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Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating β-Catenin. Silencing Wnt1 impairs mechanically induced β-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/β-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material., Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification., (©2023 American Association for Cancer Research.)

Objective: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC., Design: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC., Results: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients., Conclusion: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the "stemness" maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) displays a dismal prognosis due to late diagnosis and high chemoresistance incidence. For advanced disease stages or patients with comorbidities, treatment options are limited to gemcitabine alone or in combination with other drugs. While gemcitabine resistance has been widely attributed to the levels of one of its targets, RRM1, the molecular consequences of gemcitabine resistance in PDAC remain largely elusive. Here we sought to identify genomic, epigenomic, and transcriptomic events associated with gemcitabine resistance in PDAC and their potential clinical relevance. We found that gemcitabine-resistant cells displayed a coamplification of the adjacent RRM1 and STIM1 genes. Interestingly, RRM1, but not STIM1, was required for gemcitabine resistance, while high STIM1 levels caused an increase in cytosolic calcium concentration. Higher STIM1-dependent calcium influx led to an impaired endoplasmic reticulum stress response and a heightened nuclear factor of activated T-cell activity. Importantly, these findings were confirmed in patient and patient-derived xenograft samples. Taken together, our study uncovers previously unknown biologically relevant molecular properties of gemcitabine-resistant tumors, revealing an undescribed function of STIM1 as a rheostat directing the effects of calcium signaling and controlling epigenetic cell fate determination. It further reveals the potential benefit of targeting STIM1-controlled calcium signaling and its downstream effectors in PDAC. SIGNIFICANCE: Gemcitabine-resistant and some naïve tumors coamplify RRM1 and STIM1 , which elicit gemcitabine resistance and induce a calcium signaling shift, promoting ER stress resistance and activation of NFAT signaling., (©2021 American Association for Cancer Research.)

The role of histone ubiquitination in directing cell lineage specification is only poorly understood. Our previous work indicated a role of the histone 2B ubiquitin ligase RNF40 in controlling osteoblast differentiation in vitro. Here, we demonstrate that RNF40 has a stage-dependent function in controlling osteoblast differentiation in vivo. RNF40 expression is essential for early stages of lineage specification, but is dispensable in mature osteoblasts. Paradoxically, while osteoblast-specific RNF40 deletion led to impaired bone formation, it also resulted in increased bone mass due to impaired bone cell crosstalk. Loss of RNF40 resulted in decreased osteoclast number and function through modulation of RANKL expression in OBs. Mechanistically, we demonstrate that Tnfsf11 (encoding RANKL) is an important target gene of H2B monoubiquitination. These data reveal an important role of RNF40-mediated H2B monoubiquitination in bone formation and remodeling and provide a basis for exploring this pathway for the treatment of conditions such as osteoporosis or cancer-associated osteolysis.

Aim: Tieg1 is involved in multiple signalling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood., Methods: We have utilized Tieg1 knockout (KO) mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and extensor digitorum longus (EDL) muscles. RNA sequencing, immunoblotting, transmission electron microscopy, MRI, NMR, histochemical and mitochondrial function assays were performed., Results: Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of succinate dehydrogenase staining and a decrease in cytochrome c oxidase (COX) enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase (CS) activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in CS and respiratory chain complex activities were identified in KO soleus. 1 H-NMR spectra revealed no significant metabolic difference between wild-type and KO muscles. However, 31 P spectra revealed a significant decrease in phosphocreatine and ATPγ. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice., Conclusion: Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: Disruptor of telomeric silencing 1-like (DOT1L) is a non-SET domain containing methyltransferase known to catalyze mono-, di-, and tri-methylation of histone 3 on lysine 79 (H3K79me). DOT1L-mediated H3K79me has been implicated in chromatin-associated functions including gene transcription, heterochromatin formation, and DNA repair. Recent studies have uncovered a role for DOT1L in the initiation and progression of leukemia and other solid tumors. The development and availability of small molecule inhibitors of DOT1L may provide new and unique therapeutic options for certain types or subgroups of cancer., Methods: In this study, we examined the role of DOT1L in DNA double-strand break (DSB) response and repair by depleting DOT1L using siRNA or inhibiting its methyltransferase activity using small molecule inhibitors in colorectal cancer cells. Cells were treated with different agents to induce DNA damage in DOT1L-depleted or -inhibited cells and analyzed for DNA repair efficiency and survival. Further, rectal cancer patient samples were analyzed for H3K79me3 levels in order to determine whether it may serve as a potential marker for personalized therapy., Results: Our results indicate that DOT1L is required for a proper DNA damage response following DNA double-strand breaks by regulating the phosphorylation of the variant histone H2AX (γH2AX) and repair via homologous recombination (HR). Importantly, we show that small molecule inhibitors of DOT1L combined with chemotherapeutic agents that are used to treat colorectal cancers show additive effects. Furthermore, examination of H3K79me3 levels in rectal cancer patients demonstrates that lower levels correlate with a poorer prognosis., Conclusions: In this study, we conclude that DOT1L plays an important role in an early DNA damage response and repair of DNA double-strand breaks via the HR pathway. Moreover, DOT1L inhibition leads to increased sensitivity to chemotherapeutic agents and PARP inhibition, which further highlights its potential clinical utility. Our results further suggest that H3K79me3 can be useful as a predictive and or prognostic marker for rectal cancer patients.

The genes encoding MDM2 and CDK4 are frequently co-amplified in sarcomas, and inhibitors to both targets are approved or clinically tested for therapy. However, we show that inhibitors of MDM2 and CDK4 antagonize each other in their cytotoxicity towards sarcoma cells. CDK4 inhibition attenuates the induction of p53-responsive genes upon MDM2 inhibition. Moreover, the p53 response was also attenuated when co-depleting MDM2 and CDK4 with siRNA, compared to MDM2 single knockdown. The complexes of p53 and MDM2, as well as CDK4 and Cyclin D1, physically associated with each other, suggesting direct regulation of p53 by CDK4. Interestingly, CDK4 inhibition did not reduce p53 binding or histone acetylation at promoters, but rather attenuated the subsequent recruitment of RNA Polymerase II. Taken together, our results suggest that caution must be used when considering combined CDK4 and MDM2 inhibition for patient treatment. Moreover, they uncover a hitherto unknown role for CDK4 and Cyclin D1 in sustaining p53 activity.

Although inhibitors of bromodomain and extra terminal domain (BET) proteins show promising clinical activity in different hematologic malignancies, a systematic analysis of the consequences of pharmacological BET inhibition on healthy hematopoietic (stem) cells is urgently needed. We found that JQ1 treatment decreases the numbers of pre-, immature and mature B cells while numbers of early pro-B cells remain constant. In addition, JQ1 treatment increases apoptosis in T cells, all together leading to reduced cellularity in thymus, bone marrow and spleen. Furthermore, JQ1 induces proliferation of long-term hematopoietic stem cells, thereby increasing stem cell numbers. Due to increased numbers, JQ1-treated hematopoietic stem cells engrafted better after stem cell transplantation and repopulated the hematopoietic system significantly faster after sublethal myeloablation. As quantity and functionality of hematopoietic stem cells determine the duration of life-threatening myelosuppression, BET inhibition might benefit patients in myelosuppressive conditions., (Copyright © 2018 Ferrata Storti Foundation.)

The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a particularly dismal prognosis. Histone deacetylases (HDAC) are epigenetic modulators whose activity is frequently deregulated in various cancers including PDAC. In particular, class-I HDACs (HDAC 1, 2, 3 and 8) have been shown to play an important role in PDAC. In this study, we investigated the effects of the class I-specific HDAC inhibitor (HDACi) 4SC-202 in multiple PDAC cell lines in promoting tumor cell differentiation. We show that 4SC-202 negatively affects TGFβ signaling and inhibits TGFβ-induced epithelial-to-mesenchymal transition (EMT). Moreover, 4SC-202 markedly induced p21 (CDKN1A) expression and significantly attenuated cell proliferation. Mechanistically, genome-wide studies revealed that 4SC-202-induced genes were enriched for Bromodomain-containing Protein-4 (BRD4) and MYC occupancy. BRD4, a well-characterized acetyllysine reader, has been shown to play a major role in regulating transcription of selected subsets of genes. Importantly, BRD4 and MYC are essential for the expression of a subgroup of genes induced by class-I HDACi. Taken together, our study uncovers a previously unknown role of BRD4 and MYC in eliciting the HDACi-mediated induction of a subset of genes and provides molecular insight into the mechanisms of HDACi action in PDAC., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Targeting the Mdm2 oncoprotein by drugs has the potential of re-establishing p53 function and tumor suppression. However, Mdm2-antagonizing drug candidates, e. g. Nutlin-3a, often fail to abolish cancer cell growth sustainably. To overcome these limitations, we inhibited Mdm2 and simultaneously a second negative regulator of p53, the phosphatase Wip1/PPM1D. When combining Nutlin-3a with the Wip1 inhibitor GSK2830371 in the treatment of p53-proficient but not p53-deficient cells, we observed enhanced phosphorylation (Ser 15) and acetylation (Lys 382) of p53, increased expression of p53 target gene products, and synergistic inhibition of cell proliferation. Surprisingly, when testing the two compounds individually, largely distinct sets of genes were induced, as revealed by deep sequencing analysis of RNA. In contrast, the combination of both drugs led to an expression signature that largely comprised that of Nutlin-3a alone. Moreover, the combination of drugs, or the combination of Nutlin-3a with Wip1-depletion by siRNA, activated p53-responsive genes to a greater extent than either of the compounds alone. Simultaneous inhibition of Mdm2 and Wip1 enhanced cell senescence and G2/M accumulation. Taken together, the inhibition of Wip1 might fortify p53-mediated tumor suppression by Mdm2 antagonists., Competing Interests: The authors declare no conflict of interest.

The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The estrogen receptor α (ERα) controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to control gene transcription. A deeper understanding of these transcriptional mechanisms may uncover therapeutic targets for ERα-dependent cancers. We show that BRD4 regulates ERα-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II (RNAPII) and histone H2B monoubiquitination. Consistently, BRD4 activity is required for proliferation of ER(+) breast and endometrial cancer cells and uterine growth in mice. Genome-wide studies revealed an enrichment of BRD4 on transcriptional start sites of active genes and a requirement of BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we demonstrate that BRD4 occupancy on distal EREs enriched for H3K27ac is required for recruitment and elongation of RNAPII on EREs and the production of ERα-dependent enhancer RNAs. These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)