Radiation Leukemia Virus Common Integration at the Kis2 Locus: Simultaneous Overexpression of a Novel Noncoding RNA and of the Proximal Phf6 Gene

Abstract

Retroviral tagging has been used extensively and successfully to identify genes implicated in cancer pathways. In order to find oncogenes implicated in T-cell leukemia, we used the highly leukemogenic radiation leukemia retrovirus VL3 (RadLV/VL3). We applied the inverted PCR technique to isolate and analyze sequences flanking proviral integrations in RadLV/VL3-induced T lymphomas. We found retroviral integrations in c-myc and Pim1 as already reported but we also identified for the first time Notch1 as a RadLV common integration site. More interestingly, we found a new RadLV common integration site that is situated on mouse chromosome X (XA4 region, bp 45091000). This site has also been reported as an SL3-3 and Moloney murine leukemia virus integration site, which strengthens its implication in murine leukemia virus-induced T lymphomas. This locus, named Kis2 (Kaplan Integration Site 2), was found rearranged in 11% of the tumors analyzed. In this article, we report not only the alteration of the Kis2 gene located nearby in response to RadLV integration but also the induction of the expression of Phf6, situated about 250 kbp from the integration site. The Kis2 gene encodes five different alternatively spliced noncoding RNAs and the Phf6 gene codes for a 365-amino-acid protein which contains two plant homology domain fingers, recently implicated in the Börje-son-Forssman-Lehmann syndrome in humans. With the recent release of the mouse genome sequence, highthroughput retroviral tagging emerges as a powerful tool in the quest for oncogenes. It also allows the analysis of large DNA regions surrounding the integration locus.

The Börjeson–Forssman–Lehman syndrome (BFLS, MIM #301900)

“…However, this is only speculation, which needs further experimental substantiation. 1 More recently, Landais et al 6 reported the existence of six other isoforms of the Phf6 gene in mouse. Their function is not clear although five of them (t1, t4, t5, t6 and t7) can potentially give rise to alternative Phf6 protein isoforms.…”

“…More recently, mouse Phf6 protein was found overexpressed in tumors with rearrangements (radiation leukemia virus, RadLV integration) in the neighboring Kis2 locus (noncoding RNA; Unigene cluster Mm.277876, B250 kb proximal to the Phf6 gene). 6 Overall there is very little information on the function of the PHF6 protein available. If we dare to speculate, based on the X-inactivation skewing in carrier females, 1,2 localization of the PHF6 protein to the cell nucleus and nucleolus 1,6,8 and the latest observations of the Phf6 protein overexpression in specific mouse tumors, 6 we would suggest an important role for PHF6 in cell growth and proliferation, which may be achieved via its participation in ribosome biogenesis (at the RNA and/or protein level).…”

“…PHF6 is transcribed as a B4.5 kb mRNA, which shows a ubiquitous expression pattern as tested by Northern blot hybridization 1 and RT PCR. 6 Two major isoforms exist. These differ in the inclusion or exclusion of intron 10 from the mRNA.…”

“…Subcellular localization studies show that the PHF6 protein is localized in the cell nucleus and more prominently in the nucleolus. 1,6,8 Traditionally, this organelle has been known as the ribosome assembly factory. However, as our knowledge advances it has been suggested that the nucleolus plays an important role, among others, in nuclear export, sequestering regulatory molecules, modifying small RNAs, cell cycle regulation and processes of aging.…”

“…11 Interestingly, there is only one PHF6 mutation known, which directly affects critical residue of the first PHD finger, cytosine at position 99 (C99F). 1 In addition to the PHD fingers the PHF6 protein also contains multiple (at least four clearly recognizable and functional) 1,6 nuclear localization sequences. Subcellular localization studies show that the PHF6 protein is localized in the cell nucleus and more prominently in the nucleolus.…”