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Research Report 2: Genomic targets of Polycomb Cbx proteins in hematopoietic stem cells and differentiated blood lineages

Smit, M.J. (2014) Research Report 2: Genomic targets of Polycomb Cbx proteins in hematopoietic stem cells and differentiated blood lineages. Master's Thesis / Essay, Biology.

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A proper balance between self-renewal and differentiation of hematopoietc stem cells is crucial, because impaired self-renewal can result in tissue dysfunction while excessive self-renewal can result in blood cancer (leukemia). Adult stem cells and their differentiated progeny contain identical genetic information, but their gene expression patterns differ substantially. This is accomplished by changes in chromatin compaction by DNA methylation or histone modifications, it establishes, maintains and propagates gene expression program and drives the behaviour of cellular differentiation. Polycomb group proteins (PcGs) are a specific class of epigenetic modifiers that catalyzates histone modifications to repress gene expression of target genes. Previously, our lab showed that the balance of self-renewal and differentiation in HSCs is governed by Cbx-containing Polycomb repressive complex 1 (PRC1). Cbx7-containing PRC1 complexes induce self-renewal by repressing the expression of progenitor-specific genes, and its overexpression induces leukemia. Cbx2, Cbx4 or Cbx8 can compete with Cbx7 for integration into PRC1 and their overexpression results in differentiation and exhaustion of HSCs. To get insight into the dynamics of Polycomb PRC1 targets during normal HSC differentiation, our aim is to compare genome wide targets of endogenous Cbx proteins in different hematopoietic cell subsets. An important technique to study protein-DNA interactions is Chromatin immunoprecipitation (ChIP), which is a very versatile tool. The basic steps including fixation, sonication and immunoprecipitation requires optimization. Currently we have optimized a ChIP protocol for 100.000 cells with homemade buffers and tested several Cbx7 antibodies in different hematopoietic cell subsets. In the end, we aim to sequence DNA fragments bound by Cbx7, Cbx8, Cbx2 and Cbx4 in HSCs and multiple differentiated blood cells (T-cells, B-cells, granulocytes).

Item Type: Thesis (Master's Thesis / Essay)
Degree programme: Biology
Thesis type: Master's Thesis / Essay
Language: English
Date Deposited: 15 Feb 2018 07:58
Last Modified: 15 Feb 2018 07:58

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