Deciphering Dynamics Of Epigenome And 3D Genome During Mouse Embryonic Hematopoietic Stem Cell Formation

Hematopoietic stem cells(HSCs)are a type of pluripotent stem cells with selfrenewal and multi-lineage differentiation capabilities,which can not only maintain a stable number of HSCs in the body through self-renewal,but also rebuild the entire hematopoietic system through the function of multi-lineage differentiation.Definitive HSCs arise from aortic endothelium precursor called hemogenic endothelial cells(HEC)through endothelial-to-hematopoietic transition(EHT)in aorta-gonad-mesonephros(AGM)region in E10.5-E11.5 proved by vital imaging study and lineage tracing experiment.HEC,which is derived from a subset aortic endothelium according recent research,can give rise to pre-hematopoietic stem cells(pre-HSCs)and ultimately form mature HSCs(LT-HSCs).Endothelial-hematopoietic reprogramming is accompanied by various regulation including DNA and RNA methylation,lnc RNA,histone modification,transcriptional factor and 3D genome by reshaping gene expression.Transcription factors,interacting with chromatin-modifying and remodeling-related proteins,binds to chromatin when chromatin is accessible,thereby exerting epigenetic regulation.Runx1,the core transcription factor that regulates the ontogeny of HSC,is not only the target of epigenetic modifier that post-translationally modify Runx1 protein to influence selfexpression at the transcriptional level by altering the interaction with activating or repressive co-factors.Runx1 is also able to recruit co-activator or co-repressors to the promoter region of target genes,resulting in perturbation of the chromatin environment to regulate gene expression at the epigenetic level.In addition,previous research has found that Runx1 facilitate the chromatin loop between regulatory elements to regulate hematopoietic development at the level of chromatin architecture.Thus,understanding the regulatory mechanism of Runx1 and the hierarchical regulation of multi-omics during HSC formation is pivotal for HSC regeneration and treatment of hematopoietic malignancies.To decipher the dynamics of epigenetic regulation and change of 3D genome structure during HSC formation in mice embryos,we performed low input sis Hi-C,mini ATAC-seq and It ChIP-seq to profile the multi-omics landscape of four key cell populations related to HSC formation including early arterial endothelial cells(early AEC),hemogenic endothelial cell(HEC),pre-hematopoietic stem cells(pre-HSC)and definitive hematopoietic stem cells(LT-HSC)and decipher the regulation hierarchy of epigenetic regulation and 3D genome structure.Further,we precisely screened the potential transcription factors cooperating with Runx1 to regulate HSC formation by integrating multi-omics,which provides vital clues for the systematic analysis of the precise regulatory mechanism of HSC development.We first profiled the epigenetic landscape of early AEC,HEC,pre-HSC and LT-HSC,and identified cell-specific enhancers and potential co-factors of Runx1 such as Gata2,PU.1 and Stat4 in enhancer regions to promote HSC development.We then delineated dynamic changes in chromatin accessibility during HSC formation.By integrating information of Runx1 binding site,the region enriched in H3K27 ac modification and chromatin accessibility,we pinpointed transcription factors(TFs)that cooperated with Runx1 to bind to open enhancer regions on the genome to regulation hematopoiesis.At the same time,we also deciphered the regulation relationship between chromatin accessibility and gene expression,and found that hematopoiesis-related genes have been bound by TFs to promote low-level expression of genes before fully chromatin opening.After recruiting transcription initial complex,the degree of chromatin accessibility increased as gene transcription enhanced.In addition,by integrating the dynamic changes of 3D genome and epigenetic regulation during HSC formation,we dissected the regulatory hierarchy of histone modifications and 3D genomic changes in hematopoiesis and found that hematopoietic fate commitment has occurred in early AEC stage as enhancers of hematopoiesis-related genes have already been activated and been bound by Runx1.Under the binding of Runx1,chromatin structure has dramatic change in HEC stage,which promotes the curved folding of hematopoiesis-related enhancer towards the promoter,resulting in the increasing of enhancer-promoter interactions(EPIs),thereby regulating the hematopoietic development process.Through combining Runx1 ChIP-seq and EPI information,we also screened out TFs cooperated with Runx1 to mediated EPI to regulate hematopoiesis-related genes expression,which may provide crucial clues to reprogram endothelial cells to HSC in vitro.