ABSTRACT
Karyotypical alteration of chromosome 5 and in particular band 5q13 is a frequent finding in hairy cell leukemia (HCL). We have previously identified a number of candidate genes localized in close proximity to a constitutional inv(5)(p13.1q13.3) breakpoint in one HCL patient. These included beta-hexosaminodase HEXB, frequently mutated in the lysosomal storage disorder Sandhoff disease. We now report that the 5q13.3 breakpoint disrupts a novel evolutionary conserved alternative isoform of HEXB. This isoform directly overlaps, in a cis-antisense fashion, exon 1 of the gene for ectodermal neuronal cortex 1 ENC-1, and was thus named ENC-1AS. ENC-1 has previously been shown to be overexpressed in several malignancies, and is believed to play a critical regulatory role in malignant transformation of various tumors. Importantly, subsequent analysis of ENC-1 in purified primary HCL tumor cells revealed a striking upregulation of ENC-1 in all 26 patients examined, compared with normal peripheral blood lymphocytes from healthy donors. Upon further analysis of the ENC-1/ENC-1AS locus, we identified a complex 5' regulatory mechanism involving an inverse expression of the ENC-1 sense and the ENC-1AS transcripts in several tissues supporting the hypothesis that expression of ENC-1AS regulates ENC-1 levels. In addition, we have also found tissue-specific methylation of a 1.2 kb segment encompassing the overlapping ENC-1/ENC-1AS 5' exons, adding to the complexity of the regulation of this locus. Altogether, these results suggest that upregulation of ENC-1 contributes to the development of HCL and provides new information on the possible dysregulation of ENC-1 including expression of a novel antisense gene, ENC-1AS.
Subject(s)
Leukemia, Hairy Cell/genetics , Microfilament Proteins/genetics , Neuropeptides/genetics , Nuclear Proteins/genetics , Base Sequence , Blotting, Northern , Blotting, Southern , Chromosomes, Human, Pair 5 , DNA Primers , Hexosaminidase B , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Leukemia, Hairy Cell/enzymology , Reverse Transcriptase Polymerase Chain Reaction , beta-N-Acetylhexosaminidases/geneticsABSTRACT
Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5.
Subject(s)
DNA-Binding Proteins/genetics , Genes/genetics , Proteins/genetics , RNA, Antisense/genetics , Tumor Suppressor Proteins/genetics , Alternative Splicing/genetics , Animals , Base Composition/genetics , Cell Line, Tumor , Chromosomes/genetics , Gene Expression Profiling/methods , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Genes, Overlapping , HeLa Cells , Humans , In Situ Hybridization/methods , Kidney/cytology , Kidney/embryology , Mice , Open Reading Frames/genetics , Proteins/physiology , RNA, Long Noncoding , RNA, Messenger/genetics , RNA, Untranslated/genetics , TransferasesABSTRACT
Deletion of chromosome 13q14 is the most frequent genetic aberration in B-cell chronic lymphocytic leukemia (CLL), found in more than 50% of cases, indicating that this region contains a gene(s) involved in the development of CLL. However, the pathogenic gene in the critical 13q14 region has not yet been defined. Here, we have cloned and characterized a novel gene, DLEU7, located adjacent to the consensus deleted region, and overlapping the 3' end of DLEU1 tail to tail. Human DLEU7 encodes a putative 221 amino acid protein, with significant conservation in rodents. Mutational and expression analysis in primary CLL samples failed to demonstrate any specific mutations in DLEU7, but no DLEU7 expression could be detected in CLL cells. Methylation of a CpG island in the promoter region of DLEU7 was further analyzed as a possible mechanism for the absence of DLEU7 expression, and the promoter was found to be methylated in the majority of the CLL samples investigated.
Subject(s)
Chromosomes, Human, Pair 13/genetics , Genes, Tumor Suppressor , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Neoplasm Proteins/genetics , Amino Acid Sequence , Animals , Blotting, Northern , Cloning, Molecular , DNA Methylation , DNA Mutational Analysis , DNA Primers/genetics , Gene Deletion , Gene Expression Regulation, Leukemic , Humans , Mice , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Tumor Suppressor ProteinsABSTRACT
In the present study, we describe the human and mouse RFP2 gene structure, multiple RFP2 mRNA isoforms in the two species that have different 5' UTRs and a human-specific antisense transcript RFP2OS. Since the human RFP2 5' UTR is not conserved in mouse, these findings might indicate a different regulation of RFP2 in the two species. The predicted human and mouse RFP2 proteins are shown to contain a tripartite RING finger-B-box-coiled-coil domain (RBCC), also known as a TRIM domain, and therefore belong to a subgroup of RING finger proteins that are often involved in developmental and tumorigenic processes. Because homozygous deletions of chromosomal region 13q14.3 are found in a number of malignancies, including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), we suggest that RFP2 might be involved in tumor development. This study provides necessary information for evaluation of the role of RFP2 in malignant transformation and other biological processes.
