The Influence Of Progerin Expression On The Nature Of DNA Double‐Strand Break Repair

Abstract

Hutchinson‐Gilford Progeria Syndrome (HGPS) is a rare, fatal autosomal dominant genetic condition characterized by many features of accelerated aging in children. Children with HGPS die at an average age of fourteen, usually from heart disease. It has been known for some time that the syndrome is most commonly caused by a point mutation in the LMNA gene which normally codes for lamin A and its splice variant lamin C. The LMNA mutation commonly associated with HGPS leads to increased usage of a cryptic splice site which leads to the production of a truncated, farnesylated form of lamin A referred to as “progerin.” Interestingly, and quite significantly, progerin is also expressed at very low levels in healthy individuals and has been implicated in the normal aging process, possibly by increased expression over time. Progerin overexpression in progeria disrupts nuclear architecture and chromatin organization. Previous studies by others have indicated that progerin slows the kinetics of DNA double‐strand break (DSB) repair and leads to the accumulation of spontaneous DSBs. Strikingly, there has been little investigation into how progerin expression may alter the nature of DSB repair. DSB repair may occur through one of two ways: homologous recombination (HR), which is accurate, or non‐homologous end joining (NHEJ), which is intrinsically mutagenic. We aimed to assess whether progerin expression alters the DSB repair pathway of choice or whether it alters the nature of individual repair events. To do so, we stably expressed a GFP‐progerin fusion protein, or GFP alone, in a human cell line containing an integrated DNA substrate that served as a reporter for DSB repair. A DSB was introduced in the integrated substrate by transient expression of endonuclease I‐SceI. Events involving DSB repair via HR or NHEJ were recoverable by selection for G418‐resistant clones, and these events were analyzed at the nucleotide level and by Southern blotting analysis. We present results indicating there was an increased frequency of DSB events occurring via NHEJ in cells expressing GFP‐progerin in comparison to those expressing GFP alone, and the difference in pathway usage was highly significant (p < 0.004 by a two‐sided Fisher exact test). There was no evidence that progerin impacted the accuracy of HR events. In future work, the finding that progerin expression appears to alter the DSB repair pathway of choice within the human genome will be explored further, as will the possibility that progerin alters the homology requirements for HR. Our long‐term goal is to better understand the relationship between aging and the corruption of genome maintenance pathways.Support or Funding InformationThis work was supported by grant MCB‐1157416 from the NSF to ASW and BCW, and from an ASPIRE grant from the University of South Carolina to ASW.