Biology subjects

Lim, D. A.

Publications and source records attributed to Lim, D. A..

8 recordsLinked to original sources

bioRxiv2026

Disruption of lamina-associated genome organization activates neuronal gene programs in H3.3K27M DIPG

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor frequently driven by a histone H3 mutation that substitutes lysine 27 with methionine (H3K27M). While H3K27M is known to cause a global depletion of the repressive histone mark H3K27me3, how this epigenetic disruption contributes to oncogenic transcriptional reprogramming remains incompletely understood. Here, we show that H3.3K27M DIPG cells exhibit widespread disruption of lamina-associated domain (LAD) organization across primary tumors and patient-derived cell lines. H3.3K27M was enriched at genomic regions that lost lamina association, which acquired increased H3K27ac and showed activation of neuronal and oncogenic gene programs. Notably, LAD loss was more strongly associated with transcriptional activation than H3K27me3 depletion, linking altered LAD organization to the transcriptional consequences of H3.3K27M. Finally, expression of H3.3K27M in human embryonic stem cell-derived neural precursor cells (NPCs) was sufficient to induce LAD remodeling, predominantly through loss of lamina association, leading to activation of neuronal gene programs. Together, these findings identify disruption of lamina-associated genome architecture as a key mechanism by which the H3.3K27M oncohistone drives oncogenic transcriptional reprogramming in DIPG.

genomics↗

bioRxiv2025

Structural basis for the function of long noncoding RNA Pnky in neural stem cells

LncRNA Pnky is a trans-acting regulator of neural stem cell (NSC) differentiation, but the molecular mechanisms by which Pnky regulates neurogenesis is unknown. A fundamental step towards mechanistic understanding is to determine whether a lncRNA has folded structure that underlies biological function. Using chemical probing and high-throughput analysis, we determined the secondary structure of Pnky folded in vitro and in cellulo. Pnky adopts a compact structure in vitro with distinct modules and evidence of tertiary interactions. In cellulo, Pnky structure is remarkably similar to the in vitro conformation. We used locked nucleic acid oligonucleotides to interrogate the entire Pnky transcript for function in NSCs and identified regions that when targeted increased neurogenesis - phenocopying Pnky knockdown - without decreasing transcript abundance. Our findings provide a structural basis for the role of Pnky in neurogenesis and, more broadly, illustrate how structural maps combined with phenotypic data can advance fundamental understanding of lncRNA mechanism. HIGHLIGHTSPnky folds into highly structured conformation in vitro with distinct modules. Pnky is a compact lncRNA with evidence of tertiary interactions. Pnky structure in neural stem cells is similar to its in vitro conformation. LNA ASO perturbations identify structured regions of Pnky involved in neuronal differentiation.

molecular biology↗

bioRxiv2024

Nucleotide GPT: Sequence-Based Deep Learning Prediction of Nuclear Subcompartment-Associated Genome Architecture

The spatial organization of the genome within the nucleus is partially determined by its interactions with distinct nuclear subcompartments, such as the nuclear lamina and nuclear speckles, which play key roles in gene regulation during development. However, whether these genome-nuclear subcompartment interactions are encoded in the underlying DNA sequence remains poorly understood. The mechanisms for gene regulation are primarily encoded in noncoding DNA sequences, but deciphering how these sequence features control gene expression remains a significant challenge in genomics. Here, we present Nucleotide GPT, a transformer-based model that predicts genomic associations with spatially distinct, physical nuclear subcompartments from DNA sequence alone. Pre-trained on a diverse set of multi-species genomes, we demonstrate Nucleotide GPTs genomic understanding through evaluation on diverse prediction tasks, including histone modifications, promoter detection, and transcription factor binding sites. When finetuned to predict genome interactions with two separate nuclear subcompartments - the lamina of the inner nuclear membrane and nuclear speckles that lie more interior - Nucleotide GPT achieves an average accuracy of 73.6% for lamina-associated domains (LADs) and 79.4% accuracy for speckle-associated domains (SPADs), averaged across three cortical development cell types. Analysis of the models learned representations through Uniform Manifold Approximation and Projection (UMAP) reveals that Nucleotide GPT develops internal embeddings that effectively distinguish LADs from inter-LADs, with predicted probabilities closely corresponding to experimentally determined LAD classifications. When examining these representations in the context of cell type-invariant constitutive LADs (cLADs) compared to cell type-specific LADs, the model assigns lower confidence scores to cell type-specific LADs compared to cLADs that are conserved across neuronal differentiation, suggesting sequence features may play a stronger role in maintaining cLAD associations. Examination of the models attention patterns at correctly classified regions suggests that specific sequence elements govern model decision making about nuclear subcompartment associations. Our results demonstrate the utility of transformer architectures for studying three-dimensional (3D) genome organization and substantiate a role for DNA sequence in determining nuclear subcompartment associations.

bioinformatics↗

bioRxiv2024

Neural Stem Cell Relay from B1 to B2 cells in the adult mouse Ventricular-Subventricular Zone

Neurogenesis and gliogenesis continue in the Ventricular-Subventricular Zone (V-SVZ) of the adult rodent brain. B1 cells are astroglial cells derived from radial glia that function as primary progenitors or neural stem cells (NSCs) in the V-SVZ. B1 cells, which have a small apical contact with the ventricle, decline in numbers during early postnatal life, yet neurogenesis continues into adulthood. Here we found that a second population of V-SVZ astroglial cells (B2 cells), that do not contact the ventricle, function as NSCs in the adult brain. B2 cell numbers increase postnatally, remain constant in 12-month-old mice and decrease by 18 months. Transcriptomic analysis of ventricular-contacting and non-contacting B cells revealed key molecular differences to distinguish B1 from B2 cells. Transplantation and lineage tracing of B2 cells demonstrate their function as primary progenitors for adult neurogenesis. This study reveals how NSC function is relayed from B1 to B2 progenitors to maintain adult neurogenesis.

neuroscience↗

bioRxiv2023

Sex-specific role for the long noncoding RNA Pnky in mouse behavior

The human brain expresses thousands of different long noncoding RNAs (lncRNAs), and aberrant expression of specific lncRNAs has been associated with cognitive and psychiatric disorders. While a growing number of lncRNAs are now known to regulate neural cell development and function, relatively few have been shown to underlie animal behavior, particularly with genetic strategies that establish lncRNA function in trans. Pnky is an evolutionarily conserved, neural lncRNA that regulates brain development. Using mouse genetic strategies, we show that Pnky has sex-specific roles in mouse behavior and that this lncRNA underlies specific behavior by functioning in trans. Male Pnky-knockout (KO) mice have deficits in cued fear recall, a type of Pavlovian associative memory. In female Pnky-KO mice, the acoustic startle response (ASR) is increased and accompanied by a decrease in prepulse inhibition (PPI), both of which are behaviors altered in affective disorders. Remarkably, expression of Pnky from a bacterial artificial chromosome (BAC) transgene reverses the ASR phenotype of female Pnky-KO mice, demonstrating that Pnky underlies specific animal behavior by functioning in trans. More broadly, these data provide genetic evidence that a lncRNA gene and its function in trans can play a key role in the behavior of adult mammals, contributing fundamental knowledge to our growing understanding of the association between specific lncRNAs and disorders of cognition and mood.

neuroscience↗

bioRxiv2023

Clarifying the Transcriptional Profiles of Malignant Clones through Multiscale and Multiomic Analysis of Individual Tumors

Tumors may contain billions of cells including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that is consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

cancer biology↗

bioRxiv2022

Epigenetic reprogramming shapes the cellular landscape of schwannoma

Cell state evolution underlies tumor development and response to therapy1, but mechanisms specifying cancer cell states and intratumor heterogeneity are incompletely understood. Schwannomas are the most common tumors of the peripheral nervous system and are treated with surgery and ionizing radiation2-5. Schwannomas can oscillate in size for many years after radiotherapy6,7, suggesting treatment may reprogram schwannoma cells or the tumor microenvironment. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas. We find schwannomas are comprised of 2 molecular groups distinguished by reactivation of neural crest development pathways or misactivation of nerve injury mechanisms that specify cancer cell states and the architecture of the tumor immune microenvironment. Schwannoma molecular groups can arise independently, but ionizing radiation is sufficient for epigenetic reprogramming of neural crest to immune-enriched schwannoma by remodeling chromatin accessibility, gene expression, and metabolism to drive schwannoma cell state evolution and immune cell infiltration. To define functional genomic mechanisms underlying epigenetic reprograming of schwannomas, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of cancer evolution and establish a paradigm of epigenetic reprograming of cancer in response to radiotherapy.

cancer biology↗

bioRxiv2021

Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenic lineages

The ventricular-subventricular zone (V-SVZ) is home to the largest neurogenic niche in the adult mouse brain. Previous work has demonstrated that resident stem cells in different locations within the V-SVZ produce different subtypes of new neurons for the olfactory bulb. While great progress has been made in understanding the differences in regional stem cell potential using viral and genetic lineage tracing strategies, the core molecular heterogeneity that underlies these regional differences is largely unknown. Here we present single whole-cell and single nucleus sequencing datasets of microdissected adult mouse V-SVZ, and evidence for the existence of two broad populations of adult neural stem cells. By using spatially resolved microdissections in the single nucleus sequencing dataset as a reference, and mapping marker gene expression in the V-SVZ, we find that these two populations reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. Furthermore, we identified two subpopulations of newly born neurons that have gene expression consistent with dorsal or ventral origins. Finally, we identify genes expressed by both stem cells and the neurons they generate that specifically mark either the dorsal or ventral adult neurogenic lineage. These datasets, methods and findings will facilitate the study of region-specific regulation of adult neurogenesis.

neuroscience↗