Huntingtin triplet-repeat locus is stable under long-term Fen1 knockdown in human cells.
The influence of Fen1 loss on trinucleotide-repeat expansion varies between species. In yeast, loss or haploinsufficiency of the Fen1 homolog Rad27 leads to triplet expansion. In mice, haploinsufficiency of Fen1 leads to expansion of a Huntingtin locus CAG repeat. However, no expansion was seen of a (CTG)(n).(CAG)(n) repeat in a Myotonic dystrophy type 1 (DM1) knock-in model. In contrast, in Drosophila, a SCA7 CAG90 repeat was completely stable in a series of strains with mutations of DNA repair genes, among them PCNA, MutS and Fen1. In light of the apparent species dependence of triplet expansion, we have investigated in human cells the effect of Fen1 loss on the Huntingtin CAG repeat. We constructed a cell line, Fen-Rex, which in a reversible manner allows regulation of endogenous Fen1 expression, by using RNA interference (RNAi). Keeping the Fen1 protein knocked down 10-fold over 27 successive cell passages (10(17)-fold expansion in total) and measuring the Huntingtin triplet expansion by both length profiling of PCR products on PAGE gels, and cloning and sequencing of the repeat region, we find the Huntingtin locus completely stable. Our results argue against a role for Fen1 in triggering Huntingtin triplet expansion in human cells.
Moe
Svein Erik
SE
Centre for Molecular Biology and Neuroscience (CMBN), and Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.
Sorbo
Jan Gunnar
JG
Holen
Torgeir
T
D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D045585:Flap Endonucleases / Q000172:deficiency* / Q000235:genetics; D006801:Humans; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D013997:Time Factors; D019680:Trinucleotide Repeat Expansion
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D045585:Flap Endonucleases; C468317:FEN1 protein, human