Biology subjects

Sunkel, B. D.

Publications and source records attributed to Sunkel, B. D..

9 recordsLinked to original sources

bioRxiv2026

Hat1 Orchestrates Heterochromatin Inheritance by Regulating Localization of H3K9 Methyltransferases

Many regions of heterochromatin associate with the nuclear periphery and are known as Lamin-associated domains (LADs). Histone acetyltransferase 1 (Hat1) is a highly conserved enzyme which acetylates newly synthesized histones H4 on lysines 5 and 12 prior to their deposition on chromatin. Hat1 is required to preserve chromatin accessibility within a subset of LADs called Hat1-dependent accessibility domains (HADs). Here we profile a diverse set of histone modifications in Hat1 KO and WT immortalized mouse embryonic fibroblasts (iMEFs) and find that Hat1 regulates diverse aspects of the structure of HADs and non-HAD LADs (nhLADS). In HADs, these changes include the conversion of H3K9me2 to H3K9me3. Analysis of H3K9-specific histone methyltransferases (HMTs) shows that that Suv39h1 and Suv39h2 have distinct localization patterns, where only Suv39h2 localizes to LADs. G9a only localizes to LADs in regions enriched for H3K9me2. We find that Hat1 loss results in a redistribution of these HMTs in both HADs and nh LADs. There is a decrease in the levels of G9a with a concomitant increase in Suv39h2. These results suggest Hat1 functions to restrain the formation of a more strongly heterochromatic state and highlight a role for Hat1 as an essential regulator of heterochromatin inheritance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/713225v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@9f6238org.highwire.dtl.DTLVardef@1e95415org.highwire.dtl.DTLVardef@18f3e0aorg.highwire.dtl.DTLVardef@1322426_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Comparative modes of chromatin engagement by PAX::FOXO1 fusions in rhabdomyosarcoma

Fusion positive rhabdomyosarcoma (FP-RMS) is an aggressive soft-tissue sarcoma that most frequently affects children and adolescents. Treatment options and outcomes for children with this cancer remain poor, non-specific, and broadly toxic despite decades of research. The defining molecular drivers of the more aggressive fusion-positive subtype of the disease arise from chromosomal translocations that fuse PAX3 or PAX7 to FOXO1 to form PAX3::FOXO1 or PAX7::FOXO1, encoding fusion oncoprotein transcription factors. Despite their high degree of similarity, PAX3::FOXO1 correlates with worse patient overall survival than PAX7::FOXO1. Previous work from our groups and others has revealed evidence focused in chromatin accessibility contexts that PAX3::FOXO1 has key characteristics of a pioneer transcription factor, a specialized subclass of transcription factors that can bind nucleosomal DNA prior to generation of local accessibility. However, evidence at the genome scale for PAX3/7::FOXO1 direct nucleosome targeting, prior to the accessibility step in pioneering, has remained elusive and challenging to capture methodologically for RMS fusion oncoproteins. In this work, we compare the cellular functions of these PAX::FOXO1 fusions, including new approaches for identifying nucleosome targeting at the genome scale. We find that in zebrafish RMS initiation models, the fusions initially activate similar neural transcriptional programs but to different extents, and we further evaluate their mechanisms in RMS cells at the genome scale with modified MNase XChIP to detect nucleosome and subnucleosome fusion/chromatin binding. In establishing our cross-species comparative oncology approach, we report, to our knowledge, the first high resolution nucleosome positioning data in rhabdomyosarcoma. We find that both PAX::FOXO1 fusions bind nucleosomal DNA, but with varied motif preferences and histone mark co-localization patterns. Altogether, we establish the nucleosome targeting functions of PAX7::FOXO1 and PAX3::FOXO1 pioneering and uncover key mechanistic distinctions for chromatin engagement of the two most common RMS fusion oncoproteins. HIGHLIGHTSO_LIPartially overlapping gene signatures are activated by PAX3/7::FOXO1 in vivo C_LIO_LIModified MNase ChIP reveals PAX3/7::FOXO1 bind nucleosomal and subnucleosomal DNA C_LIO_LIPAX7::FOXO1 binds degenerate paired/homeobox motifs within nucleosome targets C_LIO_LIEach fusion engages distinct nucleosomal gene targets C_LI

cancer biology↗

bioRxiv2025

NR0B1 alters the 9-cis-retinoic acid response in Ewing Sarcoma cells

Pediatric cancers are often characterized by relatively low DNA mutation rates and are frequently driven instead by alterations in gene expression programs. In Ewing Sarcoma (EwS), these changes result from the activity of an oncogenic fusion transcription factor, EWS::FLI1, which alters the enhancer landscape and rewires transcriptional programs to promote oncogenic transformation. Among the targets directly activated by EWS::FLI1, NR0B1 has emerged as a potentially targetable transcription co-regulator and as a manifold for widespread alterations in downstream gene expression programs mediated by members of the ligand-inducible nuclear receptor superfamily of transcription factors. We have dissected the gene regulatory activity of NR0B1 in EwS models, showing its role in altering the basal gene expression programs of nuclear receptors COUP-TFII, EAR2, RXRa, and TR4. Additionally, we show that NR0B1 impacts the EwS response to retinoids, particularly 9-cis-retinoic acid signaling in part through RXRa. Our findings suggest NR0B1 silencing or inhibition offer a means of achieving a more potent response to retinoids, which may activate differentiation programs, decrease EwS gene expression signatures, and limit in vitro transformation phenotypes. Taken together, our study presents evidence of NR0B1 acting canonically as a nuclear receptor co-regulator in EwS, revealing a potential pathway to treatment of this aggressive cancer by combining NR0B1 inhibition with therapeutic nuclear receptor ligands.

cancer biology↗

bioRxiv2025

PEDIA-BRAIN: A single nuclei multiomic encyclopedia of the human pons provides a resource for normal development and disease vulnerability

The human pons relays information between the brain and the body. It is affected by pathological processes, including diffuse midline gliomas (DMGs) and multiple sclerosis (MS) which predominantly arise in childhood and middle age, respectively. Although multiple studies address these disease states, a comprehensive resource for normal pons development is lacking. Here we present the first installment of PEDIA-BRAIN, an encyclopedia of gene expression and chromatin accessibility from 140,771 human pons nuclei spanning the first trimester to early adulthood, as a resource for the scientific community. Exploration of the encyclopedia identified two trajectories to mature oligodendrocytes and developmental restriction of genes for neuron to oligodendrocyte progenitor cell synapses. To illustrate the utility of the resource, we compared single cell transcriptomes from DMG and MS tissues to the encyclopedia and identified perturbation of oligodendrocyte subtypes in both diseases. Data may be accessed at https://pediabrain.nchgenomics.org.

neuroscience↗

bioRxiv2025

Fusion transcription factor dosage controls cell state in rhabdomyosarcoma

In the fusion-positive subset of rhabdomyosarcoma, the PAX3::FOXO1 oncoprotein is the most common fusion driver. We previously established a human myoblast system for inducible expression of PAX3::FOXO1. In the current study, we modulate PAX3::FOXO1 protein expression to understand the epigenetic and phenotypic functions at different PAX3::FOXO1 levels. Proliferative and oncogenic outcomes depend on PAX3::FOXO1 dosage in this system with transformation dominant at intermediate levels and growth suppression dominant at high levels. After prolonged PAX3::FOXO1 expression, there is dosage-dependent heterogeneity in single cell gene expression profiles. We observe a dosage-specific effect for PAX3::FOXO1 chromatin recognition and identify factors that modulate PAX3::FOXO1 chromatin binding. PAX3::FOXO1 dosage affects expression signatures related to cell cycle, epithelial-mesenchymal transition, and myogenesis. Whereas intermediate PAX3::FOXO1 expression maximizes chromatin binding to modulate gene expression, high PAX3::FOXO1 expression alters S phase progression and increases accessibility behind the replication fork. We conclude that PAX3::FOXO1 exerts dosage-dependent functions to influence epigenetic heterogeneity in fusion-positive rhabdomyosarcoma.

genomics↗

bioRxiv2024

Differential Regulation of Large-scale Chromosome Conformations in Osteoblasts and Osteosarcoma

Correct chromosome organization in the cell nucleus is essential for genome function. However, dynamics and regulations of large-scale chromosomal conformations beyond single compartments at larger than ten megabases in vivo in single cells remain largely unknown. Here we use CRISPR-Sirius, a high-resolution and high-sensitivity real-time imaging technique, to directly visualize distinctions in large-scale chromosomal conformations between live osteosarcoma (OS) cells and osteoblasts, suggesting extensive chromatin reorganization during cell transformation. A surprising discovery is that chromosome 19 long arm is primarily extended in osteoblasts and maintained by H3K27me3. Extended chromosome conformation has been reported in fly and mouse but not in human cells yet. However, in OS cells, chromosome 19 primarily folded into collapsed conformations, which reshape in minutes and are regulated by the chromosome architectural proteins CTCF and cohesin in the presence of H3K27ac. Changes in chromosome conformations by knocking down the cohesin subunit RAD21 resulted in altered gene expression, including proto-oncogenes. Transcription inhibition by a small molecule inhibitor did not have detectable effects on large-scale chromosome conformation, suggesting that local transcription events have limited effects on large-scale chromosomal architecture. Our results provide unique insights into the complex regulatory mechanisms of endogenous large-scale chromosome organization in normal and transformed osteogenic tissues. One-sentence summaryRegulations of chromosome territory organization in normal and cancer bone cells visualized by CRISPR-based live-cell imaging.

cell biology↗

bioRxiv2022

The 3D chromatin landscape of rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric soft tissue cancer with a lack of precision therapy option for patients. We hypothesized that with a general paucity of known mutations in RMS, chromatin structural driving mechanisms are essential for tumor proliferation. Thus, we carried out high-depth in situ Hi-C in representative cell lines and patient-derived xenografts to understand chromatin architecture in each major RMS subtype. We report a comprehensive 3D chromatin structural analysis and characterization of fusion-positive (FP-RMS) and fusion-negative rhabdomyosarcoma (FN-RMS). We have generated spike-in in situ Hi-C chromatin interaction maps for the most common FP-RMS and FN-RMS cell lines, and compared our data with patient derived xenograft (PDX) models. In our studies we uncover common and distinct structural elements in large Mb-scale chromatin compartments, tumor-essential genes within variable topologically associating domains, and unique patterns of structural variation. Our comprehensive analysis provides high-depth chromatin interactivity maps for contextualizing gene regulation events identification of functionally critical chromatin domains in RMS. HIGHLIGHTSO_LIPAX3-FOXO1 and MYOD localize in both A- and B-compartments C_LIO_LIConserved mechanisms dictate CTCF orientation at TAD boundaries in RMS C_LIO_LIDifferential TADs in each RMS subtype encompass tumor-specific genes C_LIO_LINeo-TADs are formed from SV events in each subtype of RMS C_LIO_LIBoth major RMS subtypes have structural variation that is identifiable from Hi-C C_LIO_LIDistinct mechanisms can produce the major fusion alleles in rhabdomyosarcoma C_LIO_LIPAX3-FOXO1 and MYOD genomic binding is more enriched at regions with CNV C_LI

cancer biology↗

bioRxiv2021

Epigenetic Regulation of Nuclear Lamina-Associated Heterochromatin by HAT1 and the Acetylation of Newly Synthesized Histones

During S phase, eukaryotic cells must faithfully duplicate both the sequence of the genome and the regulatory information found in the epigenome. A central component of the epigenome is the pattern of histone post-translational modifications that play a critical role in the formation of specific chromatin states. During DNA replication, parental nucleosomes are disrupted and re-deposited on the nascent DNA near their original location to preserve the spatial memory of the epigenetic modifications. Newly synthesized histones must also be incorporated into the nascent chromatin to maintain nucleosome density. Transfer of modification patterns from parental histones to new histones is a fundamental step in epigenetic inheritance. Whether new histones play an active or passive role in epigenetic inheritance is unknown. Here we report that HAT1, which acetylates lysines 5 and 12 of newly synthesized histone H4 during replication-coupled chromatin assembly, regulates the epigenetic inheritance of chromatin states. HAT1 regulates the accessibility of large domains of heterochromatin termed HAT1-dependent Accessibility Domains (HADs). HADs are mega base-scale domains that comprise ~10% of the mouse genome. HAT1 functions as a global negative regulator of H3 K9me2/3 and HADs correspond to the regions of the genome that display HAT1-dependent increases in H3 K9me3 peak density. HADs display a high degree of overlap with a subset of Lamin-Associated Domains (LADs). HAT1 is required to maintain nuclear structure and integrity. These results indicate that HAT1 and the acetylation of newly synthesized histones are critical regulators of the epigenetic inheritance of heterochromatin and suggest a new mechanism for the epigenetic regulation of nuclear lamina-heterochromatin interactions.

genomics↗

bioRxiv2020

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma

BackgroundEwing sarcoma is an aggressive bone cancer in children and young adults that contains a pathognomonic chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the low-complexity amino-terminal portion of EWS to the carboxyl-terminus of FLI. The FLI portion contains an ETS DNA-binding domain and adjacent amino- and carboxyl-regions. Early studies using non-Ewing sarcoma cellular models provided conflicting information on the role of these adjacent regions in the oncogenic function of EWS/FLI. We therefore sought to define the specific contributions of each FLI region to EWS/FLI activity in an appropriate Ewing model, and in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. MethodsWe used a "knock-down/rescue" system to replace endogenous EWS/FLI expression with mutant forms of the protein in Ewing sarcoma cells and tested these for oncogenic transformation using soft-agar colony forming assays. These data were complemented by DNA-binding assays using fluorescence anisotropy, genomic localization assays using CUT&RUN, transcriptional regulation studies using luciferase reporter assays and RNA-sequencing, as well as chromatin accessibility assays using ATAC-sequencing. ResultsWe found that the DNA-binding domain and short flanking regions of FLI were required for oncogenic transformation, gene expression, genomic localization and chromatin accessibility when fused to the amino-terminal EWS-portion from EWS/FLI, but that the remaining regions of FLI were dispensable for these functions. Removal of a carboxyl-terminal alpha-helix from the short flanking regions of the DNA-binding domain of FLI created a hypomorphic EWS/FLI that retained normal DNA binding, genomic localization, and chromatin accessibility, but had significantly restricted transcriptional activity and a near total loss of oncogenic transformational capacity. ConclusionsThe DNA-binding domain and carboxyl-terminal short flanking region of FLI are the only portions of FLI required for EWS/FLI-mediated oncogenic transformation in a Ewing sarcoma cellular context. In addition to the well-defined DNA-binding function of FLI, this additional alpha-helix immediately downstream of the DNA-binding domain contributes a previously-undescribed function in gene regulation and oncogenic transformation. Understanding the function of this critical region could provide new therapeutic opportunities to target EWS/FLI in Ewing sarcoma.

cancer biology↗