Biology subjects

Prekovic, S.

Publications and source records attributed to Prekovic, S..

14 recordsLinked to original sources

bioRxiv2026

Extracellular matrix context shapes morphogenesis and lactation-associated states in human milk-derived mammary organoids

The mammary gland relies on reciprocal interactions between epithelial cells and their surrounding extracellular matrix (ECM) to form and maintain milk-producing tissue structures. Yet these processes remain difficult to study in human model systems. Mammary epithelial cells (MECs) can be isolated noninvasively from breast milk, but whether they generate three-dimensional organoids and respond to matrix cues has been unclear. Here, human milk-derived MECs (milk MECs) spontaneously form complex organoids, including polarized acinar and terminal duct lobular unit-like structures after isolation. To investigate how matrix composition shapes these organoids, milk MECs were cultured in decellularized mammary ECM (dECM), Matrigel, and collagen I. In dECM, milk MECs formed polarized branched networks with aligned actin organization along collagen fibrils, whereas in Matrigel they adopted a more lactation-associated state, marked by {beta}-casein expression and milk fat globules. Together, these findings establish breast milk-derived MEC organoids as a human model to study how ECM context regulates mammary morphogenesis and lactation biology.

cell biology↗

bioRxiv2026

Interdependent androgen and glucocorticoid receptor signalling shapes prostate epithelial homeostasis

Androgen signalling is essential for prostate secretory functions and epithelial cell maintenance, yet how this signal is translated into a transcriptional output remains poorly understood. Androgens functions are primarily mediated by the androgen receptor (AR), which binds hormone response elements similar to those of other steroid receptors, including the glucocorticoid receptor (GR). Here we show that AR and GR are co-expressed in the prostate epithelium, and co-located within nuclear foci. Moreover, GR promotes the formation and dynamics of AR nuclear condensates, and heterodimerizes with AR in a ligand-binding domain-dependent manner. In addition, subtle variations within the hormone response element sequence direct the co-recruitment of AR and GR into activator or repressor complexes to activate or repress gene expression. Finally, prostate-specific deletion of GR in mice disrupts AR nuclear distribution, impairs AR-dependent gene networks involved in epithelial maintenance, and promotes the expression of genes involved in metabolism and cell cycle, resulting in altered tissue homeostasis. Thus, these findings identify GR as an integral component of the AR transcriptional machinery in epithelial cells of the healthy prostate, and reveal how shared cis-regulatory elements are interpreted to generate distinct transcriptional outcomes.

cell biology↗

bioRxiv2026

Classical enhancers couple cis-regulatory logic with transcriptional condensates and 3D genome architecture

Deciphering the regulatory logic of enhancers remains a central question in understanding cell- and tissue-specific gene expression in multicellular organisms. This is particularly pertinent at multipartite enhancer clusters such as super-enhancers, where multiple enhancers contribute to the expression of a single gene. Gene expression has been studied largely through sequence-dependent recruitment of transcription factors (TF) and co-activators, whereas 3D chromatin structure has been attributed to architectural proteins such as cohesion and CTCF1-3. However, the contribution of DNA sequence encoded in enhancers to shaping higher-order genome organization remains poorly understood. Here we show that classical enhancers, embedded within multipartite super-enhancer structures, act as determinant regulatory elements that initiate the gene regulatory cascade by linking DNA sequence recognition to 3D chromatin architecture. Classical enhancers are more evolutionarily conserved and display stronger regulatory activity than facilitator elements, which lack intrinsic enhancer activity but potentiate classical enhancer function. We show that classical enhancers are selectively bound by specific TFs with strong intrinsically disordered regions, such as NFE2L2 in liver cancer cells, capable of driving transcriptional condensate formation through phase separation. NFE2L2 depletion reduced enhancer activity and induced widespread chromatin reorganization, characterized by increased cohesin and CTCF occupancy at super-enhancer boundaries and beyond. This "cohesin clogging" impaired DNA loop extrusion, led to formation of smaller topologically associated domains, and weakened enhancer-promoter contacts. These findings highlight that sequence-specific TFs have multifaceted roles beyond transcriptional control, establishing a direct mechanistic link between enhancer sequence, TF binding, condensate formation, and 3D genome organization, with the regulatory logic being encoded in the DNA sequence itself.

genomics↗

bioRxiv2025

Site-dependent Treg cell transcriptional reprograming in a metastatic colorectal cancer model holds prognostic significance

In colorectal cancer (CRC), tumor-infiltrating regulatory T (Treg) cells suppress anti-tumor immunity, promoting immune evasion and tumor progression. Effective therapies require selectively targeting tumor-infiltrating Treg (TI-Treg) cells while preserving systemic Treg cells, necessitating insight into their adaptations within the tumor microenvironment. Here, CRC-organoids were implanted in the liver of Foxp3eGFP mice to investigate location-specific phenotypic differences in TI-Treg cells. Tumor tissue exhibited an increased proportion of Treg cells and a decrease of effector CD4 and CD8 T cells compared to matched healthy tissue. RNA sequencing of Treg cells isolated from the spleen, primary liver tumor transplant, or metastases identified gene expression profiles previously associated with CRC-related Treg cells in patients. Location-specific differences included elevated expression of WNT-pathway genes in peritoneal TI-Treg cells compared to liver counterparts. Higher expression of genes upregulated in liver TI-Treg cells correlated with poor CRC prognosis. Splenic Treg cells from tumor-bearing mice displayed distinct transcriptional profiles from both their healthy counterparts and TI-Treg cells, suggesting they represent a distinct CD4+ population. Taken together, these findings highlight TI-Treg cells heterogeneity across different tumor sites and the distinct nature of splenic Treg cells in tumor-bearing hosts.

immunology↗

bioRxiv2024

Tumor-derived colorectal cancer organoids induce a unique Treg cell population through direct modulation of CD4+ T cell differentiation.

In colorectal cancer (CRC), increased numbers of tumor-infiltrating CD4+ regulatory T (Treg) cells correlate with tumor development and immunotherapy failure, leading to poor prognosis. However, the molecular and cellular mechanisms governing Treg recruitment, expansion, or differentiation remain unclear. Here, we developed an in vitro co-culture system to assess the capacity of CRC tumors to directly modulate Treg cell differentiation. CD4+ T cells from Foxp3eGFP mice were co-cultured with murine tumor-derived CRC organoids, resulting in a significant increase in Treg cell numbers. This induction of Treg cells was not due to increased proliferation, but rather through differentiation of CD4+ T cells in a TGF{beta}-dependent manner. Human CRC tumor organoids similarly induced Treg cells that exhibited enhanced suppressive capacity compared to TGF{beta}-induced Treg cells. RNA-sequencing analysis identified distinct transcriptional profiles between CRC organoid-induced Treg cells and TGF{beta}-induced Treg cells, with upregulation of key functional signature genes linked to CRC Treg cells in vivo. High expression of genes upregulated in CRC organoid-induced Treg cells correlates with shorter progression free interval and overall survival of CRC patients, highlighting their prognostic potential. Taken together, CRC tumor organoids drive CD4+ differentiation to Treg cells with a phenotype resembling tumor-infiltrating Treg cells. This model can be applied to both understand the molecular mechanisms by which tumors can directly modulate CD4+ T cell differentiation and identify approaches to disrupt Treg cell function and stimulate anti-tumor immunity.

immunology↗

bioRxiv2024

NAC-mediated ribosome localization regulates cell fate and metabolism in intestinal stem cells

Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity, however it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we show that RNA translation is at the root of this interplay. We identify the nascent polypeptide-associated complex (NAC) as a key mediator of this process, and show that it regulates ISC metabolism by relocalizing ribosomes to the mitochondria. Upon NAC inhibition, intestinal cells show decreased import of mitochondrial proteins, which are needed for oxidative phosphorylation, and, consequently, enable the cell to maintain a stem cell identity. Furthermore, we show that overexpression of NAC is sufficient to drive mitochondrial respiration and promote ISC identity. Ultimately, our results reveal the pivotal role of ribosome localization in regulating mitochondrial metabolism and ISC function. TeaserThe location of ribosomes in cells is regulated, and defines the fate of intestinal stem cells.

molecular biology↗

bioRxiv2024

Glycolytic reprogramming shapes the epigenetic landscape of activated CD4+ T Cells in Juvenile Idiopathic Arthritis

Juvenile Idiopathic Arthritis (JIA) describes a heterogeneous group of autoimmune conditions with an unknown cause and childhood onset. It is characterized by the accumulation of mononuclear cells, notably activated CD4+ memory/effector T (Tmem/Teff) cells, within the synovial fluid of affected joints. JIA CD4+ T cells exhibit a unique epigenomic signature linked to inflammation, however, the molecular mechanisms driving this remain unclear. Here we show that CD4+ T cells isolated from JIA synovial fluid (SF) exhibit abnormal intracellular metabolism marked by heightened glycolysis after activation driving transcriptional reprogramming. Epigenetic profiling between activated healthy controls and JIA patients allowed the definition of specific disease-related enhancers upregulated in SF-derived JIA CD4+ T cells. Pharmacological inhibition of glycolytic flux affected the expression of genes associated with these enhancers. When activated in the presence of JIA SF, CD4+ T cells obtained from healthy control (HC) subjects, displayed heightened glycolytic activity compared to paired plasma. Moreover, this also led to increased H3K27ac at JIA-specific genes. Increased H3K27ac was dependent on glycolytic flux, but not oxidative phosphorylation. Inhibition of glycolysis also specifically affected the transcription of genes upregulated during T cell activation in the presence of SF. Inhibiting the glycolytic enzyme pyruvate dehydrogenase (PDH) reduced JIA-associated gene expression. Taken together, these findings demonstrate that for JIA, the inflammatory microenvironment can modulate T cell activation-driven transcriptional programs through a glycolysis-mediated pathway. Specific targeting of this T cell metabolism-epigenetic axis may provide avenues for intervention during the development of autoinflammatory disease.

immunology↗

bioRxiv2023

A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator

The crosstalk between prostate cancer (PCa) cells and the tumor microenvironment plays a pivotal role in disease progression and metastasis and could provide novel opportunities for patient treatment. Macrophages are the most abundant immune cells in the prostate tumor microenvironment (TME) and are capable of killing tumor cells. To identify genes in the tumor cells that are critical for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified AR, PRKCD, and multiple components of the NF-{kappa}B pathway as hits, whose expression in the tumor cell are essential for being targeted and killed by macrophages. These data position AR signaling as an immunomodulator, and confirmed by androgen-deprivation experiments, that rendered hormone-deprived tumor cells resistant to macrophage-mediated killing. Proteomic analyses showed a downregulation of oxidative phosphorylation in the PRKCD- and IKBKG-KO cells compared to the control, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Furthermore, phosphoproteomic analyses revealed that all hits impaired ferroptosis signaling, which was validated transcriptionally using samples from a neoadjuvant clinical trial with the AR-inhibitor enzalutamide. Collectively, our data demonstrate that AR functions together with the PRKCD and the NF-{kappa}B pathway to evade macrophage-mediated killing. As hormonal intervention represents the mainstay therapy for treatment of prostate cancer patients, our findings may have direct implications and provide a plausible explanation for the clinically observed persistence of tumor cells despite androgen deprivation therapy.

cancer biology↗

bioRxiv2023

Crosstalk between the glucocorticoid and mineralocorticoid receptor boosts glucocorticoid-induced killing of multiple myeloma cells

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that culminates in improved myeloma cell killing. We show that the GR agonist Dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist Spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR-MR homodimerization, while leaving GR-GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

cancer biology↗

bioRxiv2022

PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

How steroid hormone receptors (SHRs) orchestrate transcriptional activity remains only partly understood. Upon activation, SHRs bind the genome and recruit their co-regulators, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we comprehensively dissected the Glucocorticoid Receptor (GR) co-regulatory complex involved in gene-target regulation. We describe a novel functional cross-talk between PAXIP1 and the cohesin subunit STAG2 that is critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on the genome, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. Moreover, in lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.

genomics↗

bioRxiv2021

Single-cell ATAC and RNA sequencing reveal pre-existing and persistent subpopulations of cells associated with relapse of prostate cancer

Prostate cancer is profoundly heterogeneous and patients would benefit from methods that stratify clinically indolent from more aggressive forms of the disease. We employed single-cell assay for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identified pre-existing and treatment-persistent cell subpopulations that possess transcriptional stem-like features and regenerative potential when subjected to treatment. We found distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent stem-like cells were able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal hitherto unrecognized molecular predictors of treatment response. This suggests that high analytical resolution of pre-clinical models may powerfully inform clinical decision-making.

cancer biology↗

bioRxiv2021

Perturbations in 3D genome organization can promote acquired drug resistance

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.

molecular biology↗

bioRxiv2020

Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied AR and GR in an equivalent cellular context. Analysis of chromatin and sequence features suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the results of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in selectively guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared between AR and GR shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, we find that shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

genomics↗

bioRxiv2019

Glucocorticoids regulate cancer cell dormancy

The glucocorticoid receptor directly regulates thousands of genes across the human genome in a cell-type specific manner, governing various aspects of homeostasis. The influence of the glucocorticoid receptor is also seen in various pathologies, including cancer, where it has been linked to tumorigenesis, metastasis, apoptosis resistance, and therapy bypass. Nonetheless, the direct genetic and molecular underpinnings of glucocorticoid action in cancer remain elusive. Here, we dissected the glucocorticoid receptor signalling axis and uncovered the mechanism of glucocorticoid-mediated cancer cell dormancy. Upon glucocorticoid receptor activation cancer cells undergo quiescence, subserved by cell cycle arrest through CDKN1C and reprogramming of signalling orchestrated via FOXO1/IRS2. Strikingly, co-expression of these three genes, directly regulated by glucocorticoid-induced chromatin looping, correlates with a benign molecular phenotype across human cancers, whereas triple loss is associated with increased expression of proliferation/aggressiveness markers. Finally, we show that the glucocorticoid receptor signalling axis is inactivated by alterations of either the chromatin remodelling complex or TP53 in vitro and in vivo. Our results indicate that the activation of the glucocorticoid receptor leads to cancer cell dormancy, which has several implications in terms of glucocorticoid use in cancer therapy.

cancer biology↗