NEXT-GENERATION SEQUENCING APPROACH FOR TRANSCRIPTOME AND EPIGENOME DEFINITION OF HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS AND THEIR EARLY ERYTHROID AND MYELOID COMMITTED PROGENY

Somatic stem cells are the basic tools of regenerative medicine and gene therapy, providing unique opportunities for the therapy of genetic and acquired disorders. The molecular mechanisms underlying fundamental characteristics of human somatic stem cells, such as self-renewal, commitment and differentiation, are still poorly understood. A better knowledge of these mechanisms is crucial to the understanding of stem cell biology and to the development of stem cell-based therapies. High-throughput approaches, such as next-generation sequencing technologies (NGS) became fundamental to study the transcriptome, the epigenome and the usage of regulatory elements in the genome. Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Here, we define the transcriptional and epigenetic profile of human hematopoietic stem/progenitor cells (HSPC) and early myeloid and erythroid progenitors through an integrative analysis of Cap Analysis of Gene Expression (CAGE) and chromatin immuno- precipitation (ChIP-seq) data, in order to identify transcription regulatory elements that act in HSPC lineage commitment. CAGE analysis enabled us to define more than 10,000 active promoters in HSPCs and in erythroid (EPP) and myeloid precursors (MPP). The different cell types shared most of the promoters, with only a small fraction (about 4%) being differentially transcribed, suggesting that the transcriptional state is largely maintained in early hematopoietic progenitors and precursors. Interestingly about 30% of the identified of cell-specific promoters was not annotated. These novel transcripts are possibly involved in HSPCs self- renewal, commitment and differentiation. To obtain a genome-wide description of the transcriptional regulatory regions in multipotent and lineage-restricted hematopoietic progenitors, we performed ChIP-seq analysis for histone methylations typical of promoters and enhancers, H3K4me3 and H3K4me1, respectively, and for H3K27ac to mark the active elements. Overall we identified more than 20,000 active enhancers that consistently changed upon erythroid and myeloid commitment: about 80 and 95% of the active enhancers mapped in HSPC disappeared in erytrhoid and myeloid progenitors, respectively, while novel enhancers are acquired during lineage commitment. These data indicate that enhancers are dramatically redefined during lineage commitment, and that differential enhancer usage is responsible for the differential regulation of promoter activity underlying lineage restriction. This study provided an overview of the differential transcriptional programs of HSPCs and committed myeloid and erythroid hematopoietic precursors and represents a unique source of genes and regulatory regions involved in self-renewal, commitment and differentiation of human hematopoietic stem/progenitor cells and their progeny.