Unknown,Transcriptomics,Genomics,Proteomics

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ScRNA-seq of aortic arches from smooth muscle cell–specific Hif1a knockout and control mice with atherosclerosis

ABSTRACT: Smooth muscle cells (SMCs) undergo phenotypic switching during atherosclerosis and give rise to a large fraction of plaque cells. To investigate the role of hypoxia-inducible factor 1α (HIF1α) signaling in SMC behaviour during atherogenesis, we used mice in which Hif1a is conditionally deleted in lineage-traced SMCs (Myh11-CreERT2; Rosa26-tdTomato; Hif1a-floxed). Cre recombination was induced with tamoxifen at 6 weeks of age. Atherosclerosis was induced by a single tail-vein injection of rAAV8-PCSK9 at 9 weeks of age followed by 21 weeks of high-fat diet feeding. Aortic arches were pooled by genotype from SMC-specific Hif1a knockout (Hif1aSMC-KO, n=8) and wild-type control (Hif1aWT, n=6) male mice, enzymatically dissociated, and viable cells (Draq5+/DAPI–) isolated by fluorescence-activated cell sorting. Single-cell transcriptomes were generated using the 10x Genomics Chromium Next GEM Single Cell 3′ v3.1 platform and sequenced on an Illumina HiSeq 4000. Reads were aligned to the mm10 reference transcriptome supplemented with the tdTomato transgene sequence using Cell Ranger. The dataset enables comparison of cell-type composition and of SMC-derived cell transcriptional states between genotypes, with a focus on hypoxia- and stress-response pathways underlying SMC phenotypic modulation. Only male mice were studied because the Myh11-CreERT2 transgene is Y-linked.

INSTRUMENT(S): BD FACSAria cell sorter (viable-cell sorting); Countess III cell counter (Thermo Fisher; viability check), Illumina HiSeq 4000, 10x Genomics Chromium controller; Agilent 2100 Bioanalyzer and Qubit fluorometer (Thermo Fisher) for QC, NaN, 10x Genomics Chromium controller (GEM generation on Chromium Next GEM Chip G)

ORGANISM(S): Mus musculus

SUBMITTER: Diana Sharysh

PROVIDER: E-MTAB-17142 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Different mechanisms for CBF-MYH11 function in acute myeloid Leukemia (AML) with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBF-MYH11 binding site analysis and quantitative interaction proteomics we found that CBF-MYH11 localizes to RUNX1 occupied promoters where it interacts with TAL1, FLI1 and TBP associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the co regulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knock down a subset of the CBF-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBF-MYH11 target genes, including genes implicated in hematopoietic stem cell (HSC) self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and upon fusion protein knock down repressed. Together these results suggest an essential role for CBF-MYH11 in regulating expression of genes involved in maintaining a stem cell phenotype. 17 ChIP-seq samples using antibodies recognizing the indicated proteins and one RNA-seq file from ME-1 cells were analyzed. In addition 2 ChIP-seq profiles were generated using patient AML cells. A CBFβ-MYH11 inducible U937 system (U937CM) was used to examine binding patterns before (1 profile) and after (2 profiles) induction of CBFb-MYH11. In addition, expression was measured through RNA-seq analysis of the two states. The U937CM cells were maintained in the presence of tetracyclin (Tet, 1 uM) and grown in the absence of tetracycline for 3 days to induce expression of CBFβ-MYH11. Finally, a CBFb-MYH11 knock down system was developed in ME-1 cells. Two ME-1 cell lines were created, one with a stably integrated shRNA construct that targets CBFb-MYH11 (ME-1_knockdown) and one with a scrambled shRNA construct (ME-1_SCR). Expression of the shRNA constructs was induced using doxycyclin (dox; 1 mM) treatment for 3 days.

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ScRNA-seq of aortic arches from smooth muscle cell–specific Hif1a knockout and control mice with atherosclerosis

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