Single-molecule dissection of hypomethylation-driven genome instability

The National Institute of General Medical Sciences awarded Beth Israel Deconess Medical Center, Inc. $158,600 on January 23, 2027, under the Biomedical Research and Research Training program (CFDA 93.859) to conduct single-molecule dissection of hypomethylation-driven genome instability.

The research combines technology development with mechanistic chromatin biology to address how global DNA hypomethylation—an early epigenetic alteration in human cancer—drives chromosomal instability and DNA breaks. Aim 1 develops an Oxford Nanopore Technologies-based single-molecule platform to jointly map endogenous DNA breaks and DNA methylation, overcoming limitations of existing break-mapping assays. Aim 2 uses the new method alongside INDUCE-SEQ and whole-genome bisulfite sequencing to define how DNMT1-inhibitor-induced DNA breaks relate to local methylation loss in USP48-deficient leukemia cells. Aim 3 dissects how the histone deubiquitinase USP48 regulates MACROH2A1 ubiquitination to promote DNA repair under hypomethylating stress, using CRISPR-engineered mutants, quantitative imaging, and DNA damage-response assays. The research identifies USP48 and MACROH2A1 as key regulators of genome integrity and establishes a mechanistic framework for how hypomethylation drives genome fragility, informing safer and more effective use of hypomethylating agents approved for leukemia and under investigation across other cancer types.

Performance occurs in Massachusetts. The period of performance runs from January 23, 2027, through August 23, 2029.

Abstract

Global DNA hypomethylation is one of the earliest and most pervasive epigenetic alterations in human cancer. IT is closely associated with chromosomal instability. On the other hand, DNA hypomethylating agents are approved for leukemia and are being tested across other cancer types. However, IT remains unclear whether hypomethylation at specific genomic loci directly increases DNA break susceptibility and how cancer cells repair these lesions. This GAP in knowledge limits our ability to safely and effectively use hypomethylating agents. Our preliminary data identify the histone deubiquitinase USP48 as a critical protector of genome integrity under hypomethylating stress, where IT is recruited to newly demethylated CPG islands and regulates ubiquitination of non-canonical histones such as MACROH2A1. However, the genomic localization of hypomethylation-induced DNA breaks and the molecular mechanism of USP48-dependent repair remain undefined. This F32 proposal will address these gaps by combining innovative technology development with mechanistic chromatin biology. Aim 1 will develop an oxford nanopore technologies (ONT)- based single-molecule platform to jointly map endogenous DNA breaks and DNA methylation and overcome major limitations of existing break-mapping assays. Aim 2 will use our new method as well as established methods, including induce-SEQ and whole-genome bisulfite sequencing, to define how DNMT1-inhibitor-induced DNA breaks relate to local methylation loss in USP48- deficient leukemia cells. Aim 3 will dissect how USP48 regulates MACROH2A1 ubiquitination to promote DNA repair under hypomethylating stress, using crispr-engineered mutants, quantitative imaging, and DNA damage-response assays. Together, these studies will establish a mechanistic framework for how global hypomethylation drives genome fragility and identify USP48 and MACROH2A1 as key regulators of chromatin stability during DNMT1 inhibition. The proposed work will advance understanding of how hypomethylating drugs cause DNA damage, provide transformative tools for the epigenetics and DNA repair communities, and inform strategies to improve therapeutic efficacy and patient selection. This fellowship will provide the candidate with rigorous training in single-molecule sequencing, DNA damage biology, chromatin biology, and computational analysis, supporting the transition to an independent research career focused on cancer epigenetics and genome stability.