Three Murine Leukemia Virus Integration Regions within 100 Kilobases Upstream ofc-mybAre Proximal to the 5′ Regulatory Region of the Gene through DNA Looping

Abstract

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.

Evaluating risks of insertional mutagenesis by DNA transposons in gene therapy

“…The selectivity of transcriptional regulatory proteins to interact with selective promoters may explain the pronounced lack of activation of endogenous genes that lead to cancer by enhancers in lentiviral vectors. These findings support earlier findings that murine leukemia virus integration up to 100 kbp upstream of the c- myb locus could activate a linearly distal locus through chromosomal looping loci [133]. …”

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome

“…HTLV-1 therefore has the potential to cause widespread abnormalities in chromatin structure and gene expression of the human genome by inserting ectopic CTCF-BSs. These abnormalities may, in turn, contribute to the HTLV-1-associated diseases ATL and HAM/TSP (33).…”

Stress-induced Phosphorylation of Thr486 in c-Myb by p38 Mitogen-activated Protein Kinases Attenuates Conjugation of SUMO-2/3

“…However, these alterations result mostly in increased expression of the full-length, unaltered c-MYB protein (21)(22)(23). These examples are similar to an activation of c-myb in murine leukemia, where a retrovirus integrates several kilobases upstream of the c-myb transcription start site and deregulates its expression (26,27). Nevertheless, these leukemic cells seem to be dependent on the elevated levels of c-Myb, because knocking down the gene expression is detrimental for those cells, whereas normal untransformed progenitors survive similar treatment (28,29).…”