Project Details
Projekt
Untangling site-specific histone methylation underlying microcephaly in humans
Applicant
Professorin Dr. Tanja Vogel
Subject Area
Developmental Neurobiology
Term
since 2025
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 563395927
Epigenetic processes emerged as important mechanisms for adapted gene expression impacting cell development. They regulate cell proliferation, differentiation, fate and elimination of supernumerary cells. The epigenetic processes in place during central nervous system development associate with neurodevelopmental diseases, including microcephaly and intellectual disabilities, because DNA or histone methylation balance progenitor proliferation and differentiation. During cerebral cortex development, the histone methyltransferase Disruptor of telomeric silencing 1 like (DOT1L) conferring H3K79me plays a pivotal role controlling this balance. This role of DOT1L is evolutionary conserved, as loss-of-function (LOF) in both mice and humans result in smaller brains. Interestingly, in humans a gain-of-function (GOF) of DOT1L also associates with microcephaly. To fully appreciate DOT1L functions during brain development, especially in humans, we here aim to follow up on our central hypothesis, that different H3K79me states (H3K79me1, me2, and me3) confer different functionalities at different locations. We propose that a diverse set of mechanisms controls H3K79me states, by regulating DOT1L's localization, activity, or its interactome. Molecularly, these processes can be controlled in part by signaling/phosphorylation, protein conformation and/or interacting partners. Experimentally, we will use a single-cell (sc)multiomics approach (scRNA-, scATAC-, scCUT&Tag) to untangle transcriptomic changes and the adapted epigenetic landscape during human brain development. We will investigate three human DOT1L mutations, one GOF and two LOF mutations, that we introduced into human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9. hiPSCs will be differentiated towards neural stem cells, neurons, and cortical organoids. We propose to use mass spectrometry to resolve protein complexes of DOT1L that are mediating canonical (H3K79me) and non-canonical functions of DOT1L, and posttranslational modifications of DOT1L. Thereby we aim to determine potential mechanisms of how DOT1L confers locus-specific H3K79me with different levels and states. To identify the upstream regulative layer, we will unravel signaling pathways that control DOT1L in regard to histone methylation activity or cell cycle progression. Our preliminary data revealed crosstalk to the Enhancer of zeste (EZH2), mediating H3K27me3, and we identified overlapping signaling pathways converging on both, DOT1L and EZH2. Therefore, we will focus the analysis of upstream signaling pathways mainly on these two proteins. Our screening platform can be used for studying a variety of epigenetic modifiers, as we identified a large set of pathways impinging on diverse histone methyltransferases. Making this technology available to other groups, is a major advantage of integrating this project into the SPP 2502 EPIADAPT.
DFG Programme
Priority Programmes
