Background and Significance
The overall research focus is on transcriptional regulation in hematopoietic development (blood cell development). This includes normal development, as well as defects that can lead to disease such as for example autoimmunity and malignant transformation resulting in blood cancer (leukemia). All blood cells develop from blood cell-specific stem cells in the bone marrow. Blood cancer or immunological diseases can develop if these cells have loss of function or gain of function mutations in genes that control development, function, growth or survival. A main focus of our laboratory is on the transcription factor Ikaros, encoded by the IKZF1 gene. IKZF1 is a tumor suppressor gene that is deleted or mutated in some types of human acute lymphoblastic leukemia (ALL), and when mutated, is correlated with poor prognosis (1, 2). One of the overall goals of one of the projects is to understand how Ikaros functions as a tumor suppressor, so that we in the future can better understand, and thereby better diagnose and treat, these types of blood cancer that contain Ikaros mutations (3,4). To understand the mechanism of action, we are also interested in downstream target genes of Ikaros, as well as other transcription factors that antagonize or collaborate with Ikaros in transcriptional regulation of hematopoietic development and tumor suppression.
Ikaros is a transcription factor, and thus control gene expression through direct binding to DNA and through interaction with other regulatory proteins. In addition to its function as a tumor suppressor, Ikaros is also required for proper hematopoiesis at multiple stages of development. For instance, it is absolutely required for development of B cells, as Ikaros knock-out mice (Ikzf1null) do not develop B cells at all (5). It also regulates the development of other hematopoietic lineages, demonstrated by defects in for instance red blood cells, T cells, NK cells and a specific subset of dendritic cells (pDCs) in various Ikaros-mutant mice. Interestingly, the IKZF1 gene is highly alternatively spliced, yielding several different isoforms of the Ikaros protein. However, the biological significance of the different Ikaros isoforms is incompletely understood. Furthermore, recent GWAS studies have indicated several disease-associated SNP’s annotated to both IKZF1, and to the close family member IKZF3 (encoding the protein Aiolos). However, the mechanisms for how these naturally occurring genomic variants (SNPs) might predispose for various immunological diseases, are not understood.
Experimental approach
In order to study Ikaros function in development and disease, we combine several approaches, including both mouse models and human cells. New mouse models with targeted deletions of the DNA-binding zinc fingers of Ikaros (Figure 1) have revealed distinct defects in development of various hematopoietic lineages (3). Furthermore, one of the mutants develops spontaneous thymic lymphoma, demonstrating a loss of Ikaros tumor suppressor function, and loss of Ikaros tumor suppression was also observed in a mouse model of human ALL (3,4). These mutant mice thus provide a valuable model system for further analysis of Ikaros function and elucidation of critical Ikaros target genes required for developmental regulation as well as tumor suppressor function.'
Furthermore, to specifically address the role of Ikaros as a tumor suppressor in human pre-B cell ALL, we are studying the role of Ikaros and its target genes in human pre-B ALL patient-derived cells (4). RNA-Seq, ChIP-Seq and ATAC-Seq experiments have been performed to map the genome-wide binding sites of wild-type Ikaros in human ALL cells, and to evaluate the impact of Ikaros on chromatin accessibility and downstream target genes. Similar analysis is also performed in the Ikaros-mutant mouse cells (Fig. 2). This project has resulted in several follow-up project directions that are actively being pursued. The hypothesis is that the above approaches will yield a limited list of candidate Ikaros target genes that can be future targets for therapeutic approaches for treatment of human ALL that are characterized by Ikaros mutations. As Ikaros is also involved in other processes important for normal and malignant development, the lab also works on other projects studying various aspects of Ikaros biology and
Figure 2: Example of cluster analysis of mRNA expression data by RNA-Seq (left) and visualization in the UCSC genome browser of mRNA-Seq data from cells with specific deletion of individual coding exons (right).
Project Description
The project is open for two Master students. The students will take on separate projects within the above described research areas. There are individual projects available both to study the role of Ikaros (or other transcription factors) in the development of various blood cell lineages as well as to study the roles of these factors in disease (e.g. in leukemia, autoimmunity). The projects are both follow-up of current on-going projects as well as newly developed smaller side-projects. Depending on the interest and preference of the student, different opportunities can be provided: The student can choose a smaller defined project, but there are also opportunities to be exposed to more complex projects involving a wide variety of experimental and scientific approaches. The specific details of the project(s) will depend on the status of the projects when the prospective student(s) start in lab as well as the specific interest and background of the student(s).
Figure 3: Triplex immunohistochemistry (IHC) stain on mouse spleen displaying B cells, T cells and myeloid lineage cells in organized structure in the spleen.
Methods: The student(s) will learn and perform a variety of standard molecular biology experimental methods (i.e. western (immunoblot) for protein, RT-PCR for mRNA expression, cloning) as well as standard cellular biology methods (cell culture, flow cytometry/FACS analysis, possibly also advanced cell sorting, as well as transfection/transduction for over expression and/or knock down, e.g. using CRISPR/Cas9 or dCas9-KRAB system). For some projects, we also use immunological techniques, such as IHC (Fig. 3) and ELISA. Due to the nature of the experimental models, most students will also learn about and be exposed to in vivo work with mouse models (knock out/mutant mouse models, transplantation), and there is possibility of in silico bioinformatics analysis of high throughput sequencing data (Figure 2; RNA-Seq, ChIP-Seq). Importantly, the student(s) will work in a highly productive, advanced and inspiring scientific community, both daily within the laboratory and at UCSF as a whole. In particular, the student will be encouraged to participate in the Immunology program at UCSF, including talks by invited speakers and journal club presentations. See more information about the UCSF Immunology program at this link:
Lab safety: Prior to start of work in lab, the students are required to complete an extensive series of UCSF online safety courses. In addition, the students will be trained in practical lab safety by mentor in lab for each specific method employed.
Practical information
The Master project(s) will be performed at UCSF, in San Francisco, CA, USA, and the student must be able and willing to stay for a full year, and cover the travel and potential increased living expenses. UCSF provides an outstanding environment for research and education, and in particular has an excellent Immunology program (see link above). The student will work in the group of Dr. Hilde Schjerven, who works at the UCSF Parnassus location located next to Golden Gate Park. In order to obtain a J-1 student visa to enter and stay in the US, the student must be able to document an income/salary sufficient to cover cost of living, and document health insurance coverage (contact "L?nekassa" and the Norwegian government (Folketrygden), respectively). Previous Master students in the group can also be of help in navigating the process. The student will be encouraged to seek out and apply for funding to cover travel expenses as well as potential increased cost of living due to stay abroad. Due to use of mouse models, the students should take the class “MBV4330 - Experimental animal studies” (5 stp) (e.g. early January 2018) prior to start at UCSF. The timing of start of the project in lab is dependent on approval of the J-1 visa, the processing of which could take up to 4 months. It is therefore important that anyone interested in this project initiates contact as soon as possible (see contact information above).
* Additional info: http://www.hildeschjerven.net/
And: http://schjervenlab.ucsf.edu/
References
- Mullighan, C.G., Miller, C.B., Radtke, I., Phillips, L.A., Dalton, J., Ma, J., White, D., Hughes, T.P., Le Beau, M.M., Pui, C.H., et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 453, 110-114. (2008).
- Mullighan, C.G., Su, X., Zhang, J., Radtke, I., Phillips, L.A., Miller, C.B., Ma, J., Liu, W., Cheng, C., Schulman, B.A., et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. The New England journal of medicine 360, 470-480. (2009).
- Schjerven, H., McLaughlin J., Arenzana, T.L., Frietze S., Cheng D., Wadsworth S., Lawson G.W., Bensinger S. J., Farnham P.J., Witte O.N. and Smale, S.T., Differential regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros. Nat. Immunol. 14, 1073-83. (2013).
- *Schjerven H, Ayongaba EF, Aghajanirefah A, McLaughlin J, Cheng D, Geng H, Boyd JR, Eggesb? LM, Lindeman I, Heath JL, Park E, Witte ON, Smale ST, Frietze S, Müschen M. Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre-B ALL. J Exp Med. 2017 Mar 6;214(3):793-814. doi: 10.1084/jem.20160049. PMID: 28190001 * Corresponding author.
- Wang, J.H., Nichogiannopoulou, A., Wu, L., Sun, L., Sharpe, A.H., Bigby, M., and Georgopoulos, K. Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5, 537-549. (1996).