Research project Epigenetics of genome programming and reprogramming

Giuseppe Testa

The focus of the lab is on the epigenetic mechanisms that enable lineage commitment and their aberrations in cancer.

In his classic representation of the epigenetic landscape, Conrad Waddington depicted development as the progressive channeling of pluripotency (the marble at the top of the hill) down irreversible paths of cell specification (the slopes and canyons available to the marble in its downward rolling). A current version of that same landscape brings to the fore the fate choices of embryonic and tissue-specific stem cells as key transitions for the regulation of self-renewal and differentiation that forms and maintains organisms. In order to understand these transitions we need to uncover how genomic programs are progressively deployed and what are the chromatin regulatory mechanisms that coordinate their deployment.
Among these, we have started to learn in the last decade that the methylation of histone H3 on lysine tails 4 and 27, respectively mediated by the Trithorax (Trx) and Polycomb (PcG) protein families, is central to the programming of genomes that underlies the establishment and maintenance of differentiated cell states. Not surprisingly, aberrations in these pathways have also emerged as important determinants or modulators of tumors, hinting at common regulatory circuits that preside over stem cell physiology and that are perturbed or hijacked in oncogenesis. Finally, changes in these posttranslational modifications are also prominent in the epigenetic rewiring that recently reversed Waddington’s unidirectional slopes, namely the reacquisition of pluripotency from differentiated cells through nuclear transfer or the expression of few pluripotency factors.
Hence, work in my lab is articulated in three complementary lines of research that investigate PcG and Trx function in: i) the physiology of genome programming during differentiation; ii) the aberrant genome programming that accompanies tumorigenesis; and iii) the controlled genome reprogramming that mediates induced pluripotency.


1. Polycomb and trithorax in the physiology of genome programming

The connection between histone lysine methylation and developmental fate became apparent with the realization that Ezh2, a member of the Polycomb group (PcG) of proteins first discovered in the fly as stable repressors Hox genes, catalyzes the trimethylation of histone H3 on lysine 27 (H3K27me3) while Trx (and its mammalian homologs of the Mll family), identified in the fly as a stable activator of Hox genes, catalyzes the trimethylation of histone H3 on lysine 4 (H3K4me3). In ES cells and some adult stem cells most PcG target genes are kept in a repressed state but poised for activation by a bivalent chromatin signature that features both H3K4me3 and H3K27me3. Upon differentiation many of these bivalent domains are resolved and their genes become either completely active (marked solely by H3K4me3) or definitely repressed (marked solely by H3K27me3) in a lineage specific fashion. Contrary to the long held assumption that HLM was irreversible and that this irreversibility underlined lineage stability, research over the last years revealed the existence of histone lysine demethylases (HDMs) as key effectors of the dynamic regulation of HLM, suggesting that the establishment and maintenance of cell lineages involves a regulated process of addition and removal of methyl marks. In particular, following the identification of Jmjd3 as a histone H3 lysine 27 demethylase (De Santa et al. 2007), we determined that it is required for the neural commitment of ES cells by resolving bivalent domain of key drivers and markers of neurogenesis (Burgold et al. PLoS One 2008) (Fig. 2).

Our objective is now to understand how the epigenetic axis centered on H3K27 methylation and its downstream silencing steps regulate distinct phases of differentiation as well as the maintenance of the differentiated state. To this end, we harness the power of conditional mutagenesis to modulate the activity of key members of this epigenetic cascade using neurogenesis as a model system. Convergent lines of evidence point to the key role of the PcG and Trx pathways in the acquisition of neural fates, and we are pursuing both in vitro and in vivo approaches to dissect how these epigenetic circuits orchestrate neurogenesis.


2. Polycomb in the aberrant genome programming of cancer

Consistent with the role of PcG in lineage choices, alterations in H3K27me3 are likely to be early events in the cascade of epigenetic aberrations of cancer, particularly the hypermethylation of CpG promoters that is an important mechanism of tumor suppressor inactivation. Convergent lines of evidence indicate that CpG hypermethylation in cancer cells is the result of an instructive process through which altered developmental programs determine aberrant hypermethylation at multiple loci. Throughout development de novo DNA methylation proceeds in an instructive fashion and the majority of genes that are hypermethylated in cancers are pre-marked by H3K27me3 in ES cells. The recent observation that most epithelial cancers share a core transcriptional signature with ES cells corroborates this model and suggests that the PcG-dependent gene expression program that orchestrates development in normal cells is hijacked in cancer cells as the main template for cancer DNA methylation. Finally, the oncogenic role of H3K27me3 imbalances is underscored by the direct involvement of various PcG members in human and experimental tumors, of which the two best characterized examples are Ezh2 and Bmi1, the component of Polycomb repressive complex 1 (PRC1) that stimulates ubiquitination of histone H2A lysine 119 (H2AK119) following H3K27me3 by Ezh2.
Hence, this line of research in the lab explores the proposition that loss of the physiologic regulation centered around H3K27me3 is important for the initiation and/or maintenance of tumors, combining the conditional modulation of this epigenetic axis in advanced murine models of cancer that recapitulate faithfully the pathogenesis of the corresponding human tumor.


3. Polycomb and Trithorax in controlled reprogramming and induced pluripotency

The recent derivation of pluripotent stem cells (iPS) from adult somatic cells represents a paradigmatic model of cell fate reassignment that makes the epigenetic rewiring of differentiation states amenable to genetic and biochemical studies.
Our objective is to define how the epigenetic pathways that underlie lineage acquisition operate in the context of cell fate reassignment, and what are the epigenetic windows of sensitivity during reprogramming.