ChIPping away at intestinal stemness

Abstract

In Chapter 2 we describe the quantitative ChIP-seq analysis of Polymerase II DNA occupancy. We show that levels of Polymerase II occupancy can be used as a measure for transcriptional activity. However, it also reveals additional information on transcription rates in relation to mRNA stability if it is compared to conventional techniques like microarray hybridization an RNA-seq. In Chapter 3 we describe the ChIP-seq analysis of two of the Wnt-signal executing factors: β-catenin and Tcf4. We define two classes of β-catenin peaks in primary mouse tissue and confirm their existence in two cell lines. Using a truncated, dominant negative, form of Tcf4 we find that both classes of β-catenin binding are mediated by TCF/LEFs. In Chapter 4 we investigate the transcription factor Ascl2 and the mechanism by which it performs it pivotal role in stemness. Using ChIP-seq we show that Ascl2 and β-catenin/Tcf4 converge on specific elements, and together drive the transcriptional program that defines stemness. Ascl2 adds another, very specific, layer of Wnt-signal amplification on top of previously defined Wnt enhancing mechanisms acting on intestinal stem cells. In Chapter 5 we describe the generation of a new transgenic allele. Mice harboring the Olfm4-IRES-eGFPCreERT2 allele express an eGFPCreERT2 fusion protein exclusively in the stem cells of the small intestine. Combination with a lacZ reporter strain confirmed the specific expression and the capability of Olfm4 marked cells to renew the cells of the small intestinal epithelium with the same kinetics of Lgr5+ stem cells. This mouse model improves upon the previously described Lgr5-eGFP-IRES-CreERT2 model because it is not silenced and therefore is expressed in the stem cells of all the crypts of the small intestinal epithelium. In Chapter 6 we report the results of an over-expression based screen in intestinal organoids. We show that organoids challenged by noggin withdrawal provide a valid system to screen for factors important to the intestine. By over-expressing transcription factors selected for their enrichment in stem cells we find three candidates that are able to induce insensitivity to noggin withdrawal. We show that one of these factors, Tgfβ induced factor homeobox 2 (Tgif2), is important to limit Paneth cell differentiation in vivo and in vitro. This thesis is concluded in Chapter 7 with a summarizing discussion of the different chapters.