Ambrocio, R. E. | bioRxiv Science

bioRxiv2025

Single-nucleus RNA sequencing reveals the cellular diversity of cerebrospinal fluid in the context of intraventricular hemorrhage

BackgroundIntraventricular hemorrhage (IVH) is a common and severe complication of hemorrhagic brain injury. Current treatments offer limited improvement in long-term neurological outcomes. Inflammatory responses in the cerebrospinal fluid (CSF) after IVH are thought to drive secondary injury, but the cellular mechanisms underlying this inflammation remain poorly defined. MethodsWe performed single-nucleus RNA sequencing of leukocytes isolated from CSF collected through external ventricular drains in subjects with intracerebral (n = 6) or subarachnoid (n = 1) hemorrhage. We characterized transcriptionally distinct subpopulations of neutrophils, monocytes, and lymphocytes by comparison to reference datasets. Cell-cell signaling networks were analyzed to infer cytokine-mediated communication, and a flow cytometry panel was developed to validate transcriptomic findings in independent CSF samples. ResultsWe obtained 11,191 high-quality nuclei comprising neutrophils (53.8%), monocytes (26.1%), lymphocytes (17.8%), and non-immune cells (2.4%). Neutrophils segregated into Nascent, Quiescent, and Interferon-Activated states. Monocytes exhibited classical phenotypes that include interferon-activated states (characterized by expression of VCAN or PROK2) and CXC-chemokine expressing states (characterized by expression of CXCL5 or CXCL8). Lymphocytes were mainly naive and central memory CD4 T cells. Cell-cell signaling analysis predicted strong CXC chemokine signaling from monocytes to neutrophil subsets and IL-1 family-driven inflammatory responses across multiple populations. Type I and III interferon signaling defined a neutrophil population not previously described in the central nervous system. ConclusionThis study delineates the diverse cellular immune landscape of CSF after IVH. Transcriptomic profiles reveal interferon, IL-1, and CXC chemokine signaling networks as potential therapeutic targets to mitigate secondary injury.

genomics↗

bioRxiv2021

A Budding Yeast Model and Screen to Define the Functional Consequences of Oncogenic Histone Missense Mutations

Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histone H3.3 gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only two H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine sensitive growth of H3K36 mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA binding protein and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine sensitive growth of H3K36R mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.

genetics↗