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Human Molecular Genetics Advance Access originally published online on September 30, 2004
Human Molecular Genetics 2004 13(23):2925-2936; doi:10.1093/hmg/ddh315
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Human Molecular Genetics, Vol. 13, No. 23 © Oxford University Press 2004; all rights reserved

Disruption of a novel ectodermal neural cortex 1 antisense gene, ENC-1AS and identification of ENC-1 overexpression in hairy cell leukemia

Marianne Hammarsund1, Mikael Lerner1, Chaoyong Zhu1, Mats Merup2, Monika Jansson2, Gösta Gahrton2, Hanneke Kluin-Nelemans3, Stefan Einhorn1, Dan Grandér1, Olle Sangfelt1,*,{dagger} and Martin Corcoran1,{dagger}

1Department of Oncology/Pathology, CCK, Karolinska Hospital and Institute, Stockholm, Sweden, 2Department of Medicine, Division of Hematology, Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden and 3Department of Hematology, University Hospital Groningen, Groningen, The Netherlands

Received June 24, 2004; Revised August 27, 2004; Accepted September 22, 2004

DDBJ/EMBL/GenBank accession nos{ddagger}


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder characterized by splenic and bone marrow infiltration, pancytopaenia and low numbers of circulating tumor B-cells which show distinctive ‘hair-like’ cytoplasmic projections. The disease accounts for 2% of all lymphoid leukemias and is highly sensitive to therapeutic intervention using interferon-{alpha} or purine analogues (1). The exact cell of origin of HCL remains unknown, but recent comparison of gene expression profiles of different B-cell populations shows that HCL cells may be derived from either memory B-cells (2) or splenic B-cells (3). Despite the advances in diagnosis and treatment of HCL, pathogenic molecular events associated with the development of HCL are not well understood. Cytogenetic analysis has shown that the most frequent chromosomal abnormalities of HCL cases include alterations of chromosome 5 (4), in particular band 5q13 (translocations, inversions and trisomies), and deletions of chromosome 7 (6). The presence of trisomy 5 and disruption of 5q13 in HCL cases through translocations or inversions strongly suggest that a gene of oncogenic potential may be localized to this region.

Chromosomal translocations have facilitated positional cloning of disrupted disease-causing genes, and have been instrumental in studying the structure and mechanism of chromosomal rearrangements (7–9). We have previously identified a HCL patient with a constitutional inversion, inv(5)(p13.1q13.3) (4) mapping to the same region frequently altered in sporadic HCL cases. During the search for candidate genes we mapped a number of genes, HCLG1 (10), hEFG2 and HEXB (11), on the telomeric side of the 5q13.3 inversion breakpoint, none of which were found to be rearranged in sporadic HCL cases. HCLG1 encodes a TGF-beta inducible nuclear protein (10) and hEFG2 encodes a human mitochondrial elongation factor gene (11). The beta-hexosaminodase (HEXB) gene encodes the beta element of dimeric hexosaminodase lysosomal enzymes, involved in the degradation of carbohydrate moieties of glycoproteins, glycolipids and proteoglycans (12), and which are indispensable in human metabolism for the degradation of gangliosides, an essential class of outer-layer membrane lipids. The HEXB gene has previously been found to be frequently mutated in the genetic disorder Sandhoff disease, which is characterized by the accumulation of ganglioside GM2 in neuronal lysosomes, leading to severe and, in most cases, fatal neurodegeneration (13).

The closest gene on the centromeric side of HEXB is the ectodermal neuronal cortex 1 ENC-1 gene. This gene has been shown to encode an F-actin associated protein, involved in differentiation of neural crest, colon and adipocyte cells (14). In addition, upregulation of ENC-1 has been found in medulloblastoma (15), prostate (16), glioblastomas, astrocytomas (17) and colon cancer (18) tumors, indicating that it may function in an oncogenic manner if inappropriately expressed. Interestingly, the ENC-1 gene was originally cloned as a p53-induced gene (19) and the deduced Enc-1 protein contains a BTB/POZ domain and six KELCH repeats, a domain structure recently shown to associate with Cul3/4 SCF ubiquitin ligases (20).

In this study, we report the cloning of the 5q13.3 and 5p13.1 breakpoints in a HCL patient with a constitutional inv(5)(p13.1q13.3) and demonstrate the disruption of a novel HEXB isoform, ENC-1AS, expressed as a cis-antisense transcript to the ENC-1 gene. Antisense transcripts have been found to be associated with a large number of mammalian genes, including N-MYC (21), WT1 (22), HOXA11 (23) and LEU5/RFP2 (24). Cis-acting antisense transcripts are often associated with a long (>100 bp) complementary sequences (25) and is increasingly recognized as an important regulatory mechanism affecting a variety of processes including RNA interference, translational repression, splicing, RNA transport and cytoplasmic stability, amongst others (26,27).

We show that the ENC-1 mRNA is significantly elevated in all 26 sporadic HCL patients examined, in contrast to the levels of ENC-1AS, and discuss further the potential role of ENC-1AS antisense expression and differential methylation in the regulation of ENC-1 expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To map and clone the breakpoints in the HCL patient with the constitutional inv(5)(p13.1q13.3) a strategy that combined FISH, Southern blot hybridization, PCR and DNA sequencing was used. By FISH analysis, BAC clone CTC-347N9 covering the breakpoint at 5q13.3 was identified by separately hybridizing a series of overlapping BAC clones (see Materials and Methods; Fig. 1A). Using a set of probes designed from the CTC-347N9 sequence, Southern blot analysis identified a rearranged 3.3 kb PstI fragment using probe p3 (Figs 1B and 2B). A PstI genomic library was constructed using DNA from the HCL patient with the inv(5). PCR analysis, using primers designed from the 3.3 kb fragment and vector-specific primers, amplified the rearranged fragment which was sequenced and aligned to genomic sequence using the BLAST program. This analysis revealed that the inversion breakpoint juxtaposes the 5q13.3 sequence to genomic sequence present within BAC clone CTD-2196p11, which is located at 5p13.1. This information allowed us to design primers in the vicinity of both the identified 5p13.1 and 5q13.3 breakpoint sequences, facilitating the amplification and cloning of the distal 5p13.1 inversion breakpoint. Finally, FISH analysis using BAC clones CTC-347N9 and CTD-2196p11 as probes on metaphase chromosomes confirmed the constitutional inv(5)(p13.1q13.3) (Fig. 1A).



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  		Figure 1. Mapping of inversion breakpoints at 5p13.1 and 5q13.3 by FISH and Southern blot analysis. (A) Metaphase FISH on peripheral blood lymphocytes from a HCL patient with a constitutional inv(5)(p13.1q13.3) using BAC clones CTC-347N9 (red) and CTD-2196p11 (green) as probes. A split fluorescent signal on the inverted chromosome is shown. (B) Southern blot of PstI digested DNA hybridized with probe p3. Lane 1, control human DNA; lane 2, genomic DNA from the HCL patient with the constitutional inv(5). A normal 7 kb band and a rearranged 3.3 kb band (corresponding to the 5q13.3 fusion band formed by the inversion), are marked by arrows.

 


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  		Figure 2. Schematic representation of the gene content at the 5q13.3 and 5p13.1 inversion breakpoints. Breakpoint sequences were examined using BLASTN (http://www.ncbi.nlm.nih.gov/) and the genomic region surrounding the breakpoints was analyzed by NIX (http://menu.hgmp.mrc.ac.uk/menu-bin/Nix/Nix.pl). (A) The AGRIN (XM_086178) and the ENC-1 (NM_003633) genes on the centromeric side and the HEXB (NM_000521) and hEFG2 (NM_032380) genes on the telomeric side of the 5q13.3 breakpoint are shown. The ENC-1 and HEXB genes are transcribed in opposite orientations on opposite strands and directly overlap in a cis-antisense fashion. The novel alternative antisense isoform of HEXB, termed ENC-1AS, spans the 5q13.3 breakpoint that occurs in a MER77 repetitive sequence and results in a loss of 7 nt (shown in bold), and is therefore disrupted in the patient with the inv(5)(p13.1q13.3). (B) Exon–intron structure and alternative spliceforms at the ENC-1/ENC-1AS locus. The unique first untranslated exon of ENC-1AS, located 18 kb centromeric of the 5q13.3 breakpoint, overlaps ENC-1 in a head to head manner by 252 bp, including the splice donor site of ENC-1 exon 1. Probes (p1–p3) used for Southern and northern analysis (Figs 1B, 4 and 6) are marked by black boxes. The HEXB and ENC-1AS spliceforms are shown with their putative ORF. Untranslated and coding regions are shown in white and black, respectively. The three major alternative spliceforms of ENC-1 are shown below the genomic sequence lines. The ORF is shaded dark with the 5'- and 3'-UTRs shaded gray. The hatched area indicates the retained intron 1 found in one ENC-1 spliceform. (C) The LIFR (NM_002310) and LIFRAS (AY357600S1) genes on centromeric side of the 5p13.1 breakpoint overlaps on opposite strands. The OSMR gene (NM_003999) is localized on the telomeric side of the breakpoint. The breakpoint occurs between exons 8 and 9 of LIFRAS and results in loss of 2 nt (shown in bold) and an insertion of a single T. (D) The LIFRAS gene contains 11 exons, is transcribed from the centromere towards the telomere, and overlaps two alternative untranslated 5' exons (exons 2 and 4) of the LIFR gene in an antisense fashion. The positions of polyadenylation signals in the alternative 3' exons 2, 4, 8 and 11 of LIFRAS are shown by vertical arrows.

 
Two genes, ENC-1 (18,28) and HEXB (29), were localized in close proximity to the 5q breakpoint (Fig. 2A). ENC-1 encodes a protein with C-terminal kelch repeats and an N-terminal BTB/POZ domain. The ENC-1 gene is located only 18 kb centromeric to the 5q13.3 breakpoint, whereas the HEXB gene is located 26 kb telomeric of the breakpoint (Fig. 2B). Telomeric of the HEXB gene, localized in a tail-to-tail arrangement, is the mitochondrial elongation factor gene, hEFG2, previously cloned by us (11). The availability of the orthologous mouse genomic sequence allowed us to examine the overall sequence conservation across the 5q13.3 breakpoint region in order to identify any possible conserved exons/elements in the 44 kb interval between the ENC-1 and HEXB genes. No conserved putative protein encoding domains were identified within this 44 kb segment despite extensive bioinformatic analysis including tblastx database search of the entire 44 kb segment against the GenBank nr database, nor could we identify conserved sequences of sufficient identity to indicate small protein encoding exons (30). However, the possibility still existed that additional untranslated exons or promoter/enhance/repressor element may exist for one or both of the genes flanking the 5q13.3 breakpoint. We therefore carried out detailed computer and database analyses of the breakpoint region in order to provide comprehensive transcriptional maps of the ENC-1 and HEXB genes. The ENC-1 gene contains three exons spanning 13 279 bp of genomic sequence (Fig. 2B). In addition to the published mRNAs of 4827 bp (GenBank accession no. NM_003633) encoding a protein of 589 aminoacids, the gene can be expressed in at least three alternative isoforms (Fig. 2B). As previously described (31), one alternative spliceform lacking 136 bp of exon 2, allows for the expression of a shorter open reading frame (ORF) of 516 aminoacids, partially disrupting the BTB/POZ domain of Enc-1. A third spliceform retains the entire intron 1 sequence, resulting in a 5'-untranslated region (UTR) of 4207 bp. Intron 1 of ENC-1 shows a surprisingly high level of conservation (426/638 bp 66% and 1818/2733 bp 66%) at the genomic sequence level when compared with the corresponding genomic sequence in mouse. This degree of conservation is similar to that of 5'-UTR containing exon 1 (254/367 bp 69%) and contrasts markedly with the level seen in intron 2 (2187/4691 bp 46.6%). Expression of the intron 1 containing isoform of 8588 bp is supported by dbEST, RT–PCR and northern analyses (Figs 2B and 4; data not shown). The published 1857 bp mRNA sequence for HEXB (GenBank accession no. NM_000521), spans 36 143 bp and consists of 14 exons that encode a protein of 556 aminoacids. Analysis of the GenBank EST database indicated that a novel HEXB spliceform, containing an alternative 5' exon exists (GenBank accession no. BG699110). Expression of this alternative HEXB transcript was confirmed by both northern and RT–PCR analyses in various tumor derived cell lines (Figs 4A and 5) and sequence analysis followed by BLAST2N alignments showed that the alternative 5' exon (Exon 1AS) is at least 541 bp in length and located 44 628 bp upstream of the previously described exon 1 (Figs 2B and 3) of HEXB. Importantly this novel alternative HEXB isoform spans the 5q13.3 inv(5) breakpoint and is therefore directly disrupted by the inversion breakpoint in the HCL patient. Surprisingly, exon 1AS also overlaps in opposite orientation the first exon of ENC-1 by 252 bp in a cis-antisense fashion (Figs 2 and 3). Conceptual translation of this novel alternative isoform of HEXB does not result in an ORF containing any complete functional domain of HEXB, indicating it most likely functions as a non-coding transcript. We therefore termed this gene ENC-1AS (Figs 2 and 3) and the corresponding transcript has been submitted to GenBank under accession no AY643499. Genomic sequence and RT–PCR analysis show that the ENC-1/ENC-1AS structure is conserved between human and mouse, with a murine mENC-1AS transcript also identified by RT–PCR analysis (data not shown).



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  		Figure 4. Northern blot analysis. (A) Expression analysis of ENC-1 and ENC-1AS on Multiple Tissue northern blots (MTN blots, Clontech) containing poly(A)+ RNA (2 µg/lane), using an ENC-1 exon 2-specific probe (p1) and the complete exon 1AS probe (p2). Hybridization with a control GAPDH probe confirmed equal amounts of RNA in each lane (data not shown). The same blot was used consecutively for all three probes and the locations of the probes used are shown in Figure 2B. (B) Detection of basal and induced ENC-1 and ENC-1AS expression on northern blots containing total RNA (20 µg/lane) prepared from tumor derived cell lines using the same set of probes as described earlier. The AA cell line, containing wild-type p53, was treated with adriamycin to activate p53 expression and analyzed for ENC-1 and ENC-1AS mRNA induction. The expected transcript sizes for ENC-1 and ENC-1AS are shown on the right of each panel. The lower panels detail the quantification of expression data of each hybridization by densitometry. Relative intensity values compared to the background levels are shown.

 


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  		Figure 5. Semi-quantitative RT–PCR analysis of (A) ENC-1 and ENC-1AS expression in sporadic HCL tumor cells (lanes 1–3), non-malignant peripheral blood lymphocytes from the HCL patient with the constitutional inv(5) (lane 4), CD-19+ purified B-cells from a healthy donor (lane 5), H82 cells (lane 6), AA cells (lane 7), AA cells treated with adriamycin (lane 8) and Daudi cells (lane 9) and H2O negative control (lane 10). Primers ENC-1EX1-F and ENC-1EX2-R were used for ENC-1 and primers ENC1AS-F and ENC-1ASEX2-R for ENC-1AS (Table 1). Amplification of GAPDH served as the internal control in these experiments. The expected PCR product sizes are shown on the right of each panel. (B) Strand-specific RT–PCR of ENC-1 (upper panel) and ENC-1AS (lower panel) using cDNA synthesized with oligo-dT (lanes 1 and 2) and gene-specific primers (ENC-1GSP, lanes 3 and 4 and ENC-1ASGSP, lanes 5 and 6). Lanes 1, 3 and 5: cDNA synthesized from AA cells. Lanes 2, 4 and 6: cDNA synthesized from H82 cells.

 


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  		Figure 3. ENC-1/ENC-1AScis-antisense locus on 5q13.3 region shown in detail. Overlapping ENC-1 and ENC-1AS 5' exons are transcribed in opposite orientation in a head to head manner as shown. Comparative alignment analysis of a 2650 bp human genomic sequence against corresponding mouse (M) and rat (R) genomic sequences was carried out using the VISTA program (http://www.gsd.lbl.gov/vista/). Conserved regions appear as gray peaks (>75% identity). Note the overall high conservation within the first intron of ENC-1, as well as segments upstream of the predictive transcriptional start site of ENC-1, coinciding with a putative promoter region of ENC-1. A predicted p53-binding site is marked by a thick vertical arrow. The lower panel details the results of bisulphite sequence analysis and methylation-specific PCR and restriction digestion of fragments amplified from Burkitts lymphoma derived cell line Daudi (Fig. 7). Primers ENCPF2, ENCP2, ENCPF4, ENCP4, ENCPF6, ENC-1M-F, ENC-1M-R, ENCPF8 and ENCP8 (Table 1) were used in combination to amplify bisulphite-modified DNA fragments encompassing over 2 kb of the overlapping ENC-1/ENC-1AS locus. The vertical lines correspond to the positions of potential recognition sites of the restriction enzymes, Taq1, Hha1 or HpyCh4IV. These positions were examined by either bisulphite sequence analysis and/or restriction digestion with the appropriate restriction enzymes and are indicated as M or U for methylated or unmethylated, respectively.

 
Flanking the 5p breakpoint, we identified the genes encoding the leukemia inhibitory factor (LIF) and Oncostatin M (OSM)-specific receptor subunits, LIFR (32) and OSMR (33), respectively (Fig. 2C). EST database searches, direct sequence comparison between human and mouse genomic sequences and RT–PCR analysis of computer predicted exons identified three novel 5' exons of the LIFR gene (exons 1, 2 and 4, GenBank accession nos. AY35796, AY35797 and AY35799, respectively) (Fig. 2D). These novel LIFR exons localize this gene to 40 kb centromeric of the 5p13.1 breakpoint (Fig. 2C and D) and the OSMR gene to 200 kb telomeric of the breakpoint. During the search for novel exons at the centromeric side of the 5p13.1 breakpoint, a number of ESTs were found to exactly match the corresponding genomic sequence in the interval between the 5p13.1 breakpoint and the LIFR gene. RT–PCR analysis, using primers in the most 5'- and 3'-ESTs, followed by sequence analysis revealed that these different EST clones all belong to a single spliced transcript composed of at least 11 exons covering more 114 kb of genomic sequence (Fig. 2D). Similar to the ENC-1AS transcript, this gene is also transcribed from the reverse strand and in the opposite orientation to the LIFR gene (Fig. 2D). The most 5' exon of this transcript localizes within intron 4 of the LIFR gene and also shows conservation at the genomic nucleotide level with an orthologous non-coding transcript in mouse (GenBank accession no. AK047630). BLAST2N alignment revealed that two exons from this novel mRNA directly overlap two LIFR exons (exons 1 and 4) in an antisense fashion and we therefore termed this gene, LIFRAS (Fig. 2C and D). The LIFRAS gene has been submitted to GenBank under accession nos AY357600–AY357607. Translation of the LIFRAS gene revealed no clear ORFs within either this transcript or the corresponding mouse ortholog, strongly suggesting that like ENC1-AS, it is a putative non-coding RNA, rather than a protein-encoding gene. In addition, no optimal Kozak sequence is present in either alternative 5' exons of the LIFRAS gene, and the high frequency of stop-codons in the internal exons would most likely target this transcript for the nonsense-mediated decay pathway if it was processed by the ribosomal machinery.

Given the association between chromosome 5 abnormalities and HCL (4), in addition to the finding that the ENC-1AS gene is directly disrupted in the HCL patient with the 5(inv)(p13.1q13.3), we decided to further examine the genomic status and expression levels of all the genes located close to the inversion breakpoints in a panel of 26 sporadic HCL cases. This involved analysis of OSMR, LIFR and LIFRAS located at 5p13 and ENC-1, ENC-1AS and HEXB at 5q13. Southern blot analysis of leukemic cell DNA, with cDNA probes for all six genes, showed that none were deleted, rearranged or obviously amplified (data not shown). We next examined the expression of these genes in normal tissues (human and mouse MTN blots), tumor cell lines and in HCL samples by northern blot analysis. None of the 5p13 located genes were found to be detectably expressed in hairy cell leukemic samples (data not shown). A single band of 2 kb was detected using a HEXB-specific probe in both normal tissues and HCL tumor samples (data not shown). No aberrant expression was detected in any of the HCL samples and the northern band corresponded to the size of the previously cloned cDNA for this gene (13). ENC-1 is differentially expressed in various normal tissues, with a high expression in brain and a low or absent expression in most other tissues including bone marrow (Fig. 4). Northern analysis of ENC-1 expression in purified HCL tumor cells revealed a striking elevation of ENC-1 levels in all 26 HCL cases analyzed, compared with expression in both normal B and T-lymphocytes (Fig. 6; data not shown). Weak ENC-1 expression could also be detected in total RNA isolated from non-malignant peripheral blood lymphocytes from the patient carrying the constitutional chromosome 5 inversion (Fig. 5; data not shown). Overexpression of ENC-1 in sporadic HCL cases was also confirmed by semi-quantitative RT–PCR (Fig. 5) and real-time PCR in 22 cases with available material (Fig. 6) using cDNA derived from purified HCL tumor cells. In line with the northern blot analysis, the CD19+ B-cell samples showed no detectable levels of ENC-1 in contrast to HCL cells, where ENC-1 was highly expressed (Figs 5 and 6; data not shown). These results for normal B-cells are also supported by microarray expression analysis collected from GNF (Genomics Institute of the Novartis Research Foundation) Expression Atlas Chips U133A, GNF1H and Affy U95 that shows that ENC-1 is present almost exclusively in neural tissues (http://genome.ucsc.edu/cgibin/hgGene?hgsid=31064603&db=hg16&hgg_gene=BT007392&hgg_chrom=chr5&hgg_start=74014614&hgg_end=74016383), with little or no expression in either normal lymphoid (both CD19+ B-cells and CD8+ T-cells), or leukemic cell lines, including Molt4, Daudi, Raji and K562. These results are in line with our own northern and RT–PCR analyses of various cancer cell lines. Very high ENC-1 expression was found in the small cell lung cancer cell line, H82, whereas no expression was detected in the Burkitt lymphoma cell line Daudi (Figs 4B and 5). We subsequently analyzed expression of the ENC-1AS transcript in both normal tissues and HCL cases by northern and semi-quantitative RT–PCR. A probe for the first exon of ENC-1AS, p3, detected a 2.2 kb band in most normal tissues (Fig. 4A). Interestingly, ENC-1AS is most highly expressed in placenta and almost absent in brain tissue (Fig. 4A). A comparison of ENC-1AS with ENC-1 expression in these normal tissues revealed a striking inverse correlation in their basic expression levels (Fig. 4A). Similar results were also found in tumor-derived cell lines (Figs 4B and 6; data not shown), where the melanoma cell line AA, expressing low basic levels of ENC-1, was found to express very high levels of ENC-1AS. The opposite situation, high ENC-1 and low ENC-1AS, was found in H82 cells (Figs 4B and 5A) as well as in 22 HCL cases by quantitative RT–PCR (Fig. 6B). No ENC-1AS expression was found in normal B lymphocytes from the patient with the constitutional inv(5) or in the Burkitt lymphoma cell line Daudi (Fig. 5). To confirm the transcriptional overlap and co-expression of ENC-1 and ENC-1AS, we also performed strand-specific RT–PCR for both these genes using cDNA synthesized from H82 and AA cells which express both transcripts (Fig. 5B). In order to determine whether the high levels of ENC-1 expression in HCL is associated with the expression of the TCF4 gene, which has been shown to be involved in transcriptional upregulation of this gene in colon tumor cell lines, we also carried out real-time PCR in 22 HCL cases. The results showed a higher expression of TCF4 in all 22 cases, with an average ratio of 18s/TCF4 expression of 0.528 (with a range of 0.450–0.599), compared with 0.414 seen in normal CD19 purified B-cells. These results are in agreement with the study of Basso et al. (2) who recently also showed differentially higher expression of this gene in HCL compared with normal B-cells and other lymphoproliferative disorders.



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  		Figure 6. (A) Expression analysis of ENC-1 in purified HCL tumor cells from sporadic HCL cases (lanes 1–4 and 10–14), peripheral blood lymphocytes from healthy donors (lanes 5 and 9) and tumor derived cell lines (H82 cells, lane 6; AA cells, lane 7; adriamycin treated AA cells, lane 8). Northern blots containing total RNA (20 µg/lane) were hybridized with probe p1 (Fig. 2B). 18S rRNA is shown as a loading control. (B) Quantitative RT–PCR analysis of ENC-1 (primer pair ENC1-ex2For1/ENC1-ex3Rev1) and ENC-1AS (primer pair ENC-ASF3/ENC-1ASEX2REV) expression in sporadic HCL tumor cells (lanes 1–13, HCL cases 1–13; lanes 14–22, HCL cases 15–23). Expression levels were normalized to GAPDH expression (primer pair GAPDH-F/GAPDH-R). Lane 23 is the cell line AA, treated with adriamycin, which has been shown by northern analysis to be a positive control for both ENC-1 and ENC-1AS expression (Fig. 4).

 
Another possibility behind the elevated ENC-1 expression found in HCL could be due to epigenetic changes including de-methylation of the ENC-1 promoter region. To test this, we analyzed a 2 kb genomic region encompassing the overlapping ENC-1/ENC-1AS 5' exons and both the ENC-1 and putative ENC-1AS promoter regions for hypermethylation in 21 HCL tumor samples, 41 tumor cell lines and in isolated normal B-cell DNA. Interestingly, while both HCL and normal B-cell samples were found to be non-methylated, hypermethylation was noted in two Burkitts lymphoma derived cell lines, Daudi and Raji (Figs 3 and 7) which also do not express ENC-1 mRNA (Fig. 4; data not shown). The methylation seen in these cell lines appears to be confined to a 1.2 kb region encompassing the overlapping ENC-1 exon 1 and ENC-1AS exon 1 region and the genomic sequence immediately upstream of ENC-1 exon 1 (Figs 3 and 7). No hypermethylation of the conserved putative ENC-1AS promoter region, located within intron 1 of ENC-1, 1497 bp downstream of the ENC-1 promoter region, was seen in either of the two Burkitt cell lines or in any of the HCL or normal B-cell samples (Figs 3 and 7). Interestingly, partial methylation of one CLL sample was found within the fragment amplified by primer pair ENCPF4–ENCPR4 that encompasses most of ENC1-AS exon 1. This is in contrast to the lack of methylation at this locus found in HCL, ALL and normal CD19 purified B-cells and the complete methylation seen in the cell line Daudi (Fig. 7). Bisulphite sequence analysis indicated that the methylation of this fragment in Daudi is confined to the 3' region, resulting in a hypomethylation pattern within ENC-1 intron 1 in contrast to the hypermethylation found in the overlap region. Bisulphite sequence analysis of fragments ENCPF2–ENCPR2 and ENCPF8–ENCPR8 also confirmed the restriction results that the hypermethylation is confined within the 1.2 kb segment shown in Fig. 3 although there is evidence of partial methylation within the latter fragment in Daudi (Fig. 7, lane 19; data not shown).



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  		Figure 7. Methylation analysis of the ENC1 promoter region in various B-cell derived tissues and cell lines. Bisulphite treated DNA from HCL patients (P20 and P21), Burkitts lymphoma tumor cell lines DAUDI (termed DAU) and RAJI (termed RAJ), normal CD19 separated B-cells (labelled B, or B-cell) and two other leukemic cases with CLL and ALL, respectively, was PCR amplified using primers pairs ENCPF2/ENCPR2 (627 bp), ENCPF4/ENCPR4 (601 bp), ENC1MF/ENC1MR (130 bp) and ENCPF8/ENCPR8 (273 bp) (Fig. 3 and Table 1). This was followed by restriction digestion with the restriction enzymes HpyCH4IV (lanes 2, 4, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20, 22, 25, 28, 31, 34 and 37), Taq1 (lanes 3, 5, 7, 9, 11, 13, 23, 26, 29, 32, 35 and 38) and Hha1 (lanes 24, 27, 30, 33, 36 and 39) All three restriction enzymes recognition sites include the dinucleotide CG, only present in methylated samples. M=100 bp marker, Lanes 40 and 48, negative control. Lanes 1, 14 and 21 are undigested positive PCR controls. Note the complete digestion of samples 34, 45 and 46 by HpyCH4IV, and samples 35 and 36 by Taq1 and Hha1, indicating preservation of the CpG dinucleotide at these positions owing to methylation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Genetic changes and molecular events associated with malignant transformation and tumor progression of HCL have eluded researchers to date. Chromosomal abnormalities such as trisomy of chromosome 5, translocations and inversions involving 5q13 are known to occur in HCL (4) yet no strong candidate gene, dysregulated in HCL, has been characterized to date at this genomic locus. In the present study, we have cloned and sequenced both breakpoints in a HCL patient with a constitutional inv(5)(p13.1q13.3) and identified two novel antisense genes, ENC-1AS at 5q13.3 and LIFRAS at 5p13.1, both disrupted by the inversion. These results have also demonstrated that the ENC-1AS gene is a novel alternative isoform of the previously mapped HEXB gene (13) directly overlapping in a cis-antisense fashion the ENC-1 gene. We have shown that ENC-1 is significantly overexpressed in all 26 sporadic HCL cases analyzed and has a complicated regulatory mechanism that may involve the inverse expression of the cis-acting ENC-1AS transcript. Together, these observations indicate that ENC-1 should be considered as a formal candidate gene for susceptibility to development of HCL, possibly acting as a gene with oncogenic potential when inappropriately expressed or regulated.

A number of factors support the hypothesis that ENC-1 may have a role in malignant transformation in multiple tumor types. ENC-1 is identical to PIG-10 (p53-induced protein 10), which was initially identified during a search for transcripts induced by p53 prior to the onset of apoptosis in DLD1 colon cancer cells (19). Subsequently, ENC-1 was isolated as a gene encoding a nuclear matrix protein associating with pRB (17), termed nuclear-restricted protein/brain NRP/B, possibly involved in neural differentiation (17,34). Alteration of its expression is suspected to contribute to colorectal carcinogenesis by suppressing the differentiation of colonic cells, and elevated ENC-1 expression was also found in primary colon cancer cells (18). In a recent study of differentially expressed genes in prostate carcinoma, ENC-1 was found to be overexpressed compared with normal prostate tissue. In that study, the expression pattern of ENC-1 was very similar to other genes associated with advanced stage and short time to recurrence (16). Moreover, expression profiling of multiple tumor and normal tissue samples has identified ENC-1 as being highly expressed, in addition to brain, colon and prostate tumors, in cns, ovarian and pancreatic carcinomas (http://genome-www5.stanford.edu/cgibin/source/expressionSearch?option=cluster&criteria=Hs.104925&dataset=2&organism=Hs) (35), indicating that deregulated expression of the ENC-1 gene might play an important role in the pathogenesis of various malignancies. Our novel finding that ENC-1 is overexpressed also in HCL was confirmed by several techniques, including northern analysis and real-time PCR analysis (Fig. 6), with a significant elevation of ENC-1 found in all sporadic HCL cases compared with expression in normal lymphocytes from healthy donors. Exhaustive genetic- and expression analysis of the genes flanking and spanning both the 5p13.3 and the 5q13.3 breakpoints, did not identify any aberrant expression in tumor cells from sporadic HCL patients, with the notable exception of the significant elevation in ENC-1 expression (Fig. 6). None of the three genes OSMR, LIFR or LIFR-AS, located in the 5p13 region were found to be expressed at detectable levels in HCL material by northern analysis (data not shown).

Previous identification of upregulation of ENC-1 in various malignancies and its critical role in the appropriate differentiation of an increasing number of cell types leads to the largely unexplored question of how the gene is regulated. The finding that the first exon of ENC-1 directly overlaps the cis-antisense ENC-1AS transcript, is intriguing and presents a novel mechanism by which ENC-1 expression may be regulated. Antisense RNAs have been suggested to affect the expression of the sense gene at various levels, including transcription, RNA processing, nuclear export, RNA stability or translation (25). Although ENC-1AS contains exons 2–14 of the HEXB gene it is unlikely that ENC-1AS encodes an alternative isoform of the Hexb protein. Conceptual translation of ENC-1AS results in an ORF of 331 aminoacids utilizing the fifth methionine initiation codon, present in exon 6 of HEXB, and translates into a putative Enc-1as ORF lacking the first 225 aminoacids of the N-terminal domain of HexB, The absence of any complete functional domain in the resulting Enc-1as ORF strongly suggests that ENC-1AS functions at the RNA sequence level as a regulator of ENC-1, rather than through its protein coding potential. In addition, we also identified a murine form of ENC-1AS, mENC-1AS, encoding an mHexB ORF, also lacking any complete functional domain of mHexB (data not shown). Furthermore, the 918 bp 5'-UTR of ENC-1AS is predicted to result in a transcript refractory to ribosomal scanning based translation (37) and result in non-translation of the Enc-1as ORF, again leading to the conclusion that ENC-1AS functions as a non-coding transcript. Furthermore, the fact that there is an inverse correlation in expression of ENC-1 and ENC-1AS in both normal tissues, tumor derived cell lines and HCL material (Figs 4, 5 and 6B) strongly suggest that these genes reciprocally regulate each other and suggest that ENC-1AS might affect ENC-1 expression. Another possibility is that expression of ENC-1AS could affect the splicing of ENC-1, as ENC-1AS exon 1 covers the first splice donor site of ENC-1. Indeed, ENC-1 has been shown to be alternatively spliced to produce transcripts with alternative 5'-UTRs leading to the expression of two isoforms of the Enc-1 protein with different intracellular localizations although the functional mechanisms behind this alternative splicing have not been described (31).

Disruption of ENC-1AS has only been found in one HCL case, namely in the constitutional inv(5) case described earlier. Extensive analysis revealed no disruption, deletion or amplifications of ENC-1AS in the other 26 cases of sporadic HCL, yet high levels of ENC-1 expression were found in all cases. Although aberrant regulation of ENC-1 through the disruption of ENC-1AS may predispose towards the development of HCL, other possibilities than physical disruption of ENC-1AS exists for upregulation of ENC-1 expression in sporadic HCL cases. Similar to the situation in normal tissues and cell lines, a distinct difference in basal expression levels between ENC-1 and ENC-1AS was also found in HCL tumor cells (Fig. 5; data not shown). Whether the low expression of ENC-1AS seen in HCL cases causes increased expression of ENC-1 awaits further analysis. Other reasons for upregulation of ENC-1 in HCL cases might exist. One reason for upregulation of ENC-1 has recently been described in colon cancer cells, where ENC-1 was shown to be regulated by the ß-catenin/T-cell factor (Tcf) pathway (18). In this study, ENC-1 expression was significantly elevated by transfection of an activated form of ß-catenin together with wild-type TCF4. Induction of ENC-1 through TCF4 expression is an interesting concept in regards to HCL. Unlike solid tumors such as colonic and breast cancer, dysregulation of elements of the wnt/ß-catenin signaling pathway have rarely been reported in B-cell malignancies. However, a recent microarray based study of gene expression in HCL has shown the unexpected finding that the TCF4 gene is one of the most differentially expressed genes in HCL cells compared with normal B-cells or other B-cell malignancies (2), a finding we have confirmed by real-time PCR analysis in our HCL patient material. In addition to ENC-1, TCF4 target genes include CD44, vimentin, and Cyclin D1, all of which have previously been found highly expressed in HCL cases (38–40). Dysregulation of the wnt/ß-catenin pathway in HCL, including inappropriate expression of TCF4 target genes, is currently under investigation.

In studying further possible regulatory mechanisms affecting ENC-1 expression, we identified a differentially methylated region, encompassing 1.2 kb of genomic sequence which includes the ENC-1 exon 1, ENC-1AS exon 1 overlap segment (Figs 3 and 7). In the case of ENC-1, we found that its 5' upstream promoter region was non-methylated in all 21 HCL tumor cases tested, but methylated in two Burkitt lymphoma cell lines, a finding consistent with the expression of ENC-1 in these samples (Figs 4 and 7; data not shown). The 1.2 kb region encompasses a number of potential binding sites for transcriptional factors that may be important for the regulation of ENC-1. We have identified a putative p53-binding site in the beginning of ENC-1 intron 1, within exon 1AS of ENC-1AS (Fig. 3). Interestingly, unlike the situation with ENC-1, ENC-1AS expression was not induced by adriamycin treatment in AA cells (Fig. 4), indicating that ENC-1AS expression occurs in a p53 independent manner. In addition, activation of the p53 pathway did not lead to a further increase in ENC-1 expression in HCL cells suggesting that other mechanisms are behind ENC-1 upregulation in HCL tumor cells.

Most studies suggest that ENC-1 may play a crucial role during differentiation in a variety of cell lineages. Recently Katayama et al. (41) showed that ENC-1 is significantly upregulated in the myeloma cell line U266 following interaction between CD27, a marker for memory B-cells, and its ligand CD70. CD27–CD70 interaction is believed to be essential for the differentiation of plasma cells from memory B-cells (41). Interestingly, both CD27 and its ligand CD70 are widely expressed by HCL cells (2,42), indicating a possible mechanism for ENC-1 expression in HCL, along with a putative function for ENC-1 in B-cell differentiation. Differentiation events commonly include changes in cell shape and reorganization of the actin cytoskeleton. One of the major constituents in the characteristic cell surface projections found on hairy cells is F-actin (43). Upregulation of ENC-1 in HCL is particularly interesting in this regard, as ENC-1 is also known to be an F-actin-binding protein (28,34). It remains to be determined whether ENC-1 is involved in the morphogenesis of HCL tumor cells through reorganization of the actin cytoskeleton and protein analysis of Enc-1 in HCL tumor cells should answer this question.

To summarize, our discovery that ENC-1 is overexpressed in patients with HCL brings new insight into the development of hairy cell leukemogenesis. The data presented here have also described novel ENC-1 regulatory mechanisms, including expression of the cis-antisense gene, ENC-1AS, as well as tissue-specific methylation of the ENC-1 promoter locus. Further investigation on the mechanisms involved in regulation of ENC-1 expression and functional analysis of the Enc-1 protein should provide a more profound understanding of the role of ENC-1 in the pathogenesis of HCL and other neoplasms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Primary tumor material and cell lines
Leukemic cells were collected with informed consent and local ethical committee approval from 26 patients with sporadic HCL, and non-malignant lymphocytes were obtained from healthy individuals. HCL tumor cells were collected from the spleen in all patients. The identification of a patient with a constitutional inv(5)(p13.1q13.3) has been reported previously (4). Leukemic cells were purified as previously described (5) and cytogenetic analyses were performed according to published protocols (4,45). In the large majority of the samples, the purity of the suspension exceeded 80–90%, as measured by flow cytometry for HCL-specific markers (CD19, CD103) and for T-cell markers (CD2 and CD3) (5). Purified normal B- and T-lymphocytes from healthy donors were prepared as previously described (45). Tumor-derived cell lines including H82 (small cell lung cancer), AA (melanoma), Daudi and Raji (Burkitt lymphomas) were cultured and grown in RPMI-1640 medium supplemented with 10% fetal calf serum and antibiotics. The melanoma cell line AA, containing wild-type p53, was treated with adriamycin (0.5 µg/ml) and used as a positive control for ENC-1 mRNA expression (see Results).

Bioinformatic sequences analysis
Genomic DNA sequences were obtained from the human and mouse assemblies available through the UCSC genome browser home page (http://genome.ucsc.edu/) and analyzed using the NIX (Nucleotide Identify X) program package (http://menu.hgmp.mrc.ac.uk/menu-bin/Nix/Nix.pl). Direct sequence comparison between the human and mouse sequences was performed using BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) after removal of repetitive sequences with repeatmasker2 (http://ftp.genome.washington.edu/). Direct sequence comparison of human and mouse sequence was carried out using the Consite analysis program (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite?rm=t_input) and human, mouse and rat genomic sequence using the VISTA tools program (http://pipeline.lbl.gov/cgi-bin/gateway2).

FISH analysis
Metaphase spreads were prepared using standard methodology and dual-color FISH was performed as previously described (10). A series of overlapping BAC clones obtained from BACPAC resources http://bacpac.chori.org/, containing inserts from the 5q13 region were selected based on their map location from the UCSC database and separately hybridized to metaphase slides prepared from the patient with the constitutional inv(5)(p13.1q13.3). BAC clones CTC-347N9 and CTD-2196p11 (covering the 5q13.3 and 5p13.1 breakpoints, respectively), were subsequently used for hybridizing interphase slides prepared from the HCL patients described earlier. Labelling of probes and hybridization conditions for FISH was performed as previously described (45). Peripheral mononuclear blood cells from healthy donors and non-malignant cells from the patient with the constitutional inversion on chromosome 5 were analyzed in parallel.

Breakpoint cloning and library construction
Basic molecular biology procedures, such as isolation of DNA and Southern blots were performed as previously described (47,48). Cloning of the 5q13.3 and 5p13.1 breakpoints was essentially as previously described (48). Briefly, a PstI library was constructed using DNA from the patient with the constitutional inv(5)(p13.1q13.3), and the 5q13.3 breakpoint was cloned by PCR with vector-specific primers (pBluescript II, Stratagene, USA) in combination with primers designed from the sequence of a DNA probe (termed p3) that detects a 3.3 kb rearranged band on Southern blot (Figs 1B and 2). PCR products from these reactions were cloned into the pCRII-TopoTA vector (Invitrogen) and sequenced using vector-specific primers. Sequence reactions were performed as previously described (11). All primers used for breakpoint cloning are listed in Table 1. All PCR analysis was performed with the Advantage 2 polymerase mix according to the manufacturer's instructions (Clontech, Palo Alto).


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  		Table 1. Primer sequences
 
Northern hybridization and RT–PCR
Total RNA was extracted using the Trizol reagent (Life Technologies) from HCL patient samples, tumor-derived cell lines and CD19+ purified B-cells of healthy donors (CD19 Dynabeads, Dynal Biotech ASA, Norway). The RNA was reverse transcribed with First-Strand cDNA synthesis using Superscript II (Invitrogen, Life Technologies) and real-time PCR was carried out in a BioRad iCycler using the Sybr-Green-PCR-kit (BioRad Laboratories, Hercules, CA, USA) using primers specific for ENC-1, ENC-1AS and TCF4. Strand-specific RT–PCR for ENC-1 and ENC-1AS was performed on cDNA synthesized with gene-specific primers (ENC-1GSP and ENC-1ASGSP) from AA and H82 cells using the ThermoScript RT–PCR System as described by the manufacturer (Invitrogen, Life Technologies). mRNA (MTN blots, Clontech) and total RNA blots (20 µg/lane) were hybridized with PCR derived probes from ENC-1 (probe p1), ENC-1AS (probe p2), HEXB, hEFG2, LIFR, LIFR-AS and the OSMR gene sequences. Labelling of all probes was performed as previously described (49). 18S RNA or GAPDH expression was used to measure the total amount of RNA in each sample. PCR conditions for semi-quantitative and quantitative RT–PCR analyses are available from the authors on request. All primers used for RT–PCR are shown in Table 1.

Bisulphite treatment and methylation analysis
Bisulphite treatment, PCR, followed by restriction digestion and sequencing was used to determine the methylation status of both the ENC-1 and ENC-1AS genes in purified HCL-samples, cell lines and normal B-cell samples as previously described (46). Predicted conserved promoter regions were identified using the program Consite (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite?rm=t_input) and primers specific for bisulphite-modified DNA were designed using the program Methprimer (http://www.ucsf.edu/urogene/methprimer/index1.html). Primers ENCPF2, ENCPR2, ENCPF4, ENCPR4, ENCPF6, ENC1MF, ENC1MR, ENCPF8 and ENCPR8 (Fig. 3 and Table 1) were used to amplify segments encompassing over 2 kb of sequence covering the 5' exons and predicted promoter regions of the ENC-1/ENC-1AS locus.

Methylated samples could be identified by the presence of the recognitions sites for the restriction enzymes HpyCH4IV, Taq1 and Hha1 that recognize the CpG containing sequences ACGT, TCGA and GCGC, respectively, in bisulphite-modified genomic DNA.

In addition, the methylation status at these positions was confirmed by either direct bisulphite sequence analysis of PCR fragments or of fragments subcloned into the pCRII-TopoTA plasmid vector (Invitrogen).


   ACKNOWLEDGEMENTS
 
The authors acknowledge the technical support provided by Mrs Ruth Detlofsson. This study was supported by grants from Kay Kendall Leukaemia Research Fund UK, the King Gustav V Jubilee Fund, the Swedish Cancer Foundation, Alex and Eva Wallströms foundation, The Wallenberg Foundation and the Cancer Society of Sweden.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Oncology/Pathology, CCK R8:03, 171 76, Karolinska Hospital, Stockholm, Sweden. Tel: +46 851773738; Fax: +46 8339031; Email: olle.sangfelt{at}cck.ki.se

{dagger} These authors contributed equally to this work. Back

{ddagger} ENC-1AS; AY643499, LIFRAS exons 8–11; AY643500, AY643501, AY643502 and AY643503, respectively. Back


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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