We have begun to look for distal c-myb transcriptional regulators in the region 25-100 kb upstream region. Since MuLV-induced myeloid leukemias have integrated proviruses upstream of c-myb in three regions named Mml1, 2, and 3 (25, 50 and 70kb upstream, respectively), we hypothesized that the retrovirus accesses regulatory elements. Accordingly, the provirus locations might be able to assist us in identifying regulatory regions that are important to normal cells as well as leukemic cells that have dysregulated c-myb expression. We first looked by ChIP-on-chip for histone modifications implicating gene activation in normal cells, because this might help in locating regulatory sites. H3K4me3, H3K4me1 and H3K9/14ac, which are known to be enriched at promoters that are activated, were enriched at Mml1 and/ or Mml2 in the c-myb-expressing myeloblastic cell line, M1. The enrichment of all of these histone marks decreased with IL-6 induced differentiation and down-regulation of the gene in M1 cells. However, these marks were increased and spread in tumor cells containing integrated provirus. To examine the enhancer activity of sequences representing three peaks of H3Kme4 in Mml1, 3 fragments were cloned upstream of the c-myb promoter controlling a firefly luciferase reporter gene. We were able to show in Luciferase assays that sequences within one of the regions increased luciferase activity, indicating the presence of an enhancer element. Importantly, using Chromosome Conformation Capture (3C) q-PCR assays, interactions between the 5 prime region including the promoter and all Mml sites (Mml1, 2 and 3) were detected due to DNA looping in M1 cells and tumor cells with provirus in Mml1, 2 or 3. The Mml1 region contains 2 loops giving a total of 4 loops and provirus was found to be inserted in each of these loops. Interestingly, M1 cells, that are induced to differentiate with IL-6 and do not express appreciable levels of c-myb, still maintain the same chromatin looping structure observed in cells with active transcription. Therefore, this chromatin looping structure is insufficient by itself for c-myb expression and may require addition of transcription factors to result in expression. Examination of the chromatin conformation in a non-hematopoietic cell line, NIH3T3, which does not express c-myb, revealed that differences in the looping structure at the c-myb locus exist in different cell types. Apparently, a novel interaction peak, not found in the hematopoietic cells, was present very close the promoter at approximately -11 kb. We can conclude that 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 prime regulatory region of the gene. Although such a model could be predicted based upon recent studies that show that transcriptional activation in higher eukaryotes frequently involves the long range action by regulatory elements, this is the first time it has actually been shown that the provirus is in a position to interact with the promoter and/or control region in intron1 through a 3-dimensional chromatin structure. Our study has, in addition, delineated some important regulatory regions that are distal to the c-myb coding region and can know be studied in more detail in future studies. Our investigation also showed that CTCF binds in the vicinity of the loop structures, which is not surprising because it is known that CTCF contributes to looping associated with gene activation. Although we discovered 4 dominant promoter interacting loops, only 3 were associated with CTCF. The most proximal loop was not associated with this DNA binding protein. Interestingly, others have shown that enhancer and promoter sites are often bound by cohesin and mediator complexes in the absence of CTCF. The MARWIZ online program was used to predict, within 100 kb region upstream of the c-myb in M1 cells, potential MARs which are evolutionarily conserved genome sequences that anchor DNA to the nuclear matrix and are reported to act as boundary elements for chromatin functional domains. In silico data showed that potential MARs are located at the boundaries of the loops identified in the 3C assay, thus assisting in our development of a hypothetical chromatin structure of the c-myb extended locus. In summary, retrovirus insertional mutagenesis has assisted in the identification of several potential regulatory regions in the far upstream region of c-myb that likely to be important in normal hematopoietic cell development and could be mutated or altered epigenetically in leukemia.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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