Chromatin and gene regulation during development
- Vasco Meneghini
- Chiara Antoniani
- Oriana Romano
- Tristan Félix
- Giacomo Frati
- Megane Brusson
- Anne Chalumeau
- Charlotte Calvo
- Clara Wollenschlaeger
Miccio A, In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia Proc Natl Acad Sci U S A 2008 105(30):10547-52
Miccio A.*, NuRD mediates activating and repressive functions of GATA-1 and FOG-1 during blood development EMBO J 2010 2010 Jan 20;29(2):442-56. Epub 2009 Nov 19
BLOBEL GA., Role of the GATA-1/FOG-1/NuRD pathway in the expression of human beta-like globin genes Mol Cell Biol 2010 30 (14) :3460-70
Cavazza A., Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation. Stem Cell Reports. 2016 Mar 23. pii: S2213-6711(16)00084-9. doi: 10.1016/j.stemcr.2016.03.003.
Romano O., Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment. Scientific Reports, 2016, Apr 20;6:24724. doi: 10.1038/srep24724
Dynamics of transcriptional and epigenetic networks during hematopoietic development
The rapidly expanding information on the structural and functional characteristics of the human genome allows the development of comprehensive, genome-wide approaches to the understanding of the molecular circuitry wiring stem cell genetic and epigenetic programs. The aim of our project is the genome-wide definition of the entire set of regulatory sequences used by rare human hematopoietic stem/progenitor cells and their lineage-restricted erythroid progeny at different stages of differentiation.
The definition of the genetic and epigenetic programs is achieved through the use of a number of genomic and bioinformatic tools, including RNA-seq, deepCAGE, Retroviral scanning and ChIP-Seq. We analyze genome-wide the binding sites for hematopoietic transcription factors (e.g., GATA1 and GATA2) and their co-factors, and the epigenetic histone modifications associated to transcription or silencing to define regulatory regions involved in hematopoietic stem cell biology and in erythroid commitment and differentiation.
Validation of putative regulatory regions is performed by CRISPR-Cas9 targeted disruption and chromatic conformation capture assays. The expected outcome of this research is a better understanding of the molecular basis of stemness and erythroid commitment of clinically relevant stem cells, which will provide a knowledge basis for safer and more efficient usage of stem cells in cell and gene therapy.
Molecular-based approaches for the treatment of β-Hemoglobinopathies
The definition of regulatory regions controlling the gene expression programs is fundamental for understanding the molecular mechanisms underlying many diseases and for the development of novel therapeutic approaches. As an example, numerous disease-associated sequence variations occur in cis regulatory elements, which represent in some cases potential therapeutic targets.
Sickle cell disease (SCD) and β-thalassemias are genetic diseases caused by mutations in the gene coding for the adult hemoglobin β-chain. They represent the most common monogenic disorders worldwide, affecting thousands of newborn annually. In β-thalassemia, the reduced production of adult β-chains causes α-globin precipitation and red blood cell death. In SCD, a single aminoacid substitution in the β-globin chain leads to polymerization of the sickle hemoglobin (HbS) and red blood cell deformation. β-globin disorders may lead to a severe clinical phenotype characterized by anemia, pain crises, and organ damage. So far, the only curative treatment is represented by bone marrow transplantation from a compatible donor, which, however, is available to less than 30% of the patients. Experimental treatments include gene therapy and pharmacological intervention. In the latter approach, efforts are underway to identify compounds that raise the expression of the fetal gamma-globin genes. The rational for this treatment is based on the long-standing observation that patients harboring mutations that trigger elevated gamma-globin expression, experience a more benign clinical course of the disease. However, pharmacological treatments are not equally effective for all patients, are associated with a considerable toxicity and do not represent a definitive treatment. Several nuclear factors, such as the erythroid master regulator GATA1, its cofactors FOG1 and BCL11A and the NuRD repressor complex, are implicated in the silencing of gamma-globin expression. However, their role in erythroid development and hemoglobin switching has yet to be completely elucidated.
The goal of our research is to provide the basic scientific knowledge for developing safe therapies for SCD and β-thalassemias based on genome editing approaches aimed at increasing gamma-globin expression. We aim at characterizing the transcription factors and the regulatory genomic elements that control the switch from fetal to adult globin gene expression. The fine mapping of regulatory elements involved in hemoglobin switching will provide potential targets for therapeutic induction of fetal hemoglobin. Our studies are focused on the molecular mechanisms underlying the β-to-gamma-globin switching as well as on the evaluation of the efficacy and safety of these therapeutic approaches. We apply novel molecular techniques (e.g. genome-wide genomic analyses and CRISPR/Cas9 technology) by using different cellular models, including clinically relevant hematopoietic stem cells.