Biology subjects

Lee, G. G.

Publications and source records attributed to Lee, G. G..

7 recordsLinked to original sources

bioRxiv2026

Histone modifications define distinct nucleosome positions in cell-free DNA

Circulating cell-free DNA (cfDNA) fragmentation patterns reflect nucleosome positioning. We asked whether variation in nucleosome positions in cfDNA can reveal chromatin states and associated histone modifications. Using cell-free chromatin immunoprecipitation sequencing (cfChIP-seq) in plasma from patients with prostate cancer, we identified 35,120 modification-associated positions (MAPs), loci where nucleosomes bearing active (H3K4me2) and repressive (H3K9me3) histone marks occupy reproducibly offset positions. These offsets recurred across histone marks, an independent cfChIP-seq cohort, and nuclease-digested chromatin from cell lines. MAPs were enriched in transcribed genes but depleted near transcription start sites and CTCF-binding sites where nucleosome positioning is constrained. Across 2,535 cfDNA sequencing datasets, nucleosome protection at a subset of MAPs correlated with tumor fraction (TF), reflecting differences in histone modification status between cancer-derived and non-malignant chromatin. In targeted sequencing panels, a 50-MAP model using fragments from < 0.001% of the genome distinguished samples with tumor fractions [≥]3% vs 0% (AUCs = 0.91-0.96 across prostate, breast, and lung cancers) and outperformed a state-of-the-art fragmentomics-based tumor fraction predictor. MAP-derived estimates correlated with reference TFs (r = 0.89) and longitudinal changes in TF (r = 0.91). MAP protection at ASCL1 binding sites identified neuroendocrine transformation of prostate cancer from cfDNA. These findings establish histone-modification-associated nucleosome positioning as a mutation-independent readout of tumor fraction and cancer state from routine cfDNA sequencing.

cancer biology↗

bioRxiv2026

Transcription Factor Subtype Governs Response and Resistance to DLL3-Directed T-Cell Engagement in Small Cell Lung Cancer

Although small cell lung cancer (SCLC) comprises transcription factor (TF)-defined molecular subtypes (ASCL1, NEUROD1, POU2F3), the extent to which these subtypes predict response to clinically effective therapy in patients--and whether therapy can select for subtype switching--remains unknown. The recent approval of the DLL3xCD3 bispecific T-cell engager tarlatamab represents one of the first meaningful advances in relapsed small cell lung cancer (SCLC) in decades, yet responses remain heterogeneous and resistance is inevitable. Here, we inferred SCLC gene expression from circulating chromatin in prospectively collected patient plasma (46 patients; 167 samples), enabling interrogation of response and acquired resistance to tarlatamab. Parallel development of the first immunocompetent syngeneic mouse model to study tarlatamab response and resistance enabled functional validation. Across species, findings converged on a central principle: TF subtype governs both initial response and acquired resistance. Therapeutic response was significantly associated with ASCL1-subtype tumors, whereas NEUROD1-subtype tumors exhibited inferior responses and POU2F3-subtype tumors were uniformly resistant, consistent with DLL3 being a direct ASCL1 transcriptional target and most highly expressed in ASCL1-positive tumors. Strikingly, one mode of acquired resistance revealed therapeutic selection for a NEUROD1-high state with concomitant DLL3 downregulation. Other resistant tumors exhibited enrichment of regulatory and exhausted T-cell programs, highlighting tarlatamabs dual-targeting mechanism of action. Together, these results reveal that tarlatamab exerts selective pressure against ASCL1-driven lineages, facilitating resistance through loss of an antigen intrinsically linked to that state. These findings underscore the clinical relevance of TF-defined molecular subtypes in human SCLC. More broadly, they highlight the power of integrating longitudinal in vivo plasma transcriptional profiling from patient plasma with functional mouse modeling to uncover clinical and biological mechanisms of response and resistance to cell-surface-targeted therapies.

cancer biology↗

bioRxiv2025

SNAP: Streamlined Nextflow Analysis Pipeline for Immunoprecipitation-Based Epigenomic Profiling of Circulating Chromatin

Epigenomic profiling of circulating chromatin is a powerful and minimally invasive approach for detecting and monitoring disease, but there are no bioinformatics pipelines tailored to the unique characteristics of cell-free chromatin. We present SNAP (Streamlined Nextflow Analysis Pipeline), a reproducible, scalable, and modular workflow specifically designed for immunoprecipitation-based methods for profiling cell-free chromatin. SNAP incorporates quality control metrics optimized for circulating chromatin, including enrichment score and fragment count thresholds, as well as direct estimation of circulating tumor DNA (ctDNA) content from fragment length distributions. It also includes SNP fingerprinting to enable sample identity verification. When applied to cfChIP-seq and cfMeDIP-seq data across multiple cancer types, SNAPs quality filters significantly improved classification performance while maintaining high data retention. Independent validation using plasma from patients with osteosarcoma confirmed the detection of tumor-associated epigenomic signatures that correlated with ctDNA levels and reflected disease biology. SNAPs modular architecture enables straightforward extension to additional cell-free immunoprecipitation-based assays, providing a robust framework to support studies of circulating chromatin broadly. SNAP is compatible with cloud and high-performance computing environments and is publicly available at https://github.com/prc992/SNAP/. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/694452v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@47c734org.highwire.dtl.DTLVardef@674af8org.highwire.dtl.DTLVardef@16ae938org.highwire.dtl.DTLVardef@1f57a01_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

A chromatin fiber model explains cell-free DNA fragmentation signatures of active regulatory elements

Circulating cell-free DNA (cfDNA) assays are being widely adopted in oncology and maternal-fetal medicine. Patterns of cfDNA fragmentation can provide useful information about gene regulation and expression in human disease from a blood draw. Here, we demonstrate that enhancer RNA expression - a marker of enhancer activity - can be inferred from local patterns of cfDNA fragmentation. We define a transcriptional activation score (TAS) that predicts expression of enhancers and genes based on cfDNA fragment sizes and positions near transcriptional start sites (TSSs). The TAS identifies activity of cancer-associated enhancers in patients with cancer, distinguishes clinically relevant cancer subtypes, and identifies activation of enhancers associated with treatment resistance and therapy response. We propose a simple model to account for our findings based on chromatin fiber structure and the depletion of H1 histone proteins near active TSSs. Our model provides a unified framework that reconciles seemingly conflicting observations from prior fragmentomics studies. Broadly, this work enables blood-based assessments of gene regulation in cancer and non-oncologic diseases to inform pathobiology, diagnosis, and treatment selection.

genomics↗

bioRxiv2024

Multiple Isoforms of the Activin-like receptor Baboon differentially regulate proliferation and conversion behaviors of neuroblasts and neuroepithelial cells in the Drosophila larval brain

In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor {beta} (TGF-{beta}) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. In this study we show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, we further demonstrate differential contributions of the isoforms for these cellular events with BaboA playing the major role. Our data show that stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, our study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase. Author SummaryEvolutionarily conserved TGF-{beta} signaling pathway is widely utilized as a regulator of diverse processes of brain development in both vertebrates and invertebrates. A key element in Drosophila Activin type TGF-{beta} signaling pathway is the type-I receptor Babo. The babo gene produces three isoforms, each with a unique ligand preference. Our study uncovers that Babo-mediated signaling promotes proper proliferation of NBs and NEs and conversion of NEs, together responsible for the magnitude of larval brain size growth. Three isoforms act individually or together to regulate these cellular events in coordination with developmental status. Our findings emphasize that Babo-mediated signaling is a crucial regulator of postembryonic neurogenesis that generates 90% of neuronal population for the adult brain.

developmental biology↗

bioRxiv2024

Biogenesis of circular RNAs in vitro and in vivo from the Drosophila Nk2.1/scarecrow gene

scarecrow (scro) encodes a fly homolog of mammalian Nkx2.1 that is vital for early fly development as well as for optic lobe development. Interestingly, scro was reported to produce a circular RNA (circRNA). In this study, we identified 12 different scro circRNAs, which are either mono- or multi-exonic forms. The most abundant forms are circE2 carrying the second exon only and bi-exonic circE3-E4. Levels of circE2 show an age-dependent increase in adult heads, supporting a general trend of high accumulation of circRNAs in aged fly brains. Aligning sequences of introns flanking exons uncovered two pairs of intronic complementary sequences (ICSs); one pair residing in introns 1 and 2 and the other in introns 2 and 4. The first pair was demonstrated to be essential for the circE2 production in cell-based assays; furthermore, deletion of the region including potential ICS components in the intron-2 reduced in vivo production of circE2 and circE3-E4 by 80%, indicating them to be essential for the biogenesis of these isoforms. Besides the ICS, the intron regions immediately abutting exons seemed to be responsible for a basal level of circRNA formation. Moreover, the replacement of scro-ICS with those derived from laccase2 was comparably effective in scro-circRNA production, buttressing the importance of the hairpin-loop structure formed by ICS for the biogenesis of circRNA. Lastly, overexpressed scro affected outcomes of both linear and circular RNAs from the endogenous scro locus, suggesting that Scro plays a direct or indirect role in regulating expression levels of either or both forms.

genetics↗

bioRxiv2022

DNA methylation of the endothelin receptor B makes blue fish yellow

Natural selection shapes traits during evolution including animal coloration known to be important for concealment and communication and color has been particularly salient in the explosive radiation of cichlid fish species in the rift valley lakes of East Africa. Though selection can produce variation in color via genetic substrates during early development, plasticity in coloration can occur through endocrine, neural and transcriptional cues in response to various environmental stimuli. It is well known that some animals often change color to match their visual ecology. Adult male cichlid fish (Astatotilapia burtoni, Lake Tanganyika) can switch between blue and yellow body colors. Different colors result from the expression of pigment-bearing cells, which differ in density and function between these two color morphs. We show that A. burtoni switches from yellow to blue depending on their visual environment by downregulating endothelin receptor B (EdnRB) mRNA via DNA hypermethylation at a single cytosine residue within its promoter. EdnRB functions in yellow chromatophores to signal the aggregation of yellow pigments, making yellow less visible. Taken together, the regulation of EdnRB through DNA methylation in yellow chromatophores, in part, contributes to pigmentation changes from blue to yellow, depending on visual environment.

genetics↗