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23-Feb-2018 12:47

PU.1 is crucial for lymphomyeloid development, and its stage-specific expression is critical in preventing leukemic transformation.

However, such direct repression mechanisms are not the only way to down-regulate alternate lineage expression programs.

I restriction site) ttcctcgagccagcatgtttaatgattgctt. The correct insertion of the pre-mi R was confirmed by sequencing.

The mi R expression plasmids were then cotransfected using calcium-phosphate precipitates (Invitrogen) with gag/pol and eco-env plasmids into HEK293T cells, and virus-containing supernatant was harvested 3 and 4 days later, filtered, and stored at −80°C until use.

To test for such negative regulatory events, we searched for PU.1-controlled micro RNAs (mi Rs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model.

We provide evidence that PU.1 directly controls expression of at least 4 of these mi Rs (mi R-146a, mi R-342, mi R-338, and mi R-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci.

Unbiased mapping of PU.1 binding (Ch IPseq) and locus accessibility (DNase hypersensitivity) in PUER cells and primary macrophages revealed that the expression of 4 mi Rs (mi R-146a, mi R-342, mi R-338, and mi R-155) is PU.1 dependent during the earliest steps of myeloid progenitor maturation.

One of the most prominently induced mi Rs (mi R-146a) was analyzed further.

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In agreement with this, a knock-down (KD) approach in zebrafish (-KO zebrafish model.Ectopic expression of the most robustly induced PU.1 target mi R, mi R-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays.In agreement with this observation, disruption of Transcription factors play a decisive role in determining lineage fate from HSCs.In brief, cells were lysed and RNA was extracted with phenol/chloroform and then enriched with micro-columns (mi RVana micro RNA kit; Ambion).



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