טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFourier Nitsan
SubjectCharacterization of the Molecular Mechanisms Governing
Lineage-Restricted Expression of IRF-8
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ben-Zion Levi


Abstract

Interferon Regulatory Factor-8 (IRF8) plays a key role in the hierarchical differentiation towards monocyte/dendritic cell-lineages. While much is known about the essential mechanisms for its hematopoietic-specific expression, IRF8 silencing in non-hematopoietic cells is still uncharacterized. Two major molecular mechanisms might participate in IRF8-restrictive expression: chromatin remodeling and/or transcriptional regulation. These are not necessarily two mutually exclusive events. We’ve recently identified IRF8 3rd intron as a nucleation-core for chromatin remodeling in expression-restricting cells. Presumably, this intron provides a platform for cell-type specific DNA interacting-factors during differentiation. In restrictive cells, these factors initiate histone post-translational modifications (PTMs) affecting chromatin architecture. To extend these results in cell-lines to in-vivo models, primary mouse IRF8-restrictive (myeloid progenitors) and permissive cells (macrophages) were analyzed. We show that these two cell-types exhibit differential repressive H3K27me3 histone PTM signature in IRF8 3rd region. Furthermore, we demonstrate that perturbing H3K27me3 alleviates IRF8 expression in expression-restrictive cells.

A comprehensive understanding of the molecular mechanisms underlying genome functions, namely transcription or chromatin organization, requires identification of interacting-molecules with specific genomic-regions. Thus, analysis of regulatory-elements binding the endogenous IRF8 3rd intron was required. Two molecular strategies were taken; the first, targeted proteomic analysis using Engineered DNA-Binding Molecule-Mediated Chromatin-Immunoprecipitation (enChIP), is aimed at identifying DNA binding-factors with the 3rd intron. This approach provides candidates for IRF8 repressors in expression-restrictive cells, namely Purα and Purβ. These candidates awaits further validation and characterization.

The second, shRNA library-screen, enables the high-throughput interrogation of gene-functions concurrently, allowing new genes identification in a given biological process. We identify MafK as an IRF8 regulator in expression-restrictive cells. Using ChIP-seq analysis, we reveal three repressive MafK regulatory-regions (-25kb, -20kb and IRF8 6th intron) in IRF8 locus. To delineate MafK regulatory mechanism, we’ve employed transient and stable reporter-assays, each pointing to a different mechanism. While the genome-integrated MafK reporter-constructs exert their repressive effect on the reporter-gene, the plasmid constructs demonstrated no such effect. This demonstrates that MafK modulation of local chromatin-environment is essential for its activity. Additionally, removal of MafK-int6 regulatory-region from BAC-IRF8 reporter-construct is sufficient to change histone PTM signature in the region, resulting in alleviation of neighboring reporter-gene expression. Interestingly, no repression is observed in IRF8-premissive cells, suggesting MafK cell-type specific regulation. To delineate the essential MafK DNA binding-motifs, deletion-analysis of individual MafK regulatory-regions was employed. The analysis reveals that either of the MafK25 region binding-motifs can exert repressive activity, while in MafK20 only cooperative-binding of two motifs results in repression. Conversely, in MafK-int6 only the highest-score DNA binding-motif was essential for repression. We’ve also explored the possibility that Bach1, a known MafK co-repressor, is involved in IRF8 regulation. Bach1 over-expression combined with transient MafK reporter-constructs results in reporter-gene silencing, similarly to the effect observed when MafK is over-expressed. The results confirm Bach1 involvement in MafK-mediated repressive regulation of IRF8 in expression-restrictive cells via MafK25, MafK20 and MafK-int6 regulatory-regions. In conclusion, in this work we identify and characterize the regulatory mechanism of several elements in IRF8 locus mediating chromatin remodeling, resulting in restrictive expression of IRF8 in non-hematopoietic cells.