occurs in mammals is unknown. We show that cryptic transcription is elevated in aged mammalian stem cells, including murine hematopoietic stem cells (mHSCs) and neural stem cells (NSCs) and human mesenchymal stem cells (hMSCs). Precise mapping allowed quantification of age-associated cryptic transcription in hMSCs aged in vitro. Regions with significant age-associated cryptic transcription have a unique chromatin signature: decreased H3K36me3 and increased H3K4me1, H3K4me3, and H3K27ac with age. Genomic regions undergoing such changes resemble known promoter sequences and are bound by TBP even in young cells. Hence, the more permissive chromatin state at intragenic cryptic promoters likely underlies increased cryptic transcription in aged mammalian stem cells.

Evolution of gene regulatory network architectures: examples of subcircuit conservation and plasticity between classes of echinoderms.

Developmental gene regulatory networks (GRNs) explain how regulatory states are established in particular cells during development and how these states then determine the final form of the embryo. Evolutionary changes to the sequence of the genome will direct reorganization of GRN architectures, which in turn will lead to the alteration of developmental programs. A comparison of GRN architectures must consequently reveal the molecular basis for the evolution of developmental programs among different organisms. This review highlights some of the important findings that have emerged from the most extensive direct comparison of GRN architectures to date. Comparison of the orthologous GRNs for endomesodermal specification in the sea urchin and sea star, provides examples of several discrete, functional GRN subcircuits and shows that they are subject to diverse selective pressures. This demonstrates that different regulatory linkages may be more or less amenable to evolutionary change. One of the more surprising findings from this comparison is that GRN-level functions may be maintained while the factors performing the functions have changed, suggesting that GRNs have a high capacity for compensatory changes involving transcription factor binding to cis regulatory modules.

Systematic comparison of sea urchin and sea star developmental gene regulatory networks explains how novelty is incorporated in early development.

The extensive array of morphological diversity among animal taxa represents the product of millions of years of evolution. Morphology is the output of development, therefore phenotypic evolution arises from changes to the topology of the gene regulatory networks (GRNs) that control the highly coordinated process of embryogenesis. A particular challenge in understanding the origins of animal diversity lies in determining how GRNs incorporate novelty while preserving the overall stability of the network, and hence, embryonic viability. Here we assemble a comprehensive GRN for endomesoderm specification in the sea star from zygote through gastrulation that corresponds to the GRN for sea urchin development of equivalent territories and stages. Comparison of the GRNs identifies how novelty is incorporated in early development. We show how the GRN is resilient to the introduction of a transcription factor, pmar1, the inclusion of which leads to a switch between two stable modes of Delta-Notch signaling. Signaling pathways can function in multiple modes and we propose that GRN changes that lead to switches between modes may be a common evolutionary mechanism for changes in embryogenesis. Our data additionally proposes a model in which evolutionarily conserved network motifs, or kernels, may function throughout development to stabilize these signaling transitions.

Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons.

Guanine-rich DNA sequences can fold into four-stranded G-quadruplex (G4-DNA) structures. G4-DNA regulates replication and transcription, at least in cancer cells. Here, we demonstrate that, in neurons, pharmacologically stabilizing G4-DNA with G4 ligands strongly downregulates the Atg7 gene. Atg7 is a critical gene for the initiation of autophagy that exhibits decreased transcription with aging. Using an in vitro assay, we show that a putative G-quadruplex-forming sequence (PQFS) in the first intron of the Atg7 gene folds into a G4. An antibody specific to G4-DNA and the G4-DNA-binding protein PC4 bind to the Atg7 PQFS. Mice treated with a G4 stabilizer develop memory deficits. Brain samples from aged mice contain G4-DNA structures that are absent in brain samples from young mice. Overexpressing the G4-DNA helicase Pif1 in neurons exposed to the G4 stabilizer improves phenotypes associated with G4-DNA stabilization. Our findings indicate that G4-DNA is a novel pathway for regulating autophagy in neurons.

Expression of wnt and frizzled genes during early sea star development.

The Wnt signaling pathway is highly conserved across metazoa and has pleiotropic functions in the development of many animals. Binding of a secreted Wnt ligand to its Frizzled (Fz) receptor activates Dishevelled, which then drives one of three major signaling cascades, canonical (ß-catenin), calcium, or planar cell polarity signaling. These pathways have distinct developmental effects and function in different processes in different organisms. Here we report the expression of six wnt and three fz genes during embryogenesis of the sea star, Patiria miniata, as a first step in uncovering the roles of Wnt signaling in the development of this organism. wnt3, wnt4, wnt8, and wnt16 are expressed in nested domains in the endoderm and lateral ectoderm from blastula through late gastrula stages; wnt2 and wnt5 are expressed in the mesoderm and anterior endoderm. Expression of different fz paralogs is detected in the mesoderm; posterior endoderm and ectoderm; and anterior ectoderm. Taken together, this suggests that Wnt signaling can occur throughout most of the embryo and may therefore play multiple roles during sea star development.

Development of an embryonic skeletogenic mesenchyme lineage in a sea cucumber reveals the trajectory of change for the evolution of novel structures in echinoderms.

BACKGROUND: The mechanisms by which the conserved genetic "toolkit" for development generates phenotypic disparity across metazoans is poorly understood. Echinoderm larvae provide a great resource for understanding how developmental novelty arises. The sea urchin pluteus larva is dramatically different from basal echinoderm larval types, which include the auricularia-type larva of its sister taxon, the sea cucumbers, and the sea star bipinnaria larva. In particular, the pluteus has a mesodermally-derived larval skeleton that is not present in sea star larvae or any outgroup taxa. To understand the evolutionary origin of this structure, we examined the molecular development of mesoderm in the sea cucumber, Parastichopus parvimensis. RESULTS: By comparing gene expression in sea urchins, sea cucumbers and sea stars, we partially reconstructed the mesodermal regulatory state of the echinoderm ancestor. Surprisingly, we also identified expression of the transcription factor alx1 in a cryptic skeletogenic mesenchyme lineage in P. parvimensis. Orthologs of alx1 are expressed exclusively within the sea urchin skeletogenic mesenchyme, but are not expressed in the mesenchyme of the sea star, which suggests that alx1+ mesenchyme is a synapomorphy of at least sea urchins and sea cucumbers. Perturbation of Alx1 demonstrates that this protein is necessary for the formation of the sea cucumber spicule. Overexpression of the sea star alx1 ortholog in sea urchins is sufficient to induce additional skeleton, indicating that the Alx1 protein has not evolved a new function during the evolution of the larval skeleton. CONCLUSIONS: The proposed echinoderm ancestral mesoderm state is highly conserved between the morphologically similar, but evolutionarily distant, auricularia and bipinnaria larvae. However, the auricularia, but not bipinnaria, also develops a simple skelotogenic cell lineage. Our data indicate that the first step in acquiring these novel cell fates was to re-specify the ancestral mesoderm into molecularly distinct territories. These new territories likely consisted of only a few cells with few regulatory differences from the ancestral state, thereby leaving the remaining mesoderm to retain its original function. The new territories were then free to take on a new fate. Partitioning of existing gene networks was a necessary pre-requisite to establish novelty in this system.