GSE108672
Polycomb Group Protein YY1 is an Essential Regulator of Hematopoietic Stem Cell Quiescence
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to identify Yy1 regulated genetic network by comparing transcriptome in Yy1 deficient HSCs with wild-type HSCs. Methods: Total RNA was purified from bone marrow HSCs (Lin-Sca1+c-Kit+ CD48-CD150+and Lin-Sca1+c-Kit+CD48-CD150-) sorted from Yy1-/- versus Yy1+/+ mice using RNeasy Micro kit (Qiagen).  Sequencing libraries were prepared by using Ovation RNA-Seq System V2 (NuGen) according to the manufacturer's specifications and were sequenced by an Illumina HiSeq 2000. Sequencing reads were adapter and quality trimmed using the Skewer trimming program.  Quality reads were subsequently aligned to the annotated reference genome using Subjunc aligner from subread package. qRT–PCR validation was performed using SYBR Green assays. Results: This analysis identified a small cohort of 100 significantly upregulated and 49 downregulated genes in Yy1-/- HSCs (FDR<0.05, logFC>=1).  Among them, many genes involved in cell cycle were deregulated in Yy1-/- HSCs .  Gene ontology analysis of the upregulated cohort revealed a significant enrichment of genes involved in cell cycle, cell division, chromosome condensation and segregation, cytoskeleton and spindle organizations.  Gene Set Enrichment Analysis (GSEA) revealed cell cycle gene sets associated with pre-replication complex (RC) assembly, degradation of p27, DNA replication, G1 to S phase, and M to G1 phase transitions were significantly enriched.  The RNA-seq analysis revealed that YY1-deficient HSCs had an aberrant genetic network associated with G0 to G1 phase entry. Overall design: Total RNA was purified from bone marrow HSCs (Lin-Sca1+c-Kit+CD48-CD150+and Lin-Sca1+c-Kit+CD48-CD150-) sorted from Yy1-/- versus Yy1+/+ mice, and used for RNA-seq analysis.