Dissecting The Functional Specificities Of Ikaros Family Transcription Factors During B Cell Differentiation
Reference : PhD Susan CHAN
Offer publication : April 5, 2016
B cell differentiation proceeds through several stages in the bone marrow. Pro-B cells undergo rearrangements of the immunoglobulin heavy chain. These lead to expression of the pre-B cell receptor in large pre-B cells. Transient signalling by this receptor leads then to further differentiation into small pre-B cells, where rearrangement of the light chain occurs, leading to expression of the B cell receptor in immature B cells.
These various steps are orchastrated by transcription factors that control the dynamic expression of large set of genes during the differentiation. Ikaros is one of these transcription factors, and we have previously shown an essential function this factor in the control of the differentiation from the large pre-B to the small pre-B stage (Heizmann et al., J Exp Med 2013). Interestingly, the Ikaros homolog Aiolos is expressed in large pre-B cells that
are deficient for Ikaros, but this expression is unable to promote further differentiation. This observation thus suggests that Ikaros and Aiolos exert distinct functions in pre-B cells. The aim of this project is to dissect the functional specificity of Ikaros and Aiolos in pre-B cells, and identify the features that determine specificity.
Specifically, the student will:
1) Establish cellular tools based on Ikaros deficient large pre-B cell lines. We have previously described primary large pre-B cell lines that are null for Ikaros. These cell lines will be further engineered to inactivate Aiolos (Crispr/cas9), and generate inducible tetracycline-based systems to allow reexpression of Ikaros and Aiolos.
2) Compare the effects of Ikaros and Aiolos expression. The effects of induced Ikaros or Aiolos expression will be studied in the cell lines generated in (1), using several readouts: proliferation, differentiation (Igk rearrangement, pre-BCR downregulation), or global gene expression (RNA-seq). These
experiments should reveal the specific and overlapping functions of Ikaros and Aiolos.
3) Dissect the molecular domains that mediate specificity. Chimeras between the Ikaros and Aiolos proteins will be constructed, focusing specifically on the exchange of the DNA binding domains, and their functions in the above system tested. If the student finds that the DNA binding domain mediates specificity, further dissection will be done, to exchange specific zinc finger, or amino acids. These experiments will be accompanied by the mapping of the binding sites of the WT and chimeric proteins by ChIP-sequencing. These results should identify both the domains/residues that contribute to specific target gene activation, as well as architecture of target sequences specific for each factor.
- WISHED SKILLS : Solid knowledge of molecular biology and immunology
- EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING : Genomic analyses, cytometry, cellular models
Application Deadline : Dec. 31, 2016