טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKhateb Mamduh
SubjectChromatin Remodeling Mediated by IRF8 3rd Intron Restricts
its Expression in Non-Hematopoietic Cells
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ben-Zion Levi


Abstract

Interferon Regulatory Factor8 (IRF8) is a nuclear transcription factor, critical in the hierarchical differentiation of HSC towards monocyte/dendritic cell lineages. IRF8 expression is mainly restricted to immune cells and it is activated by the combination of IFN-γ in macrophages and DCs cells. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF8 expression in non-hematopoietic cells is still unknown. In order to identify the molecular mechanisms leading to this lineage-restricted expression of IRF8, first, we used IRF8 Bacterial Artificial Chromosome (BAC) constructs. These reporter constructs were transfected to either macrophage cell line (RAW264.7) or to non-hematopoietic fibroblast cell line (NIH3T3). Our data clearly indicate that the repression of IRF8 expression in restrictive cells is mediated by its 3rd intron. In-vitro deletion of this intron alleviates the repression of transfected Bacterial Artificial Chromosome (BAC) IRF8 reporter gene in these cells. Conversely, no reporter expression was observed upon in-situ removal of the 3rd intron in the transfected BAC reporter construct to NIH3T3 cells

 To demonstrate the independent repression and chromatin remodeling ability of the 3rd intron, we have employed Luciferase retroviral and dsRED lentiviral reporter assays where the 3rd intron was cloned upstream to the reporter gene. Enrichment of a repressive H3K27me3 post-translational modification (PTM) was observed over the reporter gene only with the 3rd intron constructs resulting in selective reporter gene silencing. Additionally, removal of this 3rd intron from the dsRED lentiviral reporter assay using VCre recombines did not abrogate its repression effect.  These results points to the 3rd intron role as an initiator of sustainable gene silencing inducing chromatin condensation characterized by suppressive PTM.

Bioinformatics analysis of the 3rd intron revealed three conserved non coding sequences (CNSs). Fine deletion of these CNSs separately, elevates the reporter gene expression in BAC constructs. Whereas, unlike full-length intron, no repression effect is observed when this CNSs cloned individually to a lentiviral reporter assay. This point to a concerted manner of the CNSs to elicit a repressive effect. Finally, we determine the chromatin-state specificity of IRF8 3rd intron and its role as an initiator of repressed chromatin upon in-vitro stem cells differentiation. CRISPR/Cas9 mediated genomic deletion of IRF8 3rd intron was performed in Embryonic Stem Cells (ESC). Subsequently, these cells were triggered to differentiate into cardiomyocytes (restrictive cells) and dendritic cells (permissive cells). Genomic deletion of the 3rd intron elevates the endogenous expression of IRF8 in ES cells and cardiomyocytes clones, consistent with H3K27me3 PTM depletion in IRF8 promoter. Surprisingly, when the same clones were differentiated to dendritic cells, IRF8 expression was reduced and enrichment of H3K27me3 histone PTM was observed.

Taken together, we propose that the IRF8 lineage expression is governed by two modes of regulation, first, IRF8 3rd intron serves as a nucleation core for repressed chromatin during differentiation to IRF8 restrictive cells. Second, lineage specific enhancer that takes place upon differentiation to macrophage/dendritic lineage. To conclude, our results clearly indicate that IRF8 3rd intron is an essential element dictating lineage restricted expression via chromatin dynamics.