AIBP-mediated Cholesterol Efflux Instructs Hematopoietic Stem and Progenitor Cell Fate

Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are underexplored. Here we report that a novel somite-derived pro-hematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch upregulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide ChIP-seq, RNA-seq, and ATAC-seq indicate that Srebp2 trans-regulates Notch pathway genes required for hematopoiesis. Our studies outline a novel AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease. Overall design: The ATAC-Seq was performed using 50,000 c-Kit+CD144+ CD45.2- cells isolated from AGM region of E11.5 wild-type B6 mouse embryos. The Tn5 transposome was purified and assembled following a published protocol (46). The permeabilization was performed with 50 µl cold ATAC-RSB buffer (0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin) and the transposition was performed with 50 µl transposition mix (10µl 5× HEPES DMF Buffer, 3 µl 5 µM Tn5 transposome, 37 µl PBS, 0.5 µl 10% Tween-20, and 0.5 µl 1% digitonin) and incubated at 37° for 30 min. After transposition, the cleaned-up DNA fragments were pre-amplified for 5 cycles using NEB Q5 master mix. Each reaction contains 2.5 µl of 25 µM i5 primer, 2.5 µl of 25 µM i7 primer, 25 µl 2× NEB Q5 master mix, and 20 µl cleaned up samples. PCR settings were 5 min at 72°C, 30 sec at 98°C, and followed by additional 5 cycles (98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min). After pre-amplification, 1 µl of the pre-amplified mixture was used to run a 10 µl qPCR test to determine the optimal amplification cycle. The final amplified DNA library was purified using Qiagen DNA purification kit and sequenced on NextSeq 500 with SE75 strategy. This submission represents the ATA-Seq component of study.