Crebbp Kat Domain Missense Mutations Alter Its Dynamic Loading Onto Chromatin and Create a Transcription Factor Sink

The CREBBP gene encodes a lysine acetyltransferase (KAT) and is one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). However, the spectrum of mutations is significantly different between these two diseases, with significantly higher fractions of nonsense/frameshift mutations in DLBCL and comparatively higher fractions of lysine acetyltransferase (KAT) domain point mutations (KAT-PM) in FL. Among KAT domain mutations, multiple hotspots can be observed with R1446 (14%), Y1482 (3%) and Y1503 (7%) being the most frequently mutated residues. We have previously shown that CREBBP R1446C mutations lead to a more profound loss of histone acetylation and gene expression compared to biallelic knock-out (KO) of the CREBBP highlighting a potential dominant-negative function of R1446C via an unknown mechanism. We have also performed biochemical and structural analysis of KAT domain mutations and observed that R1446C mutations result in a partial loss of function (pLOF), whereas Y1482N and Y1503C mutations are catalytically dead. Here, we report the molecular characterization of CRISPR-engineered isogenic cell lines (RL, HT, 293T) bearing biallelic KO, R1446C, Y1482N or Y1503C mutations, or WT CREBBP, using CUT&RUN analysis of CREBBP and EP300 loading, histone marks (H3K27Ac, H3K4me1, H3K4me2, H3K27me3), and ATAC and RNA sequencing for chromatin accessibility and gene expression, respectively.

Western blot analysis showed significant reduction of H3K27Ac and H3K18Ac in all point-mutants with no alteration of CREEBP or its paralog EP300 expression. Principal component analysis (PCA) of H3K27Ac and gene expression data revealed that KO cells were most similar to WT, and all KAT-PM cells clustered separately to WT and KO cells. Notably, R1446C (partial LOF) cells reproducibly clustered separately from Y1482N and Y1503C (catalytically dead), and differential analysis identified both shared and R1446C- or Y1482N/Y1503-specific losses of H3K27Ac in all cell lines, suggesting that the degree of loss in catalytic activity rendered by different KAT domain point mutations may be associated with distinct molecular phenotypes. In order to investigate the mechanism by which KAT-PMs drive more severe molecular changes compared to biallelic KO, we investigate the redundant role of EP300 using CUT&RUN analysis of EP300 loading. CREBBP-bound active enhancers (H3K4me1+ H3K27Ac+) showed a significant increase in EP300 loading in cells with CREBBP KO compared to WT. However, we observed a slight decrease in EP300 loading within KAT-PM cells, indicating that the presence of mutant CREBBP protein prevents the redundant activity of EP300. We speculated that this may be due to mutant CREBBP residing on chromatin in an inactive state and preventing loading of EP300. We therefore analyzed CREBBP loading over CREBBP-bound active enhancers in WT cells that had significant reductions of H3K27Ac in KAT-PMs. Surprisingly, we observed a significant reduction in CREBBP loading over these regions, with a greater loss of CREBBP loading in catalytically dead mutants (Y1482N/Y1503C) compared to the partial LOF mutant (R1446C). We therefore characterized patterns of CREBBP loading during normal germinal center B-cell development by analyzing purified tonsillar naïve B-cells, centroblasts and centrocytes. We observed that CREBBP is dynamically loaded onto chromatin during the centroblast-to-centrocyte transition, indicating that loss of chromatin-bound CREBBP in KAT-PMs may be due to a failure of mutant CREBBP to load onto these sites. We next performed transcription factor (TF) motif enrichment analysis and identified a significant enrichment of key TFs such as BACH2 and IRF4 in regions with failed CREBBP loaded and EP300 compensation, indicating that CREBBP may bind these transcription factors and act as a sink that prevents redundant EP300 activity. We validated this using tetracycline-inducible over-expression of IRF4 in KAT-PM cells, which increased EP300 loading and H3K27Ac over IRF4 binding sites.

Collectively, our results indicate that CREBBP KAT-PMs prevent the redundant activity of EP300 in a manner proportionally to their relative loss of catalytic activity, by preventing the dynamic loading of CREBBP onto chromatin and acting as a transcription factor sink.

Samaniego:TG Therapeutics: Honoraria. Green:Abbvie: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Monte Rosa Therapeutics: Honoraria; Allogene: Research Funding; KDAc Therapeutics: Current holder of stock options in a privately-held company; Kite/Gilead: Research Funding; Tessa Therapeutics: Honoraria.