Leukemogenesis Section, Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA1
Molecular Oncology Laboratory, University of Glasgow, Glasgow G61 1QH, UK2
Author for correspondence: Linda Wolff. Fax +1 301 594 3996. e-mail lwolff{at}helix.nih.gov
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using this model, several loci of retrovirus integration have been identified. The most frequent target is c-myb (Mukhopadhyaya & Wolff, 1992 ; Nazarov & Wolff, 1995
; Shen-Ong & Wolff, 1987
; Wolff et al., 1991
). Its locus is rearranged and its expression at the RNA level is constitutive due to the activity of the retrovirus promoter and/or enhancer. However, many of the leukaemias induced by the 4070A virus express normal c-myb and have integrated proviruses outside the c-myb locus. In some of these tumours insertion sites were found to be clustered on chromosome 10, approximately 25 kb upstream of c-myb in a locus named Mml1 (Koller et al., 1996
).
Recently, we identified two other sites of integration, Mml2 and Mml3, in MML. Here we have reported their position 5070 kb upstream of c-myb, as well as a physical map linking Mml1, Mml2, Mml3 and Fti1, and a comparison of this region to an analogous region on human chromosome 6. Interestingly, this chromosomal region seems to be a preferred area for retroviral integrations that contribute to leukaemogenesis since Fti1, a common integration site in feline leukaemia virus (FeLV)-induced T cell lymphomas, maps to this region (Barr et al., 1999 ). Another integration site, Ahi1, in Abelson MuLV-induced lymphomas is located downstream of c-myb (Jiang et al., 1994
). For this study, we set out to determine if, in a 250 kb region upstream of c-myb, there are any genes that might be altered in expression or function thus contributing to the neoplastic disease. We concluded that, although there are several genes more than 100 kb upstream of c-myb, none appeared to be related to the oncogenic process. The region of approximately 100 kb, surrounding Mml1, 2, 3 and Fti1, is devoid of genes and presumably contains sequences involved in chromosomal structure or regulation of gene expression at a distance.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primers and probes.
The amplimers used for PCR amplification of the probes are listed in Table 1. Other probes used in this study were a c-myb genomic BglII fragment [B probe in Mukhopadhyaya & Wolff (1992)
], an Ahi1 0·8 kb genomic PstIHindIII fragment [Ahi-D probe in Poirier et al. (1988)
] and an 4070A env (RK107) 1·5 kb EcoRINheI fragment.
|
The RTPCR analysis was performed using the Titan One Tube RTPCR Kit (Roche) according to the manufacturers instructions.
Mml2, Mml3 cloning and library screening.
The Mml2 and Mml3 integration sites were cloned from the 30-2-9 and the 30-2-7 tumours, respectively. In the case of 30-2-9, genomic DNA was purified from primary tumour tissue and for 30-2-7 from an established cell line. Genomic DNA was digested with EcoRI and size-selected on a 0·8% agarose gel. Fragments from a range of about 35 kb were purified and ligated to the EcoRI cut arms of the ZAP Express vector. These
subgenomic libraries were screened with a 4070A env probe (RK107). An insert from the positive Mml2 recombinant phage (
21.1) was recloned into pBluescriptII SK(-) and sequenced. The Mml3 recombinant phage DNA (
26·2) was sequenced directly. The genomic sequences flanking the provirus were used to design amplimers for RK114 (Mml2) or MML-3 (Mml3) PCR probes.
The mouse genomic BAC library RPCI-22 (female spleen 129S6/SvEvTac, about 212000 clones, of 154 kb average insert size, and approximately 10·9-fold genomic coverage, Childrens Hospital Oakland Research Institute, Oakland, CA, USA) (Osoegawa et al., 2000 ) was screened by Southern hybridization with either an Mml1 (RK29) or an Mml2 (RK114) probe.
A genomic region containing the wild-type Mml2 locus was cloned using a BAC DNA positive clone from RPCI-22 library (#401-G9) by digestion with BamHI. Size-selected and purified fragments of 1520 kb were ligated into the DASHII vector. The subgenomic
library was screened using an Mml2 PCR probe (RK114) and the insert from a positive phage (
II-4a) was subcloned into a plasmid. Similarly, the Mml3 locus was cloned from the same BAC (#401-G9) after BamHI digestion and size selection. Fragments of 48 kb were cloned directly into the pBluescriptIISK(-) vector and the library was screened with an Mml3 PCR probe (MML-3) to obtain a positive clone (pSK-MML3).
Plasmid,
, BAC and genomic DNA isolation and Southern analysis.
Plasmid DNA was prepared with the Wizard Plus SV Miniprep System (Promega), DNA was purified from lysate using the Lambda Maxi Kit (Qiagen), BAC DNA was isolated with the Plasmid Maxi Kit (Qiagen) and genomic DNA was prepared using the Wizard Genomic DNA Purification Kit (Promega). Ten µg of DNA were digested with various restriction endonucleases, separated on a 0·8% agarose gel in a TAE system, transferred onto nylon membrane and hybridized as described for Northern analysis.
Pulsed field gel electrophoresis (PFGE).
PFGE was performed on the CHEF-DR II System (Bio-Rad) in 1% agarose in 0·5x TBE buffer at 6 V/cm for 20 h with initial switch time 50 s and final switch time 90 s.
DNA sequencing and bioinformatics.
DNA was sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and the samples were run on the ABI PRIZM 377 sequencer at the DNA Sequencing MiniCore Facility (NCI). Assembly of obtained sequence was performed by the Sequencher program (Gene Codes) and homology searches were done by the BLAST (basic local alignment search tool) program at the NCBI (Altschul et al., 1997 ). For masking the repetitive elements the RepeatMasker program was used (A. F. A. Smit and P. Green at http://ftp.genome.washington.edu/cgi-bin/RepeatMasker). Sequences were analysed by the RUMMAGE sequence annotation system (http://gen100.imb-jena.de/rummage). The search for matrix attachment regions (MAR) was carried out using the MAR-Wiz program (Singh et al., 1997
) (www.futuresoft.org/MAR-Wiz/).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
To identify potential genes in the vicinity around c-myb, Mml1, Mml2, Mml3 and Fti1, 39·6 kb in the Mml1 region was sequenced including between Mml1 and the c-myb, and sequences around the Mml2 and Mml3 (16·4 kb and 5·9 kb, respectively). To sequence the region between Mml1 and c-myb we used lambda clones subcloned from a P1 clone (#5362, Genome Systems). For sequencing around Mml2 and Mml3, plasmid clones derived from a G9 BAC (RPCI-22, #401-G9) were used. After sequencing and prior to comparative sequence analysis, repetitive elements were filtered using the RepeatMasker program (A. F. A. Smit and P. Green, unpublished data, University of Washington Genome Center, http://ftp.genome.washington.edu/cgi-bin/RepeatMasker). The extent of masking varied. It was 27·4, 28 and 38·9% for Mml1, Mml2 and Mml3, respectively (Table 2).
|
|
|
|
As indicated above, our search for exons in close proximity to Mml1, Mml2 and Mml3 did not lead to direct identification of any genes influenced by proviral integration at these loci. The lack of detectable coding regions in the vicinity might suggest that these regions harbour regulatory or structural elements. Chromatin-organizing structures, such as scaffold/matrix attachment regions (S/MARs) are found in AT-rich areas. Although the overall AT content for the 39·6 kb Mml1 region, as shown in Fig. 4A, is 56·5%, we found several areas that have a relatively high AT content, between 61 and 65%. The MAR-Wiz program (Singh et al., 1997
) (www.futuresoft.org/MAR-Wiz/) predicted the S/MAR around the position of 18·3 kb which is next to the provirus insertion site in one Mml1 tumour V4634 at a position around 17·9 kb. Since S/MARs often contain specific recognition sequences defined by a MAR/SAR recognition signature (MRS), we searched for these elements. The signature is made up of two degenerate sequences, an 8 bp sequence (AATAAYAA) and a 16 bp sequence (AWWRTAANNWWGNNNC), found within 200 bp of each other (van Drunen et al., 1999
). Although we could identify several 8 bp sequences throughout the Mml1 sequence, we could not identify the 16 bp sequences. The Mml2 sequence of 16·4 kb (Fig. 4B
) with AT content of 59·2% also contains several AT-rich areas of 6672% AT. In one of them the MAR-Wiz predicted an S/MAR around 11·3 kb, but we did not find any complete MRS. We conclude from this search for S/MARs that there is some evidence for S/MARs. Although the data are only partially predictive, they warrant further experimentation to determine directly whether or not S/MARs are present.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Repeated integration of proviruses into this chromosome 10 region, in association with tumorigenesis, suggests an important function for these sequences, despite the absence of transcribed genes in this area, that might be altered in expression due to the provirus. The proviruses at these loci are like many others that appear to be involved in neoplastic disease but have not yet been assigned a function (Jonkers & Berns, 1996 ). The fact that the region is devoid of genes, and the MAR-Wiz program predicted two potential S/MARs, might suggest that at least part of the area contains structure-related regulatory sequences. Matrix attachment elements organize chromatin topologically, allowing spatial and temporal gene regulation and the nuclear matrix is implicated in the control of development and differentiation (Nepveu, 2001
; Stein et al., 1995
). Interestingly, alterations in nuclear architecture have been associated with the neoplastic condition (Deppert et al., 2000
); there are transforming genes that have the ability to bind to nuclear matrix including SV40 Large T antigen and mutant p53, the latter of which was found to bind directly to the S/MAR (Deppert, 2000
). In addition, S/MARs are AT rich and some proteins, including the mixed lineage leukaemia (MLL) protein, contain an AT hook domain, which binds to these sequences (Broeker et al., 1996
; Caslini et al., 2000
). Moreover, MLL fusion proteins have been implicated in myeloid leukaemia in man (Dimartino & Cleary, 1999
). It is conceivable, therefore, that the virus could perturb the binding of DNA to matrix through the physical disruption or could introduce regulatory proteins such as those that bind to the LTR. Further studies will be required to determine the function of the sequences predicted to be S/MARs by the MAR-Wiz program.
One must still consider the possibility that regulatory regions associated with these integration sites could function to influence c-myb expression. Integrations have now been found in a wide region (approximately 150 kb) both upstream (Fti1, Mml3, Mml2, Mml1) of c-myb, as well as downstream (Ahi1). Until recently, downstream integrations were only found in lymphoid tumours. However, Blaydes et al. (2001) also discovered provirus insertions in this region in a series of BXH-2 myeloid leukaemias.
In the present study we considered the possibility that integrations affected Myb transcription by looking to see if, in established tumour cell lines, provirus integration in Mml1 caused increased expression of c-myb (Fig. 3A, B
) and found that some cell lines did not express it. However, our inability to find increased expression in the leukaemias may not rule out the possibility that integration into this area influenced c-myb expression during an early phase of the disease prior to differentiation. Our previous data suggest that mature MMLs (with integrations in c-myb) develop from pre-leukaemic cells in the bone marrow or spleen that are immature (Nason-Burchenal & Wolff, 1993
). If pre-leukaemic cells involving Mml1, 2, 3 are also immature cells, their mechanism of endogenous c-myb gene regulation could be very different from that of end-stage tumour cells, which are mature monocytic cells. Integrations that affect c-myb could influence early stage progression, for example by providing increased proliferation potential or an anti-apoptotic role, since these are assigned functions of c-Myb. If this is the case, one would have to predict that, at later stages of progression, these functions are provided by additional oncogenic event(s). The proximity of this proto-oncogene to the Mml integration sites continues to raise the question of whether these integration sites affect c-myb.
Overall, the data presented here indicate that the 80 kb region upstream of c-myb, which contains three separate retroviral integration sites associated with myeloid tumours, may be devoid of genes, but could possess regulatory function. Since other integration sites associated with lymphoid and myeloid tumours have also been found in this chromosomal region, it will be important to determine how sequences in this area can influence the neoplastic state.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
c Present address: Division of Human Cancer Genetics, Ohio State University, Columbus, OH 43210, USA.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barr, N. I., Stewart, M., Tsatsanis, C., Fulton, R., Hu, M., Tsujimoto, H. & Neil, J. C. (1999). The fit-1 common integration locus in human and mouse is closely linked to MYB. Mammalian Genome 10, 556-559.[Medline]
Blaydes, S. M., Kogan, S. C., Truong, B. T., Gilbert, D. J., Jenkins, N. A., Copeland, N. G., Largaespada, D. A. & Brannan, C. I. (2001). Retroviral integration at the Epi1 locus cooperates with Nf1 gene loss in the progression to acute myeloid leukemia. Journal of Virology 75, 9427-9434.
Bouck, J., Miller, W., Gorrell, J. H., Muzny, D. & Gibbs, R. A. (1998). Analysis of the quality and utility of random shotgun sequencing at low redundancies. Genome Research 8, 1074-1084.
Broeker, P. L., Harden, A., Rowley, J. D. & Zeleznik-Le, N. (1996). The mixed lineage leukemia (MLL) protein involved in 11q23 translocations contains a domain that binds cruciform DNA and scaffold attachment region (SAR) DNA. Current Topics in Microbiology and Immunology 211, 259-268.[Medline]
Burge, C. & Karlin, S. (1997). Prediction of complete gene structures in human genomic DNA. Journal of Molecular Biology 268, 78-94.[Medline]
Caslini, C., Alarcon, A. S., Hess, J. L., Tanaka, R., Murti, K. G. & Biondi, A. (2000). The amino terminus targets the mixed lineage leukemia (MLL) protein to the nucleolus, nuclear matrix and mitotic chromosomal scaffolds. Leukemia 14, 1898-1908.[Medline]
Coll, J., Saule, S., Martin, P., Raes, M. B., Lagrou, C., Graf, T., Beug, H., Simon, I. E. & Stehelin, D. (1983). The cellular oncogenes c-myc, c-myb and c-erb are transcribed in defined types of avian hematopoietic cells. Experimental Cell Research 149, 151-162.[Medline]
Deininger, P. L. (1983). Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Analytical Biochemistry 129, 216-223.[Medline]
Deppert, W. (2000). The nuclear matrix as a target for viral and cellular oncogenes. Critical Reviews in Eukaryotic Gene Expression 10, 45-61.[Medline]
Deppert, W., Gohler, T., Koga, H. & Kim, E. (2000). Mutant p53: gain of function through perturbation of nuclear structure and function? Journal of Cellular Biochemistry S35, 115-122.
Dimartino, J. F. & Cleary, M. L. (1999). Mll rearrangements in haematological malignancies: lessons from clinical and biological studies. British Journal of Haematology 106, 614-626.[Medline]
Duprey, S. P. & Boettiger, D. (1985). Developmental regulation of c-myb in normal myeloid progenitor cells. Proceedings of the National Academy of Sciences, USA 82, 6937-6941.[Abstract]
Jiang, X., Villeneuve, L., Turmel, C., Kozak, C. A. & Jolicoeur, P. (1994). The Myb and Ahi-1 genes are physically very closely linked on mouse chromosome 10. Mammalian Genome 5, 142-148.[Medline]
Jonkers, J. & Berns, A. (1996). Retroviral insertional mutagenesis as a strategy to identify cancer genes. Biochimica et Biophysica Acta 1287, 29-57.[Medline]
Koller, R., Krall, M., Mock, B., Bies, J., Nazarov, V. & Wolff, L. (1996). Mml1, a new common integration site in murine leukemia virus-induced promonocytic leukemias maps to mouse chromosome 10. Virology 224, 224-234.[Medline]
Kung, H.-J., Boerkoel, C. & Carter, T. H. (1991). Retroviral mutagenesis of cellular oncogenes: a review with insights into the mechanisms of insertional activation. Current Topics in Microbiology and Immunology 171, 1-25.[Medline]
Liebermann, D. A. & Hoffman-Liebermann, B. (1989). Proto-oncogene expression and dissection of the myeloid growth to differentiation developmental cascade. Oncogene 4, 583-592.[Medline]
Mukhopadhyaya, R. & Wolff, L. (1992). New sites of proviral integration associated with murine promonocytic leukemias and evidence for alternate modes of c-myb activation. Journal of Virology 66, 6035-6044.[Abstract]
Nason-Burchenal, K. & Wolff, L. (1993). Activation of c-myb is an early bone-marrow event in a murine model for acute promonocytic leukemia. Proceedings of the National Academy of Sciences, USA 90, 1619-1623.[Abstract]
Nazarov, V. & Wolff, L. (1995). Novel integration sites at the distal 3' end of the c-myb locus in retrovirus-induced promonocytic leukemias. Journal of Virology 69, 3885-3888.[Abstract]
Nepveu, A. (2001). Role of the multifunctional CDP/Cut/Cux homeodomain transcription factor in regulating differentiation, cell growth and development. Gene 270, 1-15.[Medline]
Osoegawa, K., Tateno, M., Woon, P. Y., Frengen, E., Mammoser, A. G., Catanese, J. J., Hayashizaki, Y. & de Jong, P. J. (2000). Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. Genome Research 10, 116-128.
Poirier, Y., Kozak, C. & Jolicoeur, P. (1988). Identification of a common helper provirus integration site in Abelson murine leukemia virus-induced lymphoma DNA. Journal of Virology 62, 3985-3992.[Medline]
Schmidt, M., Nazarov, V., Stevens, L., Watson, R. & Wolff, L. (2000). Regulation of the resident chromosomal copy of c-myc by c-Myb is involved in myeloid leukemogenesis. Molecular and Cellular Biology 20, 1970-1981.
Sheiness, D. & Gardinier, M. (1984). Expression of a proto-oncogene (proto-myb) in hemopoietic tissues of mice. Molecular and Cellular Biology 4, 1206-1212.[Medline]
Shen-Ong, G. L. & Wolff, L. (1987). Moloney murine leukemia virus-induced myeloid tumors in adult BALB/c mice: requirement of c-myb activation but lack of v-abl involvement. Journal of Virology 61, 3721-3725.[Medline]
Singh, G. B., Kramer, J. A. & Krawetz, S. A. (1997). Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Research 25, 1419-1425.
Stein, G. S., van Wijnen, A. J., Stein, J., Lian, J. B. & Montecino, M. (1995). Contributions of nuclear architecture to transcriptional control. International Review of Cytology 162A, 251-278.
Taudien, S., Rump, A., Platzer, M., Drescher, B., Schattevoy, R., Gloeckner, G., Dette, M., Baumgart, C., Weber, J., Menzel, U. & Rosenthal, A. (2000). RUMMAGE a high-throughput sequence annotation system. Trends in Genetics 16, 519-520.[Medline]
Thomas, A. & Skolnick, M. H. (1994). A probabilistic model for detecting coding regions in DNA sequences. IMA Journal of Mathematics Applied in Medicine and Biology 11, 149-160.[Medline]
Tsujimoto, H., Fulton, R., Nishigaki, K., Matsumoto, Y., Hasegawa, A., Tsujimoto, A., Cevario, S., OBrien, S. J., Terry, A., Onions, D. & Neil, J. C. (1993). A common proviral integration region, fit-1, in T-cell tumors induced by myc-containing feline leukemia viruses. Virology 196, 845-848.[Medline]
van Drunen, C. M., Sewalt, R. G., Oosterling, R. W., Weisbeek, P. J., Smeekens, S. C. & van Driel, R. (1999). A bipartite sequence element associated with matrix/scaffold attachment regions. Nucleic Acids Research 27, 2924-2930.
Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J. and others (2001). The sequence of the human genome. Science 291, 13041351.
Wallrapp, C., Verrier, S. B., Zhouravleva, G., Philippe, H., Philippe, M., Gress, T. M. & Jean-Jean, O. (1998). The product of the mammalian orthologue of the Saccharomyces cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity. FEBS Letters 440, 387-392.[Medline]
Wolff, L. (1996). Myb-induced transformation. Critical Reviews in Oncogenesis 7, 245-260.[Medline]
Wolff, L., Mushinski, J. F., Shen-Ong, G. L. & Morse III, H. C. (1988). A chronic inflammatory response. Its role in supporting the development of c-myb and c-myc related promonocytic and monocytic tumors in BALB/c mice. Journal of Immunology 141, 681-689.
Wolff, L., Koller, R. & Davidson, W. (1991). Acute myeloid leukemia induction by amphotropic murine retrovirus (4070A): clonal integrations involve c-myb in some but not all leukemias. Journal of Virology 65, 3607-3616.[Medline]
Wolff, L., Koller, R., Bies, J., Nazarov, V., Hoffman, B., Amanullah, A., Krall, M. & Mock, B. (1996). Retroviral insertional mutagenesis in murine promonocytic leukemias: c-myb and Mml1. Current Topics in Microbiology and Immunology 211, 191-199.[Medline]
Xu, Y., Mural, R., Shah, M. & Uberbacher, E. (1994). Recognizing exons in genomic sequence using GRAIL II. Genetic Engineering 16, 241-253.[Medline]
Zhang, M. Q. (1997). Identification of protein coding regions in the human genome by quadratic discriminant analysis. Proceedings of the National Academy of Sciences, USA 94, 565-568.
Received 15 October 2001;
accepted 7 December 2001.