Biology subjects

Kucinski, J.

Publications and source records attributed to Kucinski, J..

6 recordsLinked to original sources

bioRxiv2026

HES3-dependent regulatory functions in development and fusion-positive rhabdomyosarcoma

HES3/Her3 is a transcription factor that functions in non-canonical STAT3 signaling to promote the renewal of neural stem cells and has roles in multiple cancer contexts. To study its role in development and disease, we previously generated a CRISPR/Cas9 zebrafish knockout of her3, the ortholog to human HES3. HES3 is also a cooperating gene in fusion-positive rhabdomyosarcoma, an aggressive pediatric cancer, where HES3 prevents terminal myogenic differentiation, and high expression correlates with worse patient outcomes. Here, we utilize our her3/HES3 knockout model with chromatin and transcriptional profiling techniques to assess its role during early zebrafish gastrulation with the goal of understanding the function of this transcription factor and how these activities are co-opted in cancer. We found that the Her3/HES3 preferential binding motif is distinct from other HES-family members, including a CG-rich E-box motif, that it leverages to modulate the expression of genes involved in neurogenesis and WNT signaling. We also determined that motif preferences of Her3/HES3 altered its interactions with DNA, allowing it to function canonically as a transcriptional repressor with additional duality as an activator. In the context of PAX3::FOXO1, a monogenic driver of fusion-positive rhabdomyosarcoma, we find that Her3/HES3 plays an influential role in modulating the initial activities of this core oncogenic transcription factor. Upon expressing PAX3::FOXO1 in early developing zebrafish embryos, her3 knockout allowed for enhanced activation of neural programs, which are observed in the human disease, along with alterations to cell adhesion programs. Patient tumor samples could be clustered and stratified based on HES3 expression alone. We saw that patient PAX3::FOXO1-positive tumors with high levels of HES3 contained a more neural identity than those with low levels of HES3, altogether suggesting HES3 plays a critical role in regulating this neural signature during both the initial functions of PAX3::FOXO1 and in established tumors.

cancer biology↗

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↗

bioRxiv2024

Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, functions as a chimeric gain-of-function transcription factor. An incomplete understanding of PAX3::FOXO1s in vivo epigenetic mechanisms has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. We investigated PAX3::FOXO1s recognition of chromatin and subsequent transcriptional consequences. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired-box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a new mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.

cancer biology↗

bioRxiv2024

Highly quantitative measurement of differential protein-genome binding with PerCell chromatin sequencing

We report a universal strategy for 2D chromatin sequencing, to increase uniform data analyses and sharing across labs, and to facilitate highly quantitative comparisons across experimental conditions. Within our system, we provide wetlab and drylab tools for researchers to establish and analyze protein-genome binding data with PerCell ChIP-seq. Our methodology is virtually no cost and flexible, enabling rapid, quantitative, internally normalized chromatin sequencing to catalyze project development in a variety of systems, including in vivo zebrafish epigenomics and cancer cell epigenomics. While we highlight utility in these key areas, our methodology is flexible enough such that rapid comparisons of cellular spike-in versus non spike-in are possible, and generalizability to nuclease-based 2D chromatin sequencing would also be possible within the framework of our pipeline. Through the use of well-defined cellular ratios containing orthologous species chromatin, we enable cross-species comparative epigenomics and highly quantitative low-cost chromatin sequencing with utility across a range of disciplines.

genomics↗

bioRxiv2022

High-resolution map of plastid encoded polymerase binding patterns demonstrates a major role of transcription in chloroplast gene expression

Plastids are endosymbiotic organelles containing their own genomes, which are transcribed by two types of RNA polymerases. One of those enzymes is a bacterial-type, multi-subunit polymerase encoded by the plastid genome. The plastid encoded RNA polymerase (PEP) is required for efficient expression of genes encoding proteins involved in photosynthesis. Despite the importance of PEP, its DNA binding locations have not been studied on the genome-wide scale at high resolution. We established a highly specific approach to detect the genome-wide pattern of PEP binding to chloroplasts DNA using ptChIP-seq. We found that in mature Arabidopsis thaliana chloroplasts, PEP has a complex DNA binding pattern with preferential association at genes encoding rRNA, tRNA, and a subset of photosynthetic proteins. Sigma factors SIG2 and SIG6 strongly impact PEP binding to a subset of tRNA genes and have more moderate effects on PEP binding throughout the rest of the genome. PEP binding is commonly enriched on gene promoters, around transcription start sites. Finally, the levels of PEP binding to DNA are correlated with the levels of RNA accumulation, which allowed estimating the quantitative contribution of transcription to RNA accumulation.

plant biology↗

bioRxiv2019

Functional characterization of RNase H1 proteins in Arabidopsis thaliana

ABSTRACTRNase H1 is an endonuclease specific towards RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. We show that Arabidopsis thaliana contains four RNase H1-like proteins originating from two gene duplication events and alternative splicing. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for activity. Two of those proteins are nuclear, one is localized to mitochondria and one to plastids. While the nuclear RNases H1 are dispensable, the presence of at least one organellar RNase H1 is required for embryonic development. The plastid protein RNH1C affects plastid DNA copy number and sensitivity to hydroxyurea. This indicates that three genomes present in each plant cell are served by at least one specialized RNase H1 protein.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗