The research in my lab is focused on the molecular mechanisms that govern transcriptional regulation of genes in general, and during myeloid cell differentiation in particular. Bone marrow derived hematopoietic stem cells give rise to all blood cell types. This hierarchical cell fate decisions process is orchestrated by key transcription factors. These factors govern and modulate the expression of lineage specific genes. Lineage specific expression is the hallmark of this complex differentiation process, yet the molecular mechanisms that govern this lineage restricted expression are not elucidated.
In that respect, we are studying the role of a transcription factor, termed Interferon Regulatory Factor – 8 (IRF-8), in myeloid cell differentiation. IRF-8 is expre
ssed mainly in cells of the immune system and direct myeloid progenitor cell (GMP) to differentiate towards the monocyte\macrophage lineage while inhibiting differentiation towards granulocyte. In-addition, knockout mice for IRF-8 have very low levels of macrophages and certain types of dendritic cells (DCs) and overproduce granulocytes. Eventually, these mice exhibit a chronic myelogenous leukemia (CML) like syndrome. Monocytes derived from Human CML patients exhibit very low level of IRF-8 that is restored to normal levels upon treatment. Thus, IRF-8 also serves as a myeloleukemias suppressor gene.
We have shown that IRF-8 binds to target DNA sequence (ISRE) mainly upon interaction with other transcription factors and identified interacting partners and some of the target genes regulated by IRF-8. The interacting partner dictates a specific DNA recognition sequence, thus rendering IRF-8 dual transcriptional activity, that is, repression or activation.
Further, we have shown th at IRF-8 is also essential for the function of the mature macrophages such as cytokines secretion or pathogens elimination.
To identify the molecular mechanisms leading to IRF-8 lineage restricted expression, we used IRF-8 Bacterial Artificial Chromosome (BAC) reporter constructs transfected either to permissive macrophage cell line or to restrictive non-hematopoietic fibroblast cell line. Surprisingly, we have found that the control of lineage specific expression of IRF-8 is confined to an intronic segment. Deletion of this intron, 3rd intron, led 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 intron 3 between expression permissive and restrictive cell lines.
For that purpose, we employ 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 the 3rd intron of IRF-8 harbors myeloid lineage specific regulatory element that is regulated by dynamic chromatin architecture.
Current research in our laboratory is aimed at:
- Characterization of the molecular mechanisms leading to the lineage restricted expression of IRF-8
- Chromatin dynamics (effect of epigenetics)
- Repression mechanisms in IRF-8 restrictive cells
- The effect of chromosome conformation (3D structure) on IRF-8 expression
- Characterization of the regulatory network regulated by IRF-8; finding new target genes
- IRF-8 reporter mouse strain as research model