Analysis of subcellular localization of <i>ERG</i> isoforms by western blot and confocal microscopy.

<p>(A) Protein lysates from HeLa and HEK293T cells (10μg and 2μg, respectively) transiently transfected by ERG3 (3), ERG3var (v) and ERGaber (a) in pcDNA3.1 vector or by empty vector (-) were analyzed by western blot to determine subcellular localization of individual ERG isoforms (left panel). ERG3 and ERG3var were detected dominantly in the nuclear fraction of protein lysate (N) in both cell lines, while ERGaberN was found in both cytoplasmic (C) and nuclear fractions in HeLa cells and dominantly in cytoplasmic fraction in HEK293T cells. ERGaberC was not detected in any cell line using these protein loads. Using higher load of protein lysate (30μg), sensitive visualization kit and longer exposition to X-ray films ERGaberC was detected in nuclear fraction of HEK293T but not HeLa cells (right panel, black arrow points to corresponding protein band). TBP and GAPDH proteins were used to control protein load and separation of cellular fractions. (B) HEK293T cells were transiently transfected by ERG3, ERG3var and ERGaber isoforms in pcDNA3.1 vector or by empty vector. Forty-eight hours after transfection the presence and the subcellular localization of <i>ERG</i> isoforms was analyzed by confocal microscopy using Ab-N antibody. Nuclei were stained by DAPI. The scale bar represents 10μm.</p

Amplification, cloning and translation of ERG isoforms.

<p>A) Analysis of PCR-amplified <i>ERG</i> isoforms by electrophoresis on agarose gel. PCR products with length corresponding to ERG3 coding region (1458bp) were present in all samples. Calculated length of amplified coding region of predicted aberrant <i>ERG</i> isoforms was 775bp in NALM6 and ALL samples 1,2,4 and 5 (bearing the deletion of <i>ERG</i> exons 7–13) and 880bp in ALL sample 3 (bearing the deletion of <i>ERG</i> exons 7–11). PCR products corresponding to predicted aberrant <i>ERG</i> isoforms were present in all <i>ERG</i>del-positive samples and in none <i>ERG</i>del-negative samples (B-other cases: ALL-6 and ALL-7, hyperdiploid cases: ALL-8 and ALL-9, <i>ETV6/RUNX1</i>-positive case—ALL-10). (B) Schematic representation of <i>ERG</i> transcript variants cloned from NALM6 and of predicted encoded proteins. Regions encoded by alternative frames are displayed in grey. (C) Analysis of ERGaber proteins synthesized from PCR-prepared DNA templates by in vitro transcription/translation (T/T) assay. Proteins were detected by western blot using following antibodies: Anti-ERG antibody EPR3863 (Ab-N), Erg-1/2/3 Antibody C-17 (Ab-int), Erg-1/2/3 Antibody C-20 (Ab-C). In vitro T/T reaction without any DNA template served as a negative control (Neg. Ctrl.). ERGaberN was detected by Ab-N only and ERGaberC by Ab-C only. Ab-int showed only unspecific binding to proteins present in reticulocyte lysate used for T/T assay.</p

ERG gene and its transcript variants.

<p>(A) Schematic representation of <i>ERG</i> gene and its transcript variants according to the current reference sequences available in NCBI Reference Sequence Database. Exons (represented by boxes) are numbered from 5’ to 3’ within the ERG gene (RefSeq ID NG_029732.1). Alternative exon numbering used by Owczarek et al. and by Bohne et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160385#pone.0160385.ref012" target="_blank">12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160385#pone.0160385.ref014" target="_blank">14</a>] is also shown. Transcript variants are annotated with RefSeq accession numbers and names for the encoded protein isoforms commonly used in the literature (ERG3 for NM_182918.3 and ERG2 for NM_004449.4); coding exons are in green, non-coding in grey. Recurrent types of intragenic deletions are displayed in red. Forward and reverse primers (blue triangles) used for amplification of full-length coding regions are positioned across all variants which they can theoretically amplify. (B) Schematic representation of three successfully amplified and cloned <i>ERG</i> transcript variants. (C) Probe sets from HG_U95Av2 Affymetrix gene expression array annotated to <i>ERG</i> gene exons. The scheme of probe sets mapping was adapted from UCSC Genome Browser (Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. The human genome browser at UCSC. Genome Res. 2002 Jun;12(6):996–1006).</p

Intragenic <i>ERG</i> Deletions Do Not Explain the Biology of <i>ERG</i>-Related Acute Lymphoblastic Leukemia

<div><p>Intragenic <i>ERG</i> deletions occur in 3–5% of B-cell precursor acute lymphoblastic leukemia, specifically in B-other subtype lacking the classifying genetic lesions. They represent the only genetic lesion described so far present in the majority of cases clustering into a subgroup of B-other subtype characterized by a unique gene expression profile, probably sharing a common, however, not yet fully described, biological background. We aimed to elucidate whether <i>ERG</i> deletions could drive the specific biology of this <i>ERG</i>-related leukemia subgroup through expression of aberrant or decreased expression of wild type <i>ERG</i> isoforms. We showed that leukemic cells with endogenous <i>ERG</i> deletion express an aberrant transcript translated into two proteins in transfected cell lines and that one of these proteins colocalizes with wild type ERG. However, we did not confirm expression of the proteins in acute lymphoblastic leukemia cases with endogenous <i>ERG</i> deletion. <i>ERG</i> deletions resulted in significantly lower expression of wild type ERG transcripts compared to B-other cases without <i>ERG</i> deletion. However, cases with subclonal <i>ERG</i> deletion, clustering to the same <i>ERG</i> deletion associated subgroup, presented similar levels of wild type <i>ERG</i> as cases without <i>ERG</i> deletion. In conclusion, our data suggest that neither the expression of aberrant proteins from internally deleted allele nor the reduced expression of wild type <i>ERG</i> seem to provide a plausible explanation of the specific biology of <i>ERG</i> -related leukemia subgroup.</p></div

Expression of physiological <i>ERG</i> isoforms in ALL subgroups.

<p>Normalized expression levels (Y axis) of physiological <i>ERG</i> isoforms with (ERG+10) and without (ERG-10) exon 10 and their sum (ERGtotal) in 21 <i>ERG</i>del-negative B-other ALL cases (<i>ERG</i>del-), 8 <i>ERG</i>del-positive B-other ALL cases (<i>ERG</i>del+) and 13 B-other ALL cases with <i>ERG</i>del at sublocnal level (<i>ERG</i>del+sub). Horizontal lines represent means (ERG+10: 34.4±21.4, 20.6±13.0 and 41.0±17.6 for <i>ERGd</i>el-, <i>ERG</i>del+ and <i>ERG</i>del+sub, respectively; ERG-10: 34.3±22.1, 12.6±4.9 and 23.3±11.2 for <i>ERG</i>del-, <i>ERG</i>del+ and <i>ERG</i>del+sub, respectively; ERGtotal: 68.7±42.5, 33.2±16.7 and 64.3±25.5 for <i>ERG</i>del-, <i>ERG</i>del+ and <i>ERG</i>del+sub, respectively). Mann-Whitney U test was used to compare two groups.</p