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Epigenetics and embryonic stem cell development

Salvatore Oliviero - PI

Main group members

  • Andrea Lauria, Livia Caizzi Researchers

  • Isabelle Polignano, Hassan Dastsooz, Fang Yang, Annalaura Tamburrini Postdoctoral fellow

  • Mirko Giuseppe Scrivano, Claudia Vaccari, Chiara Cicconetti, Jacopo Pinto PhD students

  • Francesca Anselmi, Daniela Donna Technicians

  • Alessandro Salamone, Marco Gaspari Fellows

Research activity

Our research group combines molecular, cellular, and global genomic approaches to investigate the network of regulations that take place in response to environmental or developmental signals and that are deregulated in developmental disorders or in cancer. We study the regulation and function of epigenetic modifications to understand their role in the control of cell identity, the regulation of the correct transitions during the process of embryonic development, and they alteration in tumorigenesis. DNAme is introduced by the de novo DNMT3A and DNMT3B DNA methyltransferases together with DNMT3L, while DNMT1 together with UHRF1 are mainly involved in the propagation of the DNAme on the genome during DNA replication. The exit from pluripotency is characterised by the transition from the pre- to the post-implantation epiblast of the embryo. To analyse the gene expression patterns of the EBs at the resolution of individual cells, we collected samples at 3 and 9 days during EBs differentiation from WT and mutant cells and profled their gene expression by single-cell RNA sequencing (scRNAseq) (Figure1). 

Figure 1

During this transition genome-wide DNAme is established by the de novo DNMT3A and DNMT3B DNA methyltransferases which are strongly upregulated to establish the DNAme essential for cell fate specification during development and transcription integrity. By whole genome DNAme coupled with gene expression and cell phenotype analysis we demonstrated a specific role of DNMT3B, but not DNMT3A, in the meso-endoderm (ME) lineage specification and identified DNMT3B genomic targets that prime EpiLCs toward ME. Thus, DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provides functional characterization of the de novo DNMTs during EpiLCs lineage determination. To differentiate into meso-endoderm, EpiLCs should repress the chromatin of a number of ectoderm enhancers that at this stage are open and demethylated and should be decommissioned in the cells primed to be able to differentiate into meso-endoderm. DNMT3B-dependent DNAme establishes the meso-endodermal epigenetic landscape by repressing the expression of key TFs that would otherwise induce the default differentiation into neuroectoderm. Recently, it has become clear that nuclear long non-coding RNAs (lncRNAs) might be involved in gene transcription by interacting with nuclear factors and epigenetic modifiers. Whole-genome screens have shown that a large fraction of lncRNAs play a role in development. We recently found that histone acetyltransferase p300/ CBP, a general transcriptional coactivator that introduces the H3K27ac modification on enhancers of active genes, interacts with over a hundred of lncRNAs. We found that lncSmad7, a lncRNA specifically expressed in embryonic stem cells, is required to maintain maintains ESC self-renewal (Figure 2).  

lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. By Chromatin Isolation by RNA precipitation followed by sequencing (ChIRPseq) together with CRISPR/Cas9 mutagenesis of the target sites we demonstrated that lncSmad7 binds and recruits p300 to enhancers to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.

Figure 2

Projects in progress in the laboratory address the study of epigenetic modifications that determine embryonic stem cells fate in particular to neural development of human embryonic stem cell (hESCs) differentiation into 3D brain organoids. In particular we identified a number of lncRNAs and nuclear factors that are specifically expressed along the neural differentiation pathways or expressed in terminally differentiated neurons. By the use of CRISPR/Cas technology we will dissect their role in neural differentiation. We will analyse the network of gene regulation s and epigenetic landscape along neural differentiation. 

  • Telethon GGP19201A (2018-2023)

  • PRIN 2022: 2022RA8E3T; 2020: 2020HEFHY2; 2017: 2017P352Z4

  • AIRC: IG2017 ID 20240; IG 2022 ID 2715

Neri F, Rapelli S, Krepelova A, Incarnato D, Parlato C, Basile G, Maldotti M, Anselmi F, Oliviero S. (2017) Intragenic DNA methylation prevents spurious transcription initiation. Nature 543: 72-77. DOI: 10.1038/ nature21373 codice SCOPUS: 2-s2.0-85014546354 

Incarnato D, Morandi E, Simon LM, Oliviero S. (2018) RNA Framework: an all-in-one toolkit for the analysis of RNA structures and post-transcriptional modifications. Nucleic Acids Res. 46: e97. DOI: 10.1093/nar/ gky486 codice SCOPUS: 2-s2.0-85066849476 

Simon LM, Morandi E, Luganini A, Gribaudo G, Martinez-Sobrido L, Turner DH, Oliviero S*, Incarnato D*. (2019) In vivo analysis of influenza A mRNA secondary structures identified critical regulatory motifs. Nucleic Acids Res. 47: 7003-7017.DOI: 10.1093/nar/gkz318 codice SCOPUS: 2-s2.0-85070182306 

Lauria A, Peirone S, Giudice MD, Priante F, Rajan P, Caselle M, Oliviero S, Cereda M.U (2020) Identification of altered biological processes in heterogeneous RNA-sequencing data by discretization of expression profiles. Nucleic Acids Res. 48:1730-1747. DOI: 10.1093/nar/gkz1208 codice SCOPUS: 2-s2.0- 85081097473 

Di Timoteo G, Dattilo D, Centrón-Broco A, Colantoni A, Guarnacci M, Rossi F, Incarnato D, Oliviero S, Fatica A, Morlando M, Bozzoni I. (2020) Modulation of circRNA Metabolism by m 6 A Modification. Cell Report 31:107641. DOI: 10.1016/j.celrep.2020.107641 codice SCOPUS: 2-s2.0-85084373798 

Zorzan I, Pellegrini M, Arboit M, Incarnato D, Maldotti M, Forcato M, Tagliazucchi GM, Carbognin E, Montagner M, Oliviero S*, G. Martello* (2020) The transcriptional regulator ZNF398 mediates pluripotency and epithelial character downstream of TGF-beta in human PSCs. Nature Commun. 11: 2364. DOI: 10.1038/s41467-020-16205-9 codice SCOPUS: 2-s2.0- 85084455816 

Betto RM, Diamante L, Perrera V, Audano M, Rapelli S, Lauria A, Incarnato D, Arboit M, Pedretti S, Rigoni G, Guerineau V, Touboul D, Stirparo GG, Lohof T, Boroviak T, Grumati P, Soriano ME, Nichols J, Mitro N, Oliviero S*, G. Martello* (2021) Metabolic control of DNA methylation in naive pluripotent cells. Nature Genetics 53 :215-229.DOI: 10.1038/s41588-020-00770-2 codice SCOPUS: 2-s2.0-85100264695 

Morandi E, Manfredonia I, Simon LM, Anselmi F, van Hemert MJ, Oliviero S*, Incarnato D*. (2021) Genome-scale deconvolution of RNA structure ensembles. Nature Methods, 18: 249-252 DOI: 10.1038/ s41592-021-01075-w codice SCOPUS: 2-s2.0- 85101203392 

Maldotti M, Lauria A, Anselmi F, Molineris I, Tamburrini A, Meng G, Polignano IL, Scrivano MG, Campestre F, Simon LM, Rapelli S, Morandi E, Incarnato D, Oliviero S. (2022) The acetyltransferase p300 is recruited in trans to multiple enhancer sites by lncSmad7. Nucleic Acids Res. 50: 2587-2602. DOI: 10.1093/nar/gkac083 codice SCOPUS: 2-s2.0-85127541928 

Lauria A, Meng G, Proserpio V, Rapelli S, Maldotti M, Polignano IL, Anselmi F, Incarnato D, Krepelova A, Donna D, Levra Levron C, Donati G, Molineris I, Neri F, Oliviero S. (2023) DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells. Nat Commun. 14(1):367. DOI: 10.1038/s41467-023- 35938-x codice SCOPUS: 2-s2.0-85146771241 

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