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    • In vitro we demonstrated that the DMR can drive luciferase

    2018-10-20

    In vitro, we demonstrated that the DMR can drive luciferase expression in three different cell lines (293T,COS-7, and HepG2) and markedly enhances reporter gene activity (10- to 15-fold increase) compared with basal activity obtained with either the SIX5 promoter, the entire region that surrounds the CTG repeats (12 repeats), or the DMR in an opposite orientation. Although our in vitro assays point to the potential role of this regulatory rotenone as a promoter sequence, there is no evidence to support this in publically available datasets in hESCs or any other cell type examined. Therefore, we suggest that this element most likely acts as a proximal enhancer rather than a distal promoter for SIX5 transcription. Importantly, to establish the relevance of methylation to the regulatory function of this region, we verified that in vitro methylation of the DMR completely silenced reporter gene activity in all examined cell lines. These results indicate that aberrant methylation is, in fact, mechanistically responsible for the cis reduction in SIX5 expression. Lastly, given the frequent involvement of cardiac conduction defects in patients and in SIX5 heterozygote mice (Martorell et al., 1997; Sarkar et al., 2000; Wakimoto et al., 2002), we illustrate that the DMR is capable of driving eGFP expression in in vitro-derived transgenic cardiomyocytes. Our results are in line with the identification of this region in publically available data as a regulatory element by FAIRE (formaldehyde-assisted isolation of regulatory elements); DNaseI hypersensitive site mapping; local enrichment in monomethylation of histone 3 at lysine 4 (H3K4me1), acetylation of histone 3 at lysine 27 (H3K27Ac) and Pol2A; and the clustering of tissue-specific transcription factors. In conclusion, our functional assays suggest that SIX5 activity is finely tuned by the activity of a narrow DMR that is located upstream of the repeats, in the coding region of DMPK. This putative regulatory zone may ultimately prove to be an essential target for the treatment of DM1 clinical symptoms that are attributed to SIX5 downregulation. Together, this study shows a mechanistic association between CTG expansion size, hypermethylation, and the reduction in SIX5 expression. It demonstrates how a disease-causing mutation in one gene (CTG expansion in DMPK) can affect the expression of a different gene (SIX5) by epigenetically modifying a flanking sequence that plays a dual function: a protein coding sequence and a regulatory element for a neighboring gene. Our findings, therefore, clearly illustrate how an unstable repeat expansion leads to the spread of methylation by the loss of the 5′ boundary of a CpG island.
    Experimental Procedures
    Acknowledgments We thank Dr. Amira Gepstein for assistance with in vitro cardiac differentiation, Dr. David Zeevi for critical reading of the manuscript, Dr. Dalit Ben-Yosef for the provision of the LIS-DM-affected hESC line, Prof. Douglas Melton and Prof. Nissim Benvenisty for the provision of WT hESC lines (HES13, HES-B123, and HES-B200), and Dr. Micha Mandel for assistance with the statistical analysis. This work was partly supported by the Israel Science Foundation (to E.R., grant 711/12), a donation from the Abrasba Foundation (to E.R., Gindi family), and a Career Integration Grant (to B.R., grant 630849).
    Introduction The discovery that transcription factors (TFs) can convert somatic cells into both specialized and induced pluripotent stem cells (iPSCs) has revolutionized stem cell research and promises to have major clinical applications (Graf and Enver, 2009; Yamanaka and Blau, 2010). Lineage-instructive TFs activate and repress tissue-specific genes by recognizing sequence-specific DNA consensus motifs contained within enhancers and promoters (Ptashne, 2007). They establish gene regulatory networks (GRNs) of the novel gene expression program while dismantling those of the old program, involving the formation of feedforward, cross-inhibitory, and auto-regulatory loops (Bertrand and Hobert, 2010; Davidson, 2010; Graf and Enver, 2009; Holmberg and Perlmann, 2012). However, how these processes are coordinated and whether they recapitulate normal development remain unclear (Vierbuchen and Wernig, 2011), especially as neither TF-induced lineage conversions nor iPSC reprogramming appear to retrace normal developmental pathways (Apostolou and Hochedlinger, 2013; Di Tullio et al., 2011; Ladewig et al., 2013; Vierbuchen and Wernig, 2011).