Instructor, Department of Cell Biology
To understand the molecular mechanism of trinucleotide repeat expansion in stem cells derived from patients
Several inherited neurological and muscular disorders like the fragile X syndrome (FXS), Friedreich’s ataxia and Huntington’s disease are caused by expansion of a trinucleotide repeat (TNR). The fragile X syndrome, the most common form of inherited intellectual disability in males (one in 4000 boys are affected), is caused by an expansion of a CGG repeat tract in the 5’ untranslated region of the fragile X mental retardation (FMR1) gene. FXS patients inherit an FMR1 gene with over 200 repeats (full mutation), which is methylated and transcriptional silenced. In addition one in 230 women have expanded CGG repeats (premutation 55-200 repeats) and are at risk to have a FXS child. The mechanism for TNR expansion in human cells is not clearly understood.
TNR form stable secondary structures, while the DNA is transiently in a single stranded form like during DNA replication and DNA repair. These secondary repeat structures can trigger replication fork stalling, which potentially leads to DNA polymerase slippage, and repeat expansion. We are using a unique approach, which monitors the DNA replication in single DNA molecules by multi-color immunofluorescence microscopy (SMARD) to study the mechanism for repeat expansion in cells derived from patients with TNR diseases. We recently found that the replication fork pauses at the CGG repeats and that the direction of the replication fork is altered at the endogenous FMR1 locus in FXS human embryonic stem cells. Using newly developed stem cells derived from TNR patients and SMARD, our goal is to gain insights into the mechanism of TNR expansion and TNR stabilization.
Our studies aim to provide a better understanding of the repeat expansion mechanism for the development of therapeutic strategies to prevent repeat expansion and to stabilize the repeats in patients.
Gerhardt J. Epigenetic modifications in fragile X pluripotent stem cells; Implications in fragile X syndrome modeling. Brain Res. 2015 Oct 20, Epub ahead of print.
Gerhardt J*, Tomishima MJ, Zaninovic N, Colak D, Yan Z, Zhan Q, Rosenwaks Z, Jaffrey SR and Schildkraut CL. The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells. Mol Cell 2014, 53:19-31. Featured on the cover.
(*: co-corresponding author)
Colak D, Zaninovic N, Cohen MS, Rosenwaks Z, Yang W, Gerhardt J, Disney M and Jaffrey SR. A promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome. Science 2014, 343:1002-5.
Gerhardt J*, Zaninovic N, Zhan Q, Madireddy A, Nolin SL, Ersalesi N, Yan Z, Rosenwaks Z and Schildkraut CL. Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation. J Cell Biol 2014, 206(5):599-607. Highlighted in the journal on page 574.
(*: co-corresponding author)
Gray SJ, Gerhardt J#, Doerfler W, Small LE, Fanning E. An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene. Mol Cell Biol 2007, 27:426-37.
Zhao X, Madden-Fuentes RJ, Lou BX, Pipas JM, Gerhardt J, Rigell CJ, Fanning E. ATM- and proteasome-dependent destruction of Mre11-Rad50-Nbs1 subunits in SV40-infected primate cells. J Virol 2008, 82:5316-5328.
Gerhardt J, Jafar S, Spindler MP, Ott E, Schepers A. Identification of new human origins of DNA replication by an origin-trapping assay. Mol Cell Biol 2006, 26:7731-7746.
Ritzi M, Tillack K, Gerhardt J, Ott E, Humme S, Kremmer E, Hammerschmidt W, Schepers A. Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus. J Cell Sci 2003, 116:3971-3984.
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