A supplementary story was also being written as the World Cup moved along, a sad tale chronicling the sudden decline of European football.
However, a 50% reduction in H3K27Ac was seen at PU.1-14kb when the TALE-VP64 targeting this enhancer was expressed (supplementary material Fig. S1B), perhaps due to nucleosome displacement caused by TALE-VP64 and co-factor DNA binding.
H3K27Ac was also enriched 3.8-fold at Scl+40kb when the TALE-VP64 targeting this enhancer was expressed (supplementary material Fig. S1A).
In mouse embryonic stem cells (mESCs), in which these enhancers are not active (as determined by H3K27Ac ChIP-seq enrichment; data not shown) and target genes are weakly expressed, TALE-VP64 did not upregulate gene expression (supplementary material Fig. S1C,D).
Modest (1.5- to 8.5-fold) increases in histone H3 lysine 27 acetylation (H3K27Ac), an epigenetic modification associated with active regions of chromatin (Creyghton et al., 2010), were also seen in 416B cells at the promoters of TALE-VP64 target genes, consistent with increased transcription (supplementary material Fig. S1A,B).
Expression of TALEs targeting the PU.1-14kb partially, but not significantly, reduced the total cell numbers recovered (supplementary material Fig. S2D).
Interestingly, TALE-VP64-mediated PU.1 expression caused a loss of the Flk1+ (mesoderm) population (supplementary material Fig. S2E,F), and significantly increased the CD41+ VE-cadherin (VEcad)+ (committing haemogenic endothelial) population (Fig. 2D; supplementary material Fig. S2F).
To ensure that TALE-VP64 expression alone was not affecting CFU frequency, we assessed ESC lines carrying a non-functional TALE-VP64 [generated previously (Gao et al., 2013)], which did not affect CFU frequency (supplementary material Fig. S2B).
We next assessed the phenotypic effect of TALE-mediated modulation of gene expression by haematopoietic colony forming assays using day 6 EB cells (supplementary material Fig. S2A).
TALE-VP64-mediated PU.1 activation inhibited endothelial colony formation, whereas PU.1 repression did not (Fig. 4F; supplementary material Fig. S5).
The story book 'A tale of fishing' is a simple story for children studying in Class I to be used as supplementary reading material.
