Extended Data Figure 10. MSI2 OE represses CYP1B1 and HSP90 3'UTR Renilla Luciferase reporter activity.
a, CLIP-seq reads (replicate 1 in blue and replicate 2 in green) and clusters (purple) mapped to the 3'UTR of HSP90. Matches to the GUAG motif are shown in black. Mammal PhyloP score listed in last track. b and c, Representative data of mean per cell fluoresence for HSP90 and CYP1B1 protein in transduced CD34+ CB. Protein level in cells during in vitro culture was analyzed 3 days (D3) and 7 days (D7) after transduction and sorting for GFP. Corresponding secondary alone antibody staining is shown for each experiment. Each circle represents a cell, and greater than 200 cells were analyzed per condition. d and e, Levels of renilla luciferase activity in NIH-3T3 cells co-transfected with control or MSI2 OE vectors and the CYP1B1 or HSP90 wild type or TCC mutant 3'UTR luciferase reporter (dotted line indicates no change in renilla activity; n=4 CYP1B1 and n=3 HSP90 experiments). f, Flow plots of co-transduced CD34+ CB cells with MSI2 (GFP) and CYP1B1 (BFP) lentivirus. g, GFP+ BFP+ CFU-GEMMs generated from f were replated in to secondary CFU assays and enumerated for total number of colonies formed. A total of 24 CFU-GEMMs from MSI2-BFP and MSI2-CYP1B1 were replated (n=2 experiments). Data presented as mean ± SEM. ***p<0.001, **p<0.01. h, A model for AHR pathway attenuation through MSI2 post-transcriptional processing. MSI2 mediates the post-transcriptional down regulation of HSP90 at the outset of culture and continuously represses the prominent AHR pathway effector CYP1B1 to facilitate HSPC expansion. The resultant MSI2-mediated repression of AHR signaling enforces a self-renewal program and allows HSPC expansion ex vivo.