Gene Expression Analysis of WT and dnTCF4-expressing Short-Term Hematopoietic Stem Cells (ST-HSC)

The canonical Wnt signaling pathway is mediated by interaction of b-catenin with the TCF/LEF transcription factors and subsequent transcriptional activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by genetic manipulation of b-catenin. However, this approach does not allow to discriminate between TCF/LEF dependent or independent b-catenin activity. Here, we employed a transgenic mouse model expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) which abrogates activation of Wnt target genes even when b-catenin is stabilized and translocated into the nucleus while preserving its TCF/LEF independent activities. The disruption of the b-catenin-TCF/LEF interaction results in reduced granulocytic differentiation accompanied with an accumulation of short-term hematopoietic stem cells (ST-HSC) and granulocytic progenitors. In order to investigate the mechanism responsible for the insufficient production of granulocytes, we performed a transcriptomic analysis of ST-HSC from dnTCF4-expressing and control WT mice. Overall design: ST-HSC, defined as Lin- ckit+ CD48+ CD150- cells, were sorted from BM of 3 WT and 3 dnTCF4-expressing mice. PolyA enriched sequencing libraries were constructed using SMARTer Universal Low Input RNA Kit and Nextera XT DNA Library Prep Kit and sequenced (paired-end sequencing) on NextSeq 500 instrument from Illumina.