Long distant regulation of c-myb in normal cells and acute myeloid leukemia

Division Of Basic Sciences - Nci

Investigators

Abstract

Many of the disease-related genetic alterations in the vicinity of c-myb (e.g. retrovirus integration) involve an extended intergenic area upstream of the gene. The molecular consequences of these are unknown. However, since c-myb is highly implicated in mouse and human leukemia, we have begun to look for long distant c-myb transcriptional regulators in the region 25-100 kb upstream region. Because of recent developments in the epigenetics field we can now begin to predict the function of regions of chromatin based upon the presence of histones with specific modifications or specific DNA-binding proteins. Promoter regions and enhancers of activated genes are enriched in histone 3, mono-and tri-methylated on lysine 4 and have acetylated histone 3, for example, H3K9Ac. In an attempt to find enhancers in the upstream region of c-myb, ChIP-on-chip was carried out using antibodies specific for these marks. Preliminary data has revealed several elements in the c-myb upstream region in myeloid cells, and not in NIH-3T3 cells, that are potentially associated with transcriptional activation. Interestingly, an element with the highest abundance of H3K4me3 and H3K9Ac is located very close to the virus integration site Mml1. By comparing data from myeloid cells lines with murine acute myeloid leukemia samples with Mml1 integrations we hope in the future to be able to observe changes in the histone marks associated with the virus integrations at Mml1,2, and 3. Another approach that is being utilized in the laboratory to look for long distance regulation of c-myb is a chromosome conformation capture assay. This is allowing us to observe the genomic organization surrounding and including the c-myb locus. Using this approach, we are investigating which upstream areas through looping are in contact with the c-myb promoter in mouse cells. Our preliminary experiment revealed two upstream regions that interact with the c-myb promoter in typical myeloid cells that do not have the Mml region virus integrations. These were in close proximity to H3K4me3-rich elements identified by ChIP-on-chip. Interestingly, an experiment with one cell line carrying an Mml1 retrovirus indicated that the virus disrupts the proposed looping pattern. Although these results are preliminary they indicate that there are looping patterns involving the c-myb upstream region and c-myb that are distinct for different cell lines and that there may be enhancer like sequences upstream of c-myb.