Flournoy, J. | bioRxiv Science

bioRxiv2021

Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation

Oxidative modification of 5-methylcytosine (5mC) generates 5-hydroxymethylcytosine (5hmC), a DNA modification that exhibits unique epigenetic regulatory functions and impacts diverse biological processes. However, standard single-nucleus/cell bisulfite sequencing methods cannot resolve the base ambiguity between 5mC and 5hmC to accurately measure cell-type specific epigenomic patterns and gene regulatory functions of 5hmC or true 5mC. Here, we develop single-nucleus 5hmC sequencing (snhmC-seq) for quantitative and unbiased 5hmC profiling in single cells by harnessing differential deaminase activity of APOBEC3A towards 5mC and chemically protected 5hmC. We used snhmC-seq to profile single nuclei from cryopreserved mouse brain samples to reveal epigenetic heterogeneity of 5hmC at single-cell resolution and uncovered a non-canonical gene regulatory role of genic 5hmC in non-CG context.

genomics↗

bioRxiv2020

Single-cell multi-omics reveals elevated plasticity and stem-cell-like blasts relevant to the poor prognosis of KMT2A-rearranged leukemia

Infant ALL is a devastating malignancy caused by rearrangements of the KMT2A gene (KMT2A-r) in approximately 70% of patients. The outcome is dismal and younger age at diagnosis is associated with increased risk of relapse. To discover age-specific differences and critical drivers that mediate the poor outcome in KMT2A-r ALL, we subjected KMT2A-r leukemias and normal hematopoietic cells from patients of different ages to multi-omic single cell analysis using scRNA-Seq, scATAC-Seq and snmC-Seq2. We uncovered the following critical new insights: Leukemia cells from infants younger than 6 months have a greatly increased lineage plasticity and contain a hematopoietic stem and progenitor-like (HSPC-like) population compared to older infants. We identified an immunosuppressive signaling circuit between the HSPC-like blasts and cytotoxic lymphocytes in younger patients. Both observations offer a compelling explanation for the ability of leukemias in young infants to evade chemotherapy and immune mediated control. Our analysis also revealed pre-existing lymphomyeloid primed progenitor and myeloid blasts at initial diagnosis of B-ALL. Tracking of leukemic clones in two patients whose leukemia underwent a lineage switch documented the evolution of such clones into frank AML. These findings provide critical insights into KMT2A-r ALL and have potential clinical implications for targeted inhibitors or multi-target immunotherapy approaches. Beyond infant ALL, our study demonstrates the power of single cell multi-omics to detect tumor intrinsic and extrinsic factors affecting rare but critical subpopulations within a malignant population that ultimately determines patient outcome.

cancer biology↗