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ERP, a New Member of the Ets Transcription Factor/Oncoprotein

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MOLECULAR AND CELLULAR BIOLOGY, May 1994, p. 3292-3309 Vol. 14, No. 5 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology ERP, a New Member of the ets Transcription Factor/Oncoprotein Family: Cloning, Characterization, and Differential Expression during B-Lymphocyte Development MONICA LOPEZ, PETER OETTGEN, YASMIN AKBARALI, ULRICH DENDORFER, AND TOWIA A. LIBERMANN* Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215 Received 23 July 1993/Returned for modification 18 January 1994/Accepted 17 February 1994 The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, TT and ,uB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the w or the ,uB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer wF site, and the kck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH w site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation. The different stages of B-cell development are characterized (for a review, see reference 46). Interestingly, each of these by differential expression of a whole set of genes, most regulatory regions contains several, at least partially redundant prominently the members of the immunoglobulin gene family, B-cell-specific enhancer elements, none of them being com- as well as by differential immunoglobulin gene rearrangement pletely essential for the regulation of the IgH gene (42, 49, 58). (for a review, see reference 7). Differential expression and/or We have recently identified two new enhancer elements, ir activation of transcription factors leading to stage-specific and RB, in the intronic IgH enhancer with characteristic expression of a certain set of genes appear to be crucial for cell functional differences (47, 49). Whereas the ,uB element is differentiation in general and B-cell development in particular active throughout B-cell development (49), the 'r enhancer (for a review, see reference 46). Transcriptional regulation of element functions primarily at early stages of B-cell develop- immunoglobulin genes is of special interest for B-cell differ- ment (47). Furthermore, whereas the pRB element does not act entiation, because different classes of immunoglobulin genes as an enhancer by itself, rr alone works as a very potent are switched on at different stages of B-cell development (7, enhancer in pre-B cells (47, 49). The DNA sequences of both 83). Thus, whereas immunoglobulin heavy-chain (IgH) gene enhancer elements show striking homology to binding sites for expression occurs at the earliest stages of B-cell development transcription factors of the ets transcription factor/oncoprotein prior to IgH gene rearrangement and continues throughout family, with a GGAA sequence in the core of the binding site B-cell development, immunoglobulin light-chain genes are (55, 61, 85, 94). All members of the ets family share a highly transcribed only upon maturation of B cells after IgH gene homologous 80- to 90-amino-acid DNA-binding domain, the rearrangement (7, 83). ETS domain, although the remainder of the proteins are Analysis of the regulatory elements involved in IgH gene strikingly divergent among different members of the family (for expression has resulted in the identification and characteriza- reviews, see references 55, 61, and 85). The ETS domain is tion of at least three regulatory regions including the promoter sufficient to interact specifically with DNA sequences contain- upstream of the IgH gene, an intronic enhancer, and an a in the center of the site enhancer 3' of the IgH gene (15, 51). Several positive and ing GGA(A/T) binding (20, 53, 61, within 94, 97). Members of the ets family have been recently shown to negative regulatory elements appear to be distributed a set a see reference Most play essential roles as transcriptional regulators of whole these regulatory regions (for review, 46). such as interleu- of these elements appear to be ubiquitously active; however, of genes including many T-cell-specific genes some B-cell-specific regulatory elements have been described kin 2 (IL-2) (88), CD3 (40, 88), T-cell receptors ot, ,, and 8 (26, 40, 88, 94), human immunodeficiency virus type 2 (HIV-2) long terminal repeat (LTR) (41, 57, 94), stromelysin (96), c-fos (14, * Corresponding author. Mailing address: Department of Medicine, 25), and urokinase (60) genes. Furthermore, several ets-related Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Phone: genes have been shown to be expressed in B cells (19, 31, 72). (617) 735-3393. Fax: (617) 735-3547. As a first step in identifying putative novel members of the 3292 VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3293 ets gene family which are expressed in pre-B cells, we have agarose gel. The relevant amplified DNA fragments were applied a PCR approach using degenerate oligonucleotides eluted with glass beads (Geneclean; Bio 101) as recommended encoding conserved domains of all known members of the ets by the manufacturer. Eluted DNA was digested with NotI and family and mRNA derived from a pre-B-cell line. We now Sall (for primers A to D) or NotI alone (for primers E and F), report the isolation and characterization of a cDNA clone repurified via glass beads, and subcloned into either NotI-SaI- encoding a novel member of the ets gene family, named ERP or NotI-digested pSL1 180 (Pharmacia) or Bluescript SK+ (ets-related protein). The ERP gene is most closely related to (Stratagene). Colonies of recombinant clones were randomly SAP-i (14) and ELK-I (68), two members of the ets gene picked, and minipreparations of DNA were carried out by the family that interact with the serum response factor (SRF) (14, Magic Miniprep (Promega) procedure as recommended by the 25). ERP mRNA is expressed to different extents in a variety manufacturer. The clones were digested with SalI and XhoI or of tissues but, in B cells, is much more abundant at the NotI and analyzed on a 2% agarose gel. Double-stranded pre-B-cell stage than at later stages of B-cell development. We sequencing of positive clones was performed with the Seque- demonstrate that carboxy terminus-deleted ERP interacts with nase II kit (U.S. Biochemical) according to the manufacturer's several ets-related binding sites including the E74 and the IgH instructions. Tr sites, suggesting a potential role in IgH gene expression. 5' RACE (rapid amplification of cDNA ends) primer exten- sion. First-strand cDNA synthesis and 5' tailing with dCTP was MATERIALS AND METHODS carried out with the 5' RACE system (Bethesda Research Laboratories) according to protocols recommended by the Cell culture. S194 (murine myeloma), NIH 3T3 (murine manufacturer. Briefly, 1 jig of poly(A)+ mRNA derived from fibroblast), and P19 (murine teratocarcinoma) cells were BASC6C2 cells was reverse transcribed for 30 min at 42°C by grown in Dulbecco modified Eagle medium containing 10% using 200 U of SuperScript reverse transcriptase and 200 ng of fetal calf serum. PD31 (murine pre-B), 230-238 (murine pre- ERP-specific oligonucleotide primer 5'-GCTAGCGGCCGCT B), NFS 5.3 (murine late pre-B), BASC6C2 (murine pre-B), GAGGAGCYT7GAACTCGCC-3' (primer G). The cDNA was WEHI231 (murine mature-B), A-20 (murine mature-B), J558 RNase H treated and purified over GlassMax spin cartridges (murine myeloma), MPC1 1 (murine myeloma), P3X63 AG8 (Bethesda Research Laboratories). The cDNA was tailed at the (murine myeloma), EL.4 (murine-T), and Pu5-1.8 (murine 5' end with 10 U of terminal deoxynucleotidyl transferase (TdT) monocyte) cells were maintained in RPMI 1640 supplemented and 200 nM dCTP for 10 min at 37°C. with 10% fetal calf serum and 50 ,uM 2-mercaptoethanol. Deoxycytidine-tailed cDNA was amplified by PCR using an HAFTL (murine early-pre-B) cells were grown in RPMI 1640 anchor primer (5'-CUACUACUACUAGGCCACGCGTCG medium containing 15% fetal calf serum and 50 ,uM 2-mer- ACTAGTACGGGIIGGGIIGGGIIG-3') provided by the captoethanol. manufacturer and a nested ERP-specific primer (5'-GCCACG Isolation of poly(A)+ mRNA. Poly(A)+ mRNAs were iso- GTCGACCTCGCCATCGTTCGATGTCC-3' [primer H]) up- lated from _108 cells as described by Libermann et al. (48). stream of primer G. cDNA synthesis. First-strand cDNA synthesis was carried 5' RACE PCR amplifications were carried out in a final out with the SuperScript Choice system (Bethesda Research volume of 50 ,lI containing 10% of the tailed cDNA, 50 mM Laboratories) according to protocols recommended by the KCl, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2, 0.2 mM dNTPs, manufacturer.
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  • Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response

    Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response

    Supplementary Online Content Beltran H, Eng K, Mosquera JM, et al. Whole-exome sequencing of metastatic cancer and biomarkers of treatment response. JAMA Oncol. Published online May 28, 2015. doi:10.1001/jamaoncol.2015.1313 eMethods eFigure 1. A schematic of the IPM Computational Pipeline eFigure 2. Tumor purity analysis eFigure 3. Tumor purity estimates from Pathology team versus computationally (CLONET) estimated tumor purities values for frozen tumor specimens (Spearman correlation 0.2765327, p- value = 0.03561) eFigure 4. Sequencing metrics Fresh/frozen vs. FFPE tissue eFigure 5. Somatic copy number alteration profiles by tumor type at cytogenetic map location resolution; for each cytogenetic map location the mean genes aberration frequency is reported eFigure 6. The 20 most frequently aberrant genes with respect to copy number gains/losses detected per tumor type eFigure 7. Top 50 genes with focal and large scale copy number gains (A) and losses (B) across the cohort eFigure 8. Summary of total number of copy number alterations across PM tumors eFigure 9. An example of tumor evolution looking at serial biopsies from PM222, a patient with metastatic bladder carcinoma eFigure 10. PM12 somatic mutations by coverage and allele frequency (A) and (B) mutation correlation between primary (y- axis) and brain metastasis (x-axis) eFigure 11. Point mutations across 5 metastatic sites of a 55 year old patient with metastatic prostate cancer at time of rapid autopsy eFigure 12. CT scans from patient PM137, a patient with recurrent platinum refractory metastatic urothelial carcinoma eFigure 13. Tracking tumor genomics between primary and metastatic samples from patient PM12 eFigure 14.