Epigenetic signals and microRNAs (miRNAs) are key regulators of gene expression in developmental programs. Their alterations are at the basis of neoplastic transformation. We reported that during granulocytic differentiation of human hematopoietic stem/progenitor cells (HPC), miR-223 locates into the nucleus where it assembles a chromatin remodelling complex, including Polycomb, Argonaute and Dicer proteins, on complementary sequences of its post-transcriptional target NFI-A gene promoter. NFI-A directs erythroid differentiation, its transcriptional silencing by miR-223 induces myeloid differentiation. This evidence suggests that during HPCs lineage fate determination, miRNAs interact with matching sequences on developmentally regulated gene promoters, providing the driving force for the DNA targeting activity of chromatin modifiers. In addition, our preliminary ChIP-seq data showed an overlap between miR-223 occupancy and H3K4me3 and H3K27me3 marks in myeloid cell lines undergoing granulocytic differentiation. Among the genes marked by H3K27me3 and miR-223-Cy5 we found NFIA, according to our previous published data. Major aim of this project is to study how miRNA-DNA interactions impact in the differentiation program of HPCs and myeloid cell lines, in comparison with the corresponding acute myeloid leukemia (AML) blast phenotypes. We plan to address: i) the genes transcriptionally regulated by miR-223 and their DNA and chromatin status; ii) the biological consequences in HPCs fate decision of the expression/silencing of miR-223 and their transcriptional targets, and the efficacy of treatment with chromatin remodelling agents in normal and leukemic cells.
The identification of novel transcriptional pathways and genes epigenetically regulated by miR-223 may provide; i) novel markers for the diagnosis and prognosis of AML; ii) the basis for development of novel targeted therapies; iii) the background for further exploration and validation in other cancer models.
Research on hematological disorders has served as a pioneer for all branches of cancer research. Advances obtained in these diseases, such as chemotherapy, transplantation and targeted therapies were extended to other malignancies. The next frontier in hematological and oncological research will be to examine at the molecular level how specific chromatin signatures influence gene expression. Some of these signatures are transient while others, such as those involved in epigenetic silencing of developmentally regulated genes, are more stable and require several cell divisions to be fully implemented or reversed. The reversal of aberrant epigenetic regulation by agents influencing regulators of chromatin structure appears as a promising objective for the development of novel therapeutic strategies in onco-hematologic diseases.
We have recently shown that miRNAs have epigenetic functions and are involved in transcriptional regulation of genes directing cell lineage and fate. These data and results from other groups are suggestive of a mechanism whereby epigenetic silencing complexes are guided by miRNAs to homology-containing loci in the genome.
Our project is aimed at characterizing the functional relevance of the interaction of nuclear miRNAs with DNA in normal and neoplastic hematopoietic model systems. This work connects two previously unrelated research fiels: the nuclear function of miRNAs and Polycomb-mediated silencing of gene expression. Our research has the potential to radically change the view of miRNA functions and to identify novel roles for miRNAs in cell differentiation, lineage choice and cancer. Moreover, this research represents a technical advance in several aspects. We recently established novel methods for miRNA-chromatin immunoprecipitation/sequencing, for fluorescent miRNA transfection and intracellular detection by confocal microscopy and for miRNA in situ hybridization for the identification of miRNAs intracellular localization. We will also extend our research towards clinical applications by identifying in clinical samples the miR-223-regulated genes and exploring the effects of clinically active drugs on the transcriptional activities of miRNAs. This new area of research has immense potential with regards to targeted pharmacological interventions aimed at controlling the epigenetic state of specific genes.