Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome. In particular, we have identified intrachromosomal identical repeats that are located in reverse orientation, which may lead to chromosomal inversions. A bioinformatic workflow pathway to select appropriate regions for analysis was developed. Three such regions overlapping with known human genes, located on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard to human genomic variation is discussed.
The heat-shock protein 90 (HSP90) acts as a chaperone by ensuring proper maturation and folding of its client proteins. The HSP90 capacitor hypothesis holds that interactions with HSP90 allow proteins to accumulate mutations while maintaining function. Following this logic, HSP90 clients would be predicted to show relaxed selection compared with nonclients. In this study, we identify a new HSP90 client in the plant steroid hormone pathway: the transcription factor BES1. Its closest paralog, BZR1, is not an HSP90 client. This difference in HSP90 client status in two highly similar proteins enabled a direct test of the capacitor hypothesis. We find that BES1 shows relaxed selection compared to BZR1, hallmarks of neo-and subfunctionalization, and dynamic HSP90 client status across independent evolutionary paths. These results suggested that HSP90's influence on gene evolution may be detectable if we compare gene duplicates because duplicates share most other properties influencing evolutionary rate that might otherwise conceal the chaperone's effect. We test this hypothesis using systematically identified HSP90 clients in yeast and observe a significant trend of HSP90 clients evolving faster than their nonclient paralogs. This trend was not detected when yeast clients and nonclients were compared without considering paralog status. Our data provide evidence that HSP90 influences selection on genes encoding its clients and facilitates divergence between gene duplicates.T HE phenotypic capacitor heat-shock protein 90 (HSP90) is thought to influence evolutionary processes through its ability to both conceal and release genetic variation (Rutherford and Lindquist 1998;Queitsch et al. 2002;Yeyati et al. 2007;Jarosz and Lindquist 2010). Perturbation of this conserved and essential chaperone reveals cryptic genetic and epigenetic variation in flies, plants, fish, and yeast (Rutherford and Lindquist 1998;Queitsch et al. 2002;Sollars et al. 2003;Yeyati et al. 2007;Jarosz and Lindquist 2010). In worms, HSP90 affects the penetrance of partial loss-of-function mutations (Burga et al. 2011). As expected under the capacitor hypothesis, worms with naturally lower HSP90 levels show significantly higher mutation penetrance (Casanueva et al. 2012). HSP90-dependent variation can be revealed by moderate environmental stress alone, providing a plausible release mechanism for this concealed variation in nature (Rutherford and Lindquist 1998;Queitsch et al. 2002;Jarosz and Lindquist 2010). We showed previously that HSP90-dependent variation is common in natural plant populations, implicating the chaperone as an important player in shaping phenotype and evolutionary trajectories (Sangster et al. 2008a,b). Together, these findings have prompted a longstanding debate about the importance of HSP90 in evolutionary processes and the magnitude of its effect (Meiklejohn and Hartl 2002;Bergman and Siegal 2003;Rando and Verstrepen 2007).It is well-established that HSP90 recognizes metastable proteins and facilitates their folding and stab...
Heat-shock protein 90 (Hsp90) promotes the maturation and stability of its client proteins, including many kinases. In doing so, Hsp90 may allow its clients to accumulate mutations as previously proposed by the capacitor hypothesis. If true, Hsp90 clients should show increased evolutionary rate compared with nonclients; however, other factors, such as gene expression and protein connectivity, may confound or obscure the chaperone’s putative contribution. Here, we compared the evolutionary rates of many Hsp90 clients and nonclients in the human protein kinase superfamily. We show that Hsp90 client status promotes evolutionary rate independently of, but in a small magnitude similar to that of gene expression and protein connectivity. Hsp90’s effect on kinase evolutionary rate was detected across mammals, specifically relaxing purifying selection. Hsp90 clients also showed increased nucleotide diversity and harbored more damaging variation than nonclient kinases across humans. These results are consistent with the central argument of the capacitor hypothesis that interaction with the chaperone allows its clients to harbor genetic variation. Hsp90 client status is thought to be highly dynamic with as few as one amino acid change rendering a protein dependent on the chaperone. Contrary to this expectation, we found that across protein kinase phylogeny Hsp90 client status tends to be gained, maintained, and shared among closely related kinases. We also infer that the ancestral protein kinase was not an Hsp90 client. Taken together, our results suggest that Hsp90 played an important role in shaping the kinase superfamily.
98195-5065 (G.A.M., T.L., C.Q.)The crucial role of microRNAs in plant development is exceedingly well supported; their importance in environmental robustness is studied in less detail. Here, we describe a novel, environmentally dependent phenotype in hypomorphic argonaute1 (ago1) mutants and uncover its mechanistic underpinnings in Arabidopsis (Arabidopsis thaliana). AGO1 is a key player in microRNA-mediated gene regulation. We observed transparent lesions on embryonic leaves of ago1 mutant seedlings. These lesions increased in frequency in full-spectrum light. Notably, the lesion phenotype was most environmentally responsive in ago1-27 mutants. This allele is thought to primarily affect translational repression, which has been linked with the response to environmental perturbation. Using several lines of evidence, we found that these lesions represent dead and dying tissues due to an aberrant hypersensitive response. Although all three canonical defense hormone pathways (salicylic acid, jasmonate, and jasmonate/ethylene pathways) were up-regulated in ago1 mutants, we demonstrate that jasmonate perception drives the lesion phenotype. Double mutants of ago1 and coronatine insensitive1, the jasmonate receptor, showed greatly decreased frequency of affected seedlings. The chaperone HEAT SHOCK PROTEIN 90 (HSP90), which maintains phenotypic robustness in the face of environmental perturbations, is known to facilitate AGO1 function. HSP90 perturbation has been shown previously to upregulate jasmonate signaling and to increase plant resistance to herbivory. Although single HSP90 mutants showed subtly elevated levels of lesions, double mutant analysis disagreed with a simple epistatic model for HSP90 and AGO1 interaction; rather, both appeared to act nonadditively in producing lesions. In summary, our study identifies AGO1 as a major, largely HSP90-independent, factor in providing environmental robustness to plants. ARGONAUTE1 (AGO1) is an ancient, highly conserved essential protein. One of 10 functionally distinct AGO proteins in the plant Arabidopsis (Arabidopsis thaliana), AGO1 uses microRNAs (miRNAs) to decrease the expression of target genes. AGO1 and the miRNA pathway are crucial for many regulatory processes throughout development and in response to environmental stimuli. As frequent components of feedforward loops, miRNAs also play important roles in controlling gene expression noise and maintaining the phenotypic robustness of specific traits in several organisms, including plants ( Lempe et al., 2013).AGO1 and miRNAs also are critical for proper plant responses to abiotic stress. The expression levels of miRNAs respond to heat shock, cold, drought, oxidative stress, UV light exposure, and hypoxia (Sunkar et al., 2012). For example, in response to drought, the expression of miR169 decreases and the expression of its target gene, encoding the transcription factor NFYA5, increases in Arabidopsis (Li et al., 2008). Increased expression of NFYA5 supports plant survival in drought conditions. Transgenic plants ove...
BackgroundIdentical sequences with a minimal length of about 300 base pairs (bp) have been involved in the generation of various meiotic/mitotic genomic rearrangements through non-allelic homologous recombination (NAHR) events. Genomic disorders and structural variation, together with gene remodelling processes have been associated with many of these rearrangements. Based on these observations, we identified and integrated all the 100% identical repeats of at least 300 bp in the NCBI version 36.2 human genome reference assembly into non-overlapping regions, thus defining the Identical Repeated Backbone (IRB) of the reference human genome.ResultsThe IRB sequences are distributed all over the genome in 66,600 regions, which correspond to ~2% of the total NCBI human genome reference assembly. Important structural and functional elements such as common repeats, segmental duplications, and genes are contained in the IRB. About 80% of the IRB bp overlap with known copy-number variants (CNVs). By analyzing the genes embedded in the IRB, we were able to detect some identical genes not previously included in the Ensembl release 50 annotation of human genes. In addition, we found evidence of IRB gene copy-number polymorphisms in raw sequence reads of two diploid sequenced genomes.ConclusionsIn general, the IRB offers new insight into the complex organization of the identical repeated sequences of the human genome. It provides an accurate map of potential NAHR sites which could be used in targeting the study of novel CNVs, predicting DNA copy-number variation in newly sequenced genomes, and improve genome annotation.
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