DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the ...NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not$DNA-PK_{cs}$. We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.
RNA interference is an evolutionarily conserved process in which expression of a specific gene is post-transcriptionally inhibited by a small interfering RNA (siRNA), which recognizes a complementary ...mRNA and induces its degradation. Currently, RNA interference is being used extensively to inhibit expression of specific genes for experimental and therapeutic purposes. For applications in mammalian cells, siRNAs are designed to be <approximately 30 base pairs to avoid nonspecific effects that arise from inducing the cellular double-stranded RNA (dsRNA)-dependent protein kinase (PKR) response. Here we perform expression profiling in mammalian tissue-culture cells treated under standard conditions with conventional 21-bp siRNAs and find, unexpectedly, that >1000 genes involved in diverse cellular functions are nonspecifically stimulated or repressed. The effects on gene expression are dependent upon siRNA concentration and are stable throughout the course of siRNA treatment. Our results can be explained by previous studies showing that dsRNAs can affect multiple signaling and transcription pathways in addition to PKR. The potential for this widespread, nonspecific effect on mammalian gene expression must be carefully considered in the design of siRNA experiments and therapeutic applications.
Although genome sequencing has identified numerous noncoding alterations between primate species, which of those are regulatory and potentially relevant to the evolution of the human brain is ...unclear. Here we annotated cis-regulatory elements (CREs) in the human, rhesus macaque and chimpanzee genomes using chromatin immunoprecipitation followed by sequencing (ChIP-seq) in different anatomical regions of the adult brain. We found high similarity in the genomic positioning of rhesus macaque and human CREs, suggesting that the majority of these elements were already present in a common ancestor 25 million years ago. Most of the observed regulatory changes between humans and rhesus macaques occurred before the ancestral separation of humans and chimpanzees, leaving a modest set of regulatory elements with predicted human specificity. Our data refine previous predictions and hypotheses on the consequences of genomic changes between primate species and allow the identification of regulatory alterations relevant to the evolution of the brain.
Copy number variation (CNV) contributes to disease and has restructured the genomes of great apes. The diversity and rate of this process, however, have not been extensively explored among great ape ...lineages. We analyzed 97 deeply sequenced great ape and human genomes and estimate 16% (469 Mb) of the hominid genome has been affected by recent CNV. We identify a comprehensive set of fixed gene deletions (n = 340) and duplications (n = 405) as well as >13.5 Mb of sequence that has been specifically lost on the human lineage. We compared the diversity and rates of copy number and single nucleotide variation across the hominid phylogeny. We find that CNV diversity partially correlates with single nucleotide diversity (r(2) = 0.5) and recapitulates the phylogeny of apes with few exceptions. Duplications significantly outpace deletions (2.8-fold). The load of segregating duplications remains significantly higher in bonobos, Western chimpanzees, and Sumatran orangutans-populations that have experienced recent genetic bottlenecks (P = 0.0014, 0.02, and 0.0088, respectively). The rate of fixed deletion has been more clocklike with the exception of the chimpanzee lineage, where we observe a twofold increase in the chimpanzee-bonobo ancestor (P = 4.79 × 10(-9)) and increased deletion load among Western chimpanzees (P = 0.002). The latter includes the first genomic disorder in a chimpanzee with features resembling Smith-Magenis syndrome mediated by a chimpanzee-specific increase in segmental duplication complexity. We hypothesize that demographic effects, such as bottlenecks, have contributed to larger and more gene-rich segments being deleted in the chimpanzee lineage and that this effect, more generally, may account for episodic bursts in CNV during hominid evolution.
DNA damage provokes DNA repair, cell‐cycle regulation and apoptosis. This DNA‐damage response encompasses gene‐expression regulation at the transcriptional and post‐translational levels. We show that ...cellular responses to UV‐induced DNA damage are also regulated at the post‐transcriptional level by microRNAs. Survival and checkpoint response after UV damage was severely reduced on microRNA‐mediated gene‐silencing inhibition by knocking down essential components of the microRNA‐processing pathway (Dicer and Ago2). UV damage triggered a cell‐cycle‐dependent relocalization of Ago2 into stress granules and various microRNA‐expression changes. Ago2 relocalization required CDK activity, but was independent of ATM/ATR checkpoint signalling, whereas UV‐responsive microRNA expression was only partially ATM/ATR independent. Both microRNA‐expression changes and stress‐granule formation were most pronounced within the first hours after genotoxic stress, suggesting that microRNA‐mediated gene regulation operates earlier than most transcriptional responses. The functionality of the microRNA response is illustrated by the UV‐inducible miR‐16 that downregulates checkpoint‐gene CDC25a and regulates cell proliferation. We conclude that microRNA‐mediated gene regulation adds a new dimension to the DNA‐damage response.
The study of microRNA (miRNA) regulation in the pathogenesis of autoimmune diseases and hematopoietic malignancies provides new understanding of the mechanisms of disease and is currently the focus ...of many researchers in the field. Autoimmune disorders and cancers of immune system comprise a wide range of genetically complex diseases that share certain aspects of dysregulated genetic networks, most notably deactivation of apoptosis. miRNA mechanisms control gene expression at the post-transcriptional level, linking mRNA processing and gene function. Considerable amount of data have been accumulated that indicate that the alteration of miRNA expression closely mirrors the development of immune system diseases and is likely to play a role in their pathogenesis. However, a knowledge gap remains in our understanding of how miRNA dysregulation and the specific effects of miRNAs on target gene expression underlay the disease phenotype. Here we review a number of studies describing miRNA alterations in autoimmune diseases and hematopoietic cancers and discuss potential miRNA-regulated mechanisms that differentially influence the development of autoimmunity as compared to cancer progression.
The molecular instructions that govern gene expression regulation are encoded in the genome and ultimately determine the morphology and functional specifications of the human brain. As a consequence, ...changes in gene expression levels might be directly related to the functional decline associated with brain aging. Small noncoding RNAs, including miRNAs, comprise a group of regulatory molecules that modulate the expression of hundred of genes which play important roles in brain metabolism. Recent comparative studies in humans and nonhuman primates revealed that miRNAs regulate multiple pathways and interconnected signaling cascades that are the basis for the cognitive decline and neurodegenerative disorders during aging. Identifying the roles of miRNAs and their target genes in model organisms combined with system-level studies of the brain would provide more comprehensive understanding of the molecular basis of brain deterioration during the aging process.
The mammalian ATF/CREB family of transcription factors comprises a large group of basic-region leucine zipper (bZIP) proteins whose members mediate diverse transcriptional regulatory functions. Here ...we report that expression of a specific mouse ATF gene, ATFx, is down-regulated in a variety of cells undergoing apoptosis following growth factor deprivation. When stably expressed in an interleukin 3 (IL-3)-dependent cell line, ATFx suppresses apoptosis resulting from cytokine deprivation. Conversely, a dominant-negative ATFx mutant induces apoptosis of cells cultured in the presence of growth factors. We also show that 24p3, a secreted lipocalin that induces apoptosis when added to hematopoietic cells, represses ATFx expression. However, constitutive expression of ATFx renders cells resistant to 24p3-mediated apoptosis. Collectively, our results indicate that ATFx is an anti-apoptotic factor, a novel role for an ATF protein.
TATA-box-binding protein (TBP) is a highly conserved RNA polymerase II general transcription factor that binds to the core promoter and initiates assembly of the preinitiation complex. Two proteins ...with high homology to TBP have been found: TBP-related factor 1 (TRF1), described only in Drosophila melanogaster, and TRF2, which is broadly distributed in metazoans. Here, we report the identification and characterization of an additional TBP-related factor, TRF3. TRF3 is virtually identical to TBP in the C-terminal core domain, including all residues involved in DNA binding and interaction with other general transcription factors. Like other TBP family members, the N-terminal region of TRF3 is divergent. The TRF3 gene is present and expressed in vertebrates, from fish through humans, but absent from the genomes of the urochordate Ciona intestinalis and the lower eukaryotes D. melanogaster and Caenorhabditis elegans. TRF3 is a nuclear protein that is present in all human and mouse tissues and cell lines examined. Despite the highly homologous TBP-like C-terminal core domain, gel filtration analysis indicates that the native molecular weight of TRF3 is substantially less than that of TFIID. Interestingly, after mitosis, reimport of TRF3 into the nucleus occurs subsequent to TBP and other basal transcription factors. In summary, TRF3 is a highly conserved vertebrate-specific TRF whose phylogenetic conservation, expression pattern, and other properties are distinct from those of TBP and all other TRFs.
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