Three Murine Leukemia Virus Integration Regions within 100 Kilobases Upstream of c-myb Are Proximal to the 5 ' Regulatory Region of the Gene through DNA Looping

Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Journal of Virology (Impact Factor: 4.44). 07/2012; 86(19):10524-32. DOI: 10.1128/JVI.01077-12
Source: PubMed


Retroviruses integrated into genomic DNA participate in long-range gene activation from as far away as several hundred kilobases. Hypotheses have been put forth to account for these phenomena, but data have not been provided to support a physical mechanism that explains long-range activation. In murine leukemia virus-induced myeloid leukemia in mice, integrated proviruses have been found upstream of c-myb in three regions, named Mml1, Mml2, and Mml3 (25, 50, and 70 kb upstream, respectively). The transcription factor c-Myb is an oncogene whose dysregulation and/or mutation can lead to human leukemia. We hypothesized that the murine c-myb upstream region contains regulatory elements accessed by the retrovirus. To identify regulatory sites in the murine c-myb upstream region, we looked by chromatin immunoprecipitation with microarray technology (ChIP-on-chip) for histone modifications implicating gene activation in normal cells. H3K4me3, H3K4me1, and H3K9/14ac were enriched at Mml1 and/or Mml2 in the myeloblastic cell line M1, which expresses c-myb. The enrichment of all of these histone marks decreased with differentiation-induced downregulation of the gene in M1 cells but increased and spread in tumor cells containing integrated provirus. Importantly, using chromosome conformation capture (3C)-quantitative PCR assays, interactions between the 5' region, including the promoter and all Mml sites (Mml1, Mml2, and Mml3), were detected due to DNA looping in M1 cells and tumor cells with provirus in Mml1, Mml2, or Mml3. Therefore, our study provides a new mechanism of retrovirus insertional mutagenesis whereby spatial chromatin organization allows distally located provirus, with its own enhancer elements, to access the 5' regulatory region of the gene.

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Available from: Jan Markus, Jan 23, 2014
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    • "Taking into account the size of a provirus or a retroviral vector, it seems plausible that some degree of local structural reorganization of the genome should accompany integration. In the study by Zhang et al. [35] the authors observed positional expansion, comprising several kb, of enhancers (H3K4Me1) in tumor cells containing proviruses at the c-Myb locus. It would be interesting to address at a large-scale how the local chromatin environment changes in response to retroviral integration in tumors, as well as determine BET protein binding sites genome-wide. "
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    ABSTRACT: Background Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined. Results We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues. Conclusions We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.
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    ABSTRACT: c-Myb plays an essential role in regulation of properly balanced hematopoiesis through transcriptional regulation of genes directly controlling cellular processes such as proliferation, differentiation and apoptosis. The transcriptional activity and protein levels of c-Myb are strictly controlled through posttranslational modifications such as phosphorylation, acetylation, ubiquitination and SUMOylation. Conjugation of SUMO proteins has been shown to suppress the transcriptional activity of c-Myb. SUMO-1 modifies c-Myb under physiological conditions, while SUMO-2/3 conjugation was reported in cells under stress. Since stress also activates several cellular protein kinases, we investigated whether phosphorylation of c-Myb changes in stressed cells and whether a mutual interplay exists between phosphorylation and SUMOylation of c-Myb. Here we show that several types of environmental stress induce a rapid change in c-Myb phosphorylation. Interestingly the phosphorylation of Thr486, located in close proximity to c-Myb SUMOylation site Lys499, is detected preferentially in nonSUMOylated protein and has a negative effect on stress-induced SUMOylation of c-Myb. Stress-activated p38MAPKs phosphorylate Thr486 in c-Myb, attenuate its SUMOylation, and increase its proteolytic turnover. Stressed cells, expressing a phosphorylation-deficient Thr486Ala mutant, demonstrated decreased expression of c-Myb target genes Bcl-2 and Bcl-xL and accelerated apoptosis due to increased SUMOylation of the mutant protein. These results suggest that phosphorylation- dependent modulation of c-Myb SUMOylation may be important for proper response of cells to stress. In summary, we have identified a novel regulatory interplay between phosphorylation and SUMOylation of c-Myb that regulates its activity in stressed cells.
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