Genetically encoded multivalent sensors to detect bivalent epigenetic modifications in living stem cells

Eukaryotic DNA is organized in the form of chromatin whose basic unit is the nucleosome. The four core histones forming the nucleosome, H2A, H2B, H3 and H4 can be highly post-translationally modified, especially on their N-terminal tail protruding from the nucleosome particle. Histone post-translational modifications (PTMs) work combinatorially to establish chromatin states defined by specific gene expression status. Found in embryonic stem cells (ESCs) at promoters of key developmental genes, bivalent chromatin is the combination of the active chromatin mark, trimethylation of lysine 4 on histone H3 (H3K4me3) and the repressive mark, H3K27me3. Established and maintained by Polycomb (Pc) and Trithorax (Trx) proteins, bivalency is proposed to keep gene transcription repressed but poised for activation. How bivalent domains are organized within the nucleus and how it is installed by Pc and Trx is still unkown. In this work, we aim to answer these questions by designing probes that allow live cell imaging of bivalent domains and by studying the establishment and removal of H3K4 methylation on nucleosomes. The current lack of live cell imaging methods for PTM patterns prompt us to engineer genetically encoded sensors which bind to bivalent marks in a multivalent fashion. These sensors contain a fluorescent protein and two reader domains, joined by flexible linkers. Their selectivity for bivalent nucleosomes was tested in a pulldown assay with a library of differently modified reconstituted nucleosomes. For this, we obtained site-specifically modified histones via expressed protein ligation. The best probe was then applied in live ESCs to visualize bivalent domains. Subsequent imaging by confocal microscopy revealed the organization of bivalent chromatin into discrete and local clusters. Furthermore, this probe was employed to monitor loss of bivalency upon treatment with a small molecule epigenetic inhibitor. Then, we studied the histone-lysine N-methyltransferase Set1B and lysine-specific histone demethylase 1A (LSD1), enzymes which deposit and remove methyl groups of H3K4 respectively. We measured a mid-micromolar affinity for LSD1 to reconstituted nucleosomes using microscale thermophoresis. This interaction might have an impact on the recruitment of LSD1 to its target genes. We then measured the activity of Set1B complex on symmetrically modified H3K27me3 nucleosomes and on asymmetrically modified H3K4me3 nucleosomes. We showed that H3K27me3 does not influence the activity of Set1B complex whereas H3K4me3 activates Set1B-mediated deposition of methyl groups at K4 on the opposite H3 tail. This findings might have important implications concerning the establishment of bivalent chromatin. Together these results gave insights about multivalent binding of tandem reader domains, subnuclear bivalent chromatin organization and establishment of bivalent marks. In the future, we envisage to develop multivalent sensors for other PTM patterns of biological interest.

A Tale of Two Tumors: Unraveling the Complexities of Epigenetic Dysregulation in Cancer

Maria Christine Donaldson

Epigenetics plays an important role in cancer development and progression. Cancer cells hijack the epigenome by modifying the histone protein units responsible for packaging DNA, or by modifying the DNA itself, resulting in changes to chromatin topology and transcriptional programming within the cell. In this thesis, I report our investigation to uncover the mechanisms of epigenetic regulation and how epigenetic control of transcriptional programming goes awry in cancer. We investigate these processes in two separate contexts. In our first study, we investigate the mechanisms by which EZH2 oncogenic mutations alter structure and function of topologically associating domains in non-Hodgkin lymphoma. The process of chromatin folding leads to a systematic arrangement of hierarchical structural and regulatory elements found within the nucleus. A topologically associating domain (TAD) is a regulatory unit of self interacting DNA, where the transcription of the genes within a TAD is usually dictated by the presence of active (H3K36me3) or inactive (H3K27me3) histone marks. In cancer, the somatic point mutation in EZH2Y646X leads to a genome wide increase in H3K27me3, associated with transcriptional repression. While this alteration leads to changes in the global epigenetic status of the cell, its impact on chromatin organization and TAD-related function remain unclear. By combining transcriptomics and epigenetics with analyses of chromatin structure, we demonstrate a functional interplay between TADs and the epigenetic and transcriptional program of the genes found within them. This balance is altered by EZH2Y646X, leading to the synergistic silencing of entire domains directly targeting cell differentiation and tumor suppressive programs. A closer look reveals that the silencing of tumor suppressive TADs are coupled with structural modifications and changes in promoter interactions within EZH2Y646X target TAD6.139. Impressively, the TADâs transcriptional and epigenetic programs are restored by pharmacological inhibition of EZH2Y646X. Our results indicate that EZH2Y646X alters the topology and function of chromatin domains to promote synergistic oncogenic programs. In our second study, we dissect the epigenetic, transcriptomic, and metabolic signaling dependencies using a novel model of central nervous system primitive neural ectodermal tumors (CNS-PNETs). Central nervous system (CNS) tumors are the leading cause of cancer-associated death in children. Primitive neural-ectodermal tumors (CNS-PNETs) are a particularly aggressive subtype of embryonal CNS-tumor, with a five-year overall survival rate in less than 50% of patients. Despite sharing a similar cell of origin of other CNS tumors, CNS-PNETs have a different anatomical location, unique genetic and epigenetic features, and significantly worse clinical outcome. In addition, a lack of in vivo models for studying CNS tumors challenges the opportunity to dissect the genetic variables that underlie the origin of these tumors. We developed a novel CNS-PNET mouse model, called CNS-NPCs, using neural progenitor cells derived from human-iPS cells. Through histological, DNA methylation, and RNA sequencing analyses, we find that the CNS-NPC model recapitulates the the morphologic, epigenetic, and transcriptomic features of primary CNS-PNETs. In addition, through in vivo metabolic analyses of CNS-NPCs and the patient derived cell line, PFSK-1, we identified dysregulation in the neurotransmitter...

EPFL2019

The Pygo2-H3K4me2/3 interaction is dispensable for mouse development and Wnt signaling-dependent transcription

Michel Aguet

Pygopus has been discovered as a fundamental Wnt signaling component in Drosophila. The mouse genome encodes two Pygopus homologs, Pygo1 and Pygo2. They serve as context-dependent beta-catenin coactivators, with Pygo2 playing the more important role. All Pygo proteins share a highly conserved plant homology domain (PHD) that allows them to bind di- and trimethylated lysine 4 of histone H3 (H3K4me2/3). Despite the structural conservation of this domain, the relevance of histone binding for the role of Pygo2 as a Wnt signaling component and as a reader of chromatin modifications remains speculative. Here we generate a knock-in mouse line, homozygous for a Pygo2 mutant defective in chromatin binding. We show that even in the absence of the potentially redundant Pygo1, Pygo2 does not require the H3K4me2/3 binding activity to sustain its function during mouse development. Indeed, during tissue homeostasis, Wnt/beta-catenin-dependent transcription is largely unaffected. However, the Pygo2-chromatin interaction is relevant in testes, where, importantly, Pygo2 binds in vivo to the chromatin in a PHD-dependent manner. Its presence on regulatory regions does not affect the transcription of nearby genes; rather, it is important for the recruitment of the histone acetyltransferase Gcn5 to chromatin, consistent with a testis-specific and Wnt-unrelated role for Pygo2 as a chromatin remodeler.

Company Of Biologists Ltd2013

Protein phosphatase 1 regulates the histone code for long-term memory

Johannes Gräff

Chromatin remodeling through histone posttranslational modifications (PTMs) and DNA methylation has recently been implicated in cognitive functions, but the mechanisms involved in such epigenetic regulation remain poorly understood. Here, we show that protein phosphatase 1 (PP1) is a critical regulator of chromatin remodeling in the mammalian brain that controls histone PTMs and gene transcription associated with long-term memory. Our data show that PP1 is present at the chromatin in brain cells and interacts with enzymes of the epigenetic machinery including HDAC1 (histone deacetylase 1) and histone demethylase JMJD2A (jumonji domain-containing protein 2A). The selective inhibition of the nuclear pool of PP1 in forebrain neurons in transgenic mice is shown to induce several histone PTMs that include not only phosphorylation but also acetylation and methylation. These PTMs are residue-specific and occur at the promoter of genes important for memory formation like CREB (cAMP response element-binding protein) and NF-kappaB (nuclear factor-kappaB). These histone PTMs further co-occur with selective binding of RNA polymerase II and altered gene transcription, and are associated with improved long-term memory for objects and space. Together, these findings reveal a novel mechanism for the epigenetic control of gene transcription and long-term memory in the adult brain that depends on PP1.

Society for Neuroscience2009

A Tale of Two Tumors: Unraveling the Complexities of Epigenetic Dysregulation in Cancer

Maria Christine Donaldson

EPFL2019