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Background Chromosome conformation within the nucleus is essential for genome function. These have primarily been studied at the scale of loops and compartments, or at lower spatial resolution using traditional in situ hybridization in chemically fixed samples. However, the mesoscale organization of single chromosomes in vivo, shaped by the interplay between chromatin architectural proteins and histone modifications, remains partially understood. In this study, we interrogated the mesoscale conformations of interphase chromosomes in live human osteoblasts and transformed osteosarcoma cells, focusing on chromosome 19. Results Chromosome conformations were quantified by the aspect ratio of the principal axes of gyration tensors. In osteoblasts, approximately 81% of chromosome 19 are observed to consist of regions characterized by highly extended organizations, with aspect ratios approximately four times greater than those of spheres. In contrast, in osteosarcoma cells, the chromosome displays an extensively collapsed conformation, with aspect ratios more closely approximately that of a sphere. In both cell types, the chromosome’s conformation is bimodal and the balance between these two modes differs very significantly between the two cell types. While the mesoscopic conformation is considerably stable, it is superimposed on dynamic, smaller scale regions. Additional results reveal that this significant conformational shift is independent of the cell cycle but co-regulated by CTCF, cohesion, and H3K27 modifications. Conclusions Our findings provide new insights into the coordinated complex regulatory mechanisms governing mesoscale chromosome organization in normal and transformed osteogenic tissues.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor. Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage with an aggressive subtype caused by translocations involving PAX3- /PAX7-FOXO1 chimeric transcription factors. Here the authors show that the BRG1-containing BAF complex is overexpressed and acts largely independently of the PAX3-FOXO1 chimera on chromatin to result in a myogenic differentiation blockade in this malignancy.

The constitutively active androgen receptor (AR) splice variant 7 (AR-V7) plays an important role in the progression of castration-resistant prostate cancer (CRPC). Although biomarker studies established the role of AR-V7 in resistance to AR-targeting therapies, how AR-V7 mediates genomic functions in CRPC remains largely unknown. Using a ChIP-exo approach, we show AR-V7 binds to distinct genomic regions and recognizes a full-length androgen-responsive element in CRPC cells and patient tissues. Remarkably, we find dramatic differences in AR-V7 cistromes across diverse CRPC cells and patient tissues, regulating different target gene sets involved in CRPC progression. Surprisingly, we discover that HoxB13 is universally required for and colocalizes with AR-V7 binding to open chromatin across CRPC genomes. HoxB13 pioneers AR-V7 binding through direct physical interaction, and collaborates with AR-V7 to up-regulate target oncogenes. Transcriptional coregulation by HoxB13 and AR-V7 was further supported by their coexpression in tumors and circulating tumor cells from CRPC patients. Importantly, HoxB13 silencing significantly decreases CRPC growth through inhibition of AR-V7 oncogenic function. These results identify HoxB13 as a pivotal upstream regulator of AR-V7–driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7–driven prostate tumors.

In the past decade, we have seen the emergence of sequence-based methods to understand chromosome organization. With the confluence of in situ approaches to capture information on looping, topological domains, and larger chromatin compartments, understanding chromatin-driven disease is becoming feasible. Excitingly, recent advances in single molecule imaging with capacity to reconstruct “bulk-cell” features of chromosome conformation have revealed cell-to-cell chromatin structural variation. The fundamental question motivating our analysis of the literature is, can altered chromatin structure drive tumorigenesis? As our community learns more about rare disease, including low mutational frequency cancers, understanding “chromatin-driven” pathology will illuminate the regulatory structures of the genome. We describe recent insights into altered genome architecture in human cancer, highlighting multiple pathways toward disruptions of chromatin structure, including structural variation, noncoding mutations, metabolism, and de novo mutations to architectural regulators themselves. Our analysis of the literature reveals that deregulation of genome structure is characteristic in distinct classes of chromatin-driven tumors. As we begin to integrate the findings from single cell imaging studies and chromatin structural sequencing, we will be able to understand the diversity of cells within a common diagnosis, and begin to define structure–function relationships of the misfolded genome.

Ewing sarcoma is an aggressive bone cancer of children and young adults defined by the presence of a chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the amino-terminal domain of EWS to the carboxyl-terminus of FLI. The EWS portion is an intrinsically disordered transcriptional regulatory domain, while the FLI portion contains an ETS DNA-binding domain and two flanking regions of unknown function. Early studies using non-Ewing sarcoma models provided conflicting information on the roles of each domain of FLI in EWS/FLI oncogenic function. We therefore sought to define the specific contributions of each FLI domain to EWS/FLI activity in a well-validated Ewing sarcoma model and, in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. We analyzed a series of engineered EWS/FLI mutants with alterations in the FLI portion using a variety of assays. Fluorescence anisotropy, CUT&RUN, and ATAC-sequencing experiments revealed that the isolated ETS domain is sufficient to maintain the normal DNA-binding and chromatin accessibility function of EWS/FLI. In contrast, RNA-sequencing and soft agar colony formation assays revealed that the ETS domain alone was insufficient for transcriptional regulatory and oncogenic transformation functions of the fusion protein. We found that an additional alpha-helix immediately downstream of the ETS domain is required for full transcriptional regulation and EWS/FLI-mediated oncogenesis. These data demonstrate a previously unknown role for FLI in transcriptional regulation that is distinct from its DNA-binding activity. This activity is critical for the cancer-causing function of EWS/FLI and may lead to novel therapeutic approaches.

Glucocorticoids (GCs) have been widely used as coadjuvants in the treatment of solid tumours, but GC treatment may be associated with poor pharmacotherapeutic response or prognosis. The genomic action of GC in these tumours is largely unknown. Here we find that dexamethasone (Dex, a synthetic GC)-regulated genes in triple-negative breast cancer (TNBC) cells are associated with drug resistance. Importantly, these GC-regulated genes are aberrantly expressed in TNBC patients and are associated with unfavourable clinical outcomes. Interestingly, in TNBC cells, Compound A (CpdA, a selective GR modulator) only regulates a small number of genes not involved in carcinogenesis and therapy resistance. Mechanistic studies using a ChIP-exo approach reveal that Dex- but not CpdA-liganded glucocorticoid receptor (GR) binds to a single glucocorticoid response element (GRE), which drives the expression of pro-tumorigenic genes. Our data suggest that development of safe coadjuvant therapy should consider the distinct genomic function between Dex- and CpdA-liganded GR. Glucocorticoids are widely used as coadjuvants in the treatment of solid tumours. Here, Chen et al . show that genes regulated by dexamethasone- but not Compound A-liganded glucocorticoid receptor are associated with therapy resistance and unfavourable clinical outcomes in triple-negative breast cancer.

C omprehensive identification of driver mutations in prostate cancer can serve to enhance our understanding of the disease and expand the use of available treatment options. Two recent and comple- mentary studies from Barbieri et al. and Grasso et al.

We present a rare case of endogenous endophthalmitis caused by Citrobacter koseri. A 69-year-old woman with a history of poorly controlled diabetes and a protracted urinary tract infection (UTI) presented with a painful swollen left eye. There was no history of eye surgery or trauma. Imaging revealed an abscess in the right kidney. Although endophthalmitis is very rare in healthy patient, it is more common in the immunocompromised. In this patient, several multiple system illnesses including poorly controlled diabetes appear to have worked synergistically to make endophthalmitis a realistic complication of an otherwise isolated and remote source of infection, in this case pyelonephritis. Endophthalmitis, in the absence of an obvious exogenous cause, should be investigated thoroughly to exclude metastatic microbial spread. In addition, chronic features of UTI in a patient with poorly controlled diabetes or who is otherwise immunosuppressed warrant the exclusion of an underlying renal abscess.

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, correlates with worse patient overall survival than . 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. Partially overlapping gene signatures are activated by PAX3/7::FOXO1 in vivoModified MNase ChIP reveals PAX3/7::FOXO1 bind nucleosomal and subnucleosomal DNAPAX7::FOXO1 binds degenerate paired/homeobox motifs within nucleosome targetsEach fusion engages distinct nucleosomal gene targets.

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.