15: An intron of IRF-8 harbors myeloid lineage specific regulatory element that is regulated by dynamic chromatin architecture

Interferon Regulatory Factor-8 (IRF-8) is an IFN-gamma responsive gene that serves as a key factor in the hierarchical differentiation of bone marrow derived cells towards monocyte/macrophage lineage. In attempt to identify the molecular mechanisms leading to this lineage restricted expression of IRF-8, we are using IRF-8 Bacterial Artificial Chromosome (BAC) reporter constructs transfected either to permissive macrophage cell line or to restrictive non-hematopoietic fibroblast cell line. We present data showing that the control of lineage specific expression of IRF-8 is confined to an intronic segment. Deletion of this intron leads to loss of lineage specific expression of BAC IRF-8 reporter. Interestingly, this IRF-8 intron exhibits evolutionary conserved regions, which are not typical to “benign” intronic sequences. To gain molecular insight to this restricted expression, we compared histone modifications and nucleosome occupancy in this intron between expression permissive and restrictive cell lines. For that purpose, we employed Chromatin Immuno-Precipitation (ChIP) and Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) analyses. The ChIP data revealed subtle differences in the overall histone modification profile (”histone code”). The FAIRE analysis pointed to a significant nucleosome density difference between permissive and restrictive cell lines. Accordingly, IRF-8 repression in a restrictive myeloid progenitor cell line was partially alleviated upon treatment with histone methyltransferase inhibitors. Altogether, our data clearly suggests that an intron of IRF-8 harbors myeloid lineage specific regulatory element that is regulated by dynamic chromatin architecture. Taken together, it is most probable that this intron serves as nucleation core for chromatin condensation in restrictive cells.