The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure

David Valle-García,Emily Bernstein,Michael A Dyer,Félix Recillas-Targa,Lyra M Griffiths

doi:10.1186/2193-1801-3-222

Abstract

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-222) contains supplementary material, which is available to authorized users.

Highlights

ATRX is an ATP-dependent chromatin-remodeling helicase of the SWI/SNF family and was originally identified because mutations in its coding region give rise to a complex disorder known as α-Thalassemia, Mental Retardation X-linked (ATRX) syndrome
One of the plasmids contains the full length ATRX cDNA sequence in a pEGFP (GFP-ATRX) (Bérubé et al 2008) backbone and the other is cloned in a lentiviral backbone (ATRXYFP) and was derived from the sequence contained in GFP-ATRX (Newhart et al 2012)
We found that the ATRX cDNA sequence that has been cloned corresponds to isoform 2, which lacks exon 6 in comparison to isoform 1

Summary

Introduction

ATRX is an ATP-dependent chromatin-remodeling helicase of the SWI/SNF family and was originally identified because mutations in its coding region give rise to a complex disorder known as α-Thalassemia, Mental Retardation X-linked (ATRX) syndrome. Large-scale genome sequencing efforts have identified ATRX mutations and deletions in a range of tumor types, including glioblastoma multiforme, neuroblastoma and pancreatic neuroendocrine tumors (Jiao et al 2011; Schwartzentruber et al 2012; Cheung et al 2012; Molenaar et al 2012; Clynes et al 2013). These alterations appear to result in loss of functional ATRX, suggesting that ATRX acts as tumor suppressor. The mechanisms by which such mutations result in ATRX loss-of-function remain unclear

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Conclusion