Somatic stem cells are the basic tools of regenerative medicine and gene therapy, providing unique opportunities for the therapy of currently untreatable 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.
The rapidly expanding information on the structural and functional characteristics of the human genome allows the development of genome-wide approaches to the understanding of the molecular circuitry wiring the genetic and epigenetic programs of somatic stem cells. High-throughput approaches are essential to study the transcriptome, the epigenome and the usage of regulatory elements in the genome.
The objective of this thesis is to define the transcriptional and epigenetic profile of cord blood-derived human hematopoietic stem/progenitor cells (HSPC) and their committed, myeloid and erythroid progeny .
For this purpose, high-throughput technologies were exploited such as Cap Analysis of Gene Expression (CAGE) and deep CAGE tags sequencing, which allow the genome-wide mapping of active promoters and transcription start sites (TSSs) and the measurement of specific transcript abundance and Chromatin Immunoprecipitation coupled to deep sequencing technology (ChIP-Seq), for a genome-wide analysis of chromatin signatures typical of regulatory elements.
CAGE analysis enabled us to define >9,000 active promoters in HSPC and in erythroid and myeloid precursors. Around 500 transcripts in both HSPC and committed precursors were generated by the usage of alternative promoters, in some cases in a lineage-specific fashion.
The different cell types shared most of the promoters, suggesting that the transcriptional state is largely maintained in early hematopoietic progenitors and precursors. Therefore, only a relatively small number of differentially used promoters define the identity of hematopoietic cells at different stages of differentiation.
85% of the active promoters in each cell type were associated with known genes, whereas 15% of the transcripts identified by CAGE were classified as novel. Surprisingly, a significantly higher proportion (around 30%) of cell-specific promoters was not annotated. These novel transcripts are possibly involved in HSPC 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 active promoters and enhancers, H3K4me3 and H3K4me1 (histone H3 lysine 4 trimethylation and monomethylation), respectively. More than 15,000 H3K4me3 islands and >55,000 H3K4me1 islands (putative promoters and enhancers, respectively) were discovered in all cell types analyzed.
As expected, most of the promoters identified by CAGE overlapped with H3K4me3 peaks. Interestingly, a small fraction of TSSs was found in regions highly enriched in H3K4me1, indicating the presence of potential "enhancer RNA" recently described in a murine model of neuronal differentiation (Kim et al., Nature, 2010). Finally, a large fraction of the putative enhancers defined as H3K4me1 islands, contained binding sites for hematopoietic master regulators, such as GATA1.
Overall, this study provided an overview of the differential transcriptional programs of HSPC 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 cells and their progeny.