Genome-wide CRISPR screen to determine
the identity of the genes that mediate the functional consequences
of Hepatic Leukemia Factor (Hlf)
Masters Degree project in General Molecular Biology, 30 credits
Oguzhan Ergün
Supervisor: David Bryder
Co-supervisor: Martin Wahlestedt
The Section of Developmental
Hematopoesis
Division of Molecular Hematology
Project runs: 2015-01-26 – 2015-06-06
In this project, I will try to determine the identity of the genes that are responsible for mediating the
functional consequences of Hlf expression. Since Hlf is a DNA binding transcription factor (TF) these genes
may be either direct or secondary targets of Hlf DNA-binding, or may alternatively be interaction partners.
Background;
Hlf encodes a DNA binding transcription TF belonging to a subfamily of basic leucine zipper (bZIP) TFs that
contain proline acidic-rich (PAR) domain and bind DNA through homo- and heterodimerization with any of
its family members including Dbp, Tef and Nfil3 (Hunger et al., Molecular and Cellular Biology, 1996). Hlf
has been found to give rise to a form of a childhood B-cell leukemia when fused with the gene E2A through
the translocation t(17;19) (Hunger et al., Mol. and Cell. Biology, 1996). However, no published study has
identify the role of Hlf in normal physiological blood cell formation. Previous unpublished studies suggested
that Hlf is an important candidate regulator in early cell fate decision
To investigate this, Hlf inducible GMLPs will be isolated and transduced with a genome wide CRISPR
knockout library (GECKO v2). After transduction cells will be cultured on OP9 stromal cells and allowed to
differentiate towars B lymphocytes. Isolation of these B cells by FACS will followed by genomic DNA
purification and PCR amplification. Finally, these amplicons will be subjected to deep sequencing and
evaluated to find out which gene(s) were knocked out that mediate in this differentiation process.
Significance:
To identify the candidate genes regulated by Hlf and involve in early cell decision in hematopoiesis will may
lead better understanding of transcriptional network of hematopoiesis.
References
1. Bryder D, Rossi DJ, Weissman IL.Hematopoietic stem cells: the paradigmatic tissue-specific stem cell.
The American Society for Investigative Pathology. 2006;169(2):338-346.
2. Hunger SP, Brown R, Cleary, M L. DNA-binding and transcriptional regulatory properties of hepatic
leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF. Molecular and
Cellular Biology .1994;14(9):5986-5996.
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