Periodic Reporting for period 1 - M-Y-cro (Metabolism-YEATS-crosstalk: Elucidating metabolic dependencies of epigenetic YEATS action)
Berichtszeitraum: 2023-08-01 bis 2025-07-31
Cells and ultimately organisms tightly regulate essential pathways to support complex biological processes such as homeostasis, growth, and differentiation. Increasing evidence suggests that these pathways operate in a highly integrated and coordinated manner, rather than functioning in isolation. The overarching aim of this proposal was to deepen our understanding of the mechanistic regulation of key cellular pathways, with a particular focus on the interplay between metabolism and epigenetics (i.e. chromatin metabolism). Building on previous findings and expertise from our laboratory - specifically the discovery of a functional interaction between the folate enzyme MTHFD1 and the histone acetylation reader protein BRD4 - we hypothesized that similar metabolism-centric regulatory relationship(s) may exist with YEATS domain-containing reader proteins, which recognize histone acylation marks. Moreover, although dysregulation of YEATS proteins has been linked to various diseases, most notably in different types of cancer, their overall function, particularly the context and consequences of their histone acylation reading ability, remain only partially understood. Thus, to investigate potential functional relationships, we proposed a series of work packages (WPs) within the proposal: While WP1 focused on characterizing the baseline functions of YEATS proteins under various metabolite-enriched conditions, WP2 aimed to identify functional interactions between YEATS proteins and metabolic enzymes, that require further in-depth characterization, as proposed in WP3.
To address these WPs, we planned to develop YEATS-perturbation-dependent fluorescent reporter systems, that could respond upon genetic or chemical perturbation, thus linking these phenotypes to YEATS protein function. In the absence of suitable chemical tool compounds for all YEATS proteins, we aimed to generate dTAG-YEATS cell lines that enable targeted protein degradation upon incubation with the corresponding PROTAC.
To address these WPs, we planned to develop YEATS-perturbation-dependent fluorescent reporter systems, that could respond upon genetic or chemical perturbation, thus linking these phenotypes to YEATS protein function. In the absence of suitable chemical tool compounds for all YEATS proteins, we aimed to generate dTAG-YEATS cell lines that enable targeted protein degradation upon incubation with the corresponding PROTAC.
We successfully established model cell lines in HAP1 cells for all four YEATS domain-containing proteins by N-terminally tagging them with an FKBP12 degron (dTAG) using the PITCH knock-in strategy. We thoroughly characterized these knock-ins in terms of efficient degradation in time and dose-dependent manner. We performed RNA-seq. experiments and observed major transcriptional changes that correlated with the essentiality of YEATS proteins in the given cell line. We have established a fluorescent reporter cell line for one YEATS protein, performed perturbation experiments and validated potential hits. We are also expanding the workflow to the remaining YEATS variants.
Overall, this project and its associated protocols will provide a blueprint for generating reporter cell lines that facilitate the discovery of functional relationships between identified hit proteins and target proteins. This approach will be applied across various projects in the Kubicek lab. In addition to ongoing efforts to elucidate metabolism-centric mechanisms of YEATS function, the lab’s broader interest and integration of knowledge in chromatin biology and folate metabolism has led to the surprising discovery of a de novo purine synthesis pathway regulator. Besides implications in basic mechanisms of metabolic pathway regulation, these results provide a rationale for purine analog mediated resistances.