Cell's heavyweight! The proposed nan mustard is a model to study the pathogenesis of human nervous system diseases!

iPlants:2018 In July, 2001, cell magazine published a research paper entitled "RNA dependent epigenetic silicing directions transcribed down regulation caused by internal repeat expansions" from sureshkumar balasubramanian research group of Monash University in Australia.human Friedreich ataxia disease is a kind of nervous system related disease. The first intron of FXn (frataxin) gene contains "amplification" of GAA trinucleotide repeat, which leads to gene silencing.in this study, we studied the mechanism of GAA trinucleotide repeat silencing gene in the third intron of iil1 gene in Arabidopsis thaliana. It was found that the repeated amplification of GAA trinucleotide resulted in the accumulation and increase of 24 NT siRNA (siRNA) and heterochromatin histone modification at iil1 gene, and down regulated the transcription through RdDM pathway dependent epigenetic modification such as DNA methylation.therefore, this study provides a new insight into the possible pathogenesis of Friedreich ataxia.Background: nucleotide repeat amplification has been proved to be associated with the pathogenesis of more than 40 human diseases, most of which are nervous system related diseases.the first and most common amplification of three nucleotide repeats was found, and now four, five, six, and even twelve nucleotide repeat amplification have been found.nucleotide repeat amplification can occur in coding regions, such as repeated amplification of coding regions. For example, relatively small number of CAG trinucleotide repeats (~ 40 repeats) are seen in Huntington's disease, and mainly lead to polyglutamines extension in coding proteins, which may be toxic and pathogenic; at the same time, it can also occur in non coding regions, such as Friedreich ataxia or embrittlement The amplification of non coding regions in sex X syndrome is large-scale (about 2000 repeats), and it is generally believed that the mechanism of amplification in non coding regions affecting gene function seems to be different among different diseases (see figure below).Fig. 1: the majority of patients with Friedreich ataxia (FRDA) caused by nucleotide repeat amplification are related to the "amplification" of the GAA trinucleotide repeat in the first intron of the gene encoding FXn (frataxin) protein on chromosome 9 (9q13-q21.1).FXn (frataxin) protein is involved in the transport of iron into mitochondria, which prevents the accumulation of iron in mitochondria by the intermediate peptidase in mitochondria.in the normal population, there are about 6-34 GAA repeat units in this locus.however, the number of GAA in patients with Friedreich's ataxia can range from 67 to 1700, with 800-1000 GAA repeat units being the most common.this "amplified" GAA repeat sequence seriously reduces the expression of FXn (frataxin), which is widely distributed in the spinal cord, cerebellum and cerebral cortex of the central nervous system, as well as the heart, skeletal muscle, liver, kidney and pancreas cells in non nervous system.therefore, the disease is characterized by progressive ataxia, pyramidal tract sign, dysphonia, deep sensory abnormalities, scoliosis, arcuate foot and heart damage. Therefore, we should try to increase the expression of FXn as a potential therapeutic option for FRDA.previous studies have shown that the epigenetic silencing of FXn locus may be related to antisense FXn transcription.if the gene transcribes both sense and antisense transcripts, in principle, it may produce small RNAs that cause RNA interference and gene silencing.however, the small RNA at FXn site is still not detected.although these results suggest that GAA / TTC repeat amplification may lead to gene transcription down regulation through epigenetic gene silencing, it is still unclear how this occurs.interestingly, our team previously found that in Arabidopsis ecotype bur-0, there were also a large number of GAA / TTC repeat amplification sequences in intron 3 of iil1 gene; Moreover, it also seriously reduced the transcription level of iil1 gene, which caused the species to show growth defects under high temperature, which is called "irregular damaged leaf" (IIL) phenotype, while the natural suppressor of bur-0 background 15 (ns15, iil1-ns15), partial loss of repeat amplification (about 100 repeats), and only 23 repeats (iil1 Col) in Col-0. The above two plants have no IIL phenotype (as shown in the figure below), which indicates that the number of repeat amplification sequences is related to the degree of gene silencing.however, there is still no answer to how repeated amplification leads to down regulation of iil1 locus.in addition, it was found that the repeat amplification of GAA / TTC containing iil1 in Arabidopsis thaliana is similar to that of FXn which leads to human FRDA.both of them are caused by intron repeat amplification, which leads to the decrease of gene expression.in addition, the repeated amplification of both genes shows somatic variability and genetic instability, which indicates that at least some potential mechanisms may be conservative in the whole species. Therefore, it is helpful to understand the pathogenesis of FRDA by studying how repeat amplification leads to down regulation of iil1 locus! Fig. 2 phenotypic differences between bur-0 and Col-0, ns15 and the number of repeat amplification sequences of iil1 gene. Research methods and conclusion: firstly, it was confirmed that the transcription decline of iil1 induced by trinucleotide repeat amplification sequence was related to epigenetic changes.studies have shown that, compared with ns15 and Col-0 controls, the accumulation of heterochromatin histone H3K27me3 modification of iil1 gene intron repeat amplification sequence in bur-0 plants is increased, while the modification level of active chromatin histone h3k36me3 is decreased, which indicates that iil1-bur-0 locus is an epigenetic silence in trinucleotide repeat amplification sequence dependent manner (see figure below).on the other hand, hlp1 and CLF, the components of PRC2 complex that mediate H3K27me3, are knocked down by RNAi, showing inhibition of IIL phenotype.Second, it is necessary to confirm that the trinucleotide repeat amplification sequence can produce 24 NT siRNA and is necessary for gene silencing.the study found that compared with Col-0 or ns15, the number of 24nt siRNA at iil1 locus in bur-0 increased significantly at 23 ° C and 27 ° C, consistent with phenotype.at the same time, it was found that 24 NT siRNA diffused upstream and downstream of the repeat sequence in bur-0 plants grown at 27 ° C compared with 23 ° C.in conclusion, the accumulation of iil1 24nt siRNA in bur-0 was temperature dependent and increased site specificity, which was related to the number of repeat amplification sequences.further knockout of dcl3 in bur-0, which is responsible for the production of 24nt siRNA, showed a significant increase in h3k36me3 level at bur-0 and inhibited the IIL phenotype (see figure below). Therefore, dcl3 protein and its 24nt siRNA are necessary for down regulation of gene expression mediated by trinucleotide repeat amplification sequence.Third, we confirmed that the siRNA of iil1 gene in bur-0 mediated gene silencing through the RdDM pathway.the RdDM pathway is one of the main pathways of de novo mediated DNA methylation in plants (for details, please click the latest research to reveal the role of RdDM pathway in plant seed development, and the latest research of Nature Genetics | reveals the new initiation mechanism of DNA methylation!).by knocking down the core proteins of all the RdDM pathways in bur-0, almost all of them showed inhibitory effect on IIL phenotype, indicating that the RdDM pathway is necessary for the down regulation of iil1 transcription caused by repeated amplification.as the RdDM pathway mainly affects DNA methylation, it was found that compared with Col-0, bur-0 had high methylation from 5 'region to exon 8 of iil1 gene, including repeat amplification, especially DNA methylation of CHG and CHH. in conclusion, the results suggest that, like FRDA in humans, down regulation of transcription caused by repeated amplification of GAA / TTC trinucleotide is associated with the increase of inhibitory epigenetic markers at iil1 gene. at the same time, this study also demonstrated that repeated amplification of GAA / TTC at iil1 increased the accumulation of 24 NT siRNA, which was temperature dependent and related to IIL phenotype. on the other hand, this study further demonstrated that siRNA mediated DNA methylation via the RdDM pathway and subsequently led to an increase in the level of heterochromatin histone modifications such as H3K27me3, which played an important role in gene silencing induced by repeated amplification sequence of intron GAA / TTC in plant genes (as shown in the figure below). iplants: in view of the early discovery of antisense transcripts and histone modifications in FXn, this study suggests that RNA dependent transcriptional gene silencing pathway should be further evaluated as a possible mechanism of epigenetic silencing of FXn expression in FRDA. due to the existence of some conservative mechanisms among species, Arabidopsis as a model can also provide another insight into the pathogenesis of human diseases. As Arabidopsis research is easier to carry out genetic screening and obtain mutants, the basic research of Arabidopsis deserves attention! At the same time, this study also provides the molecular mechanism of repeat amplification of silencing gene by GAA / TTC trinucleotide in plant gene introns, so it is worth recommending! Link to the original text: