The Notch signaling pathway is pivotal in regulating cell differentiation, stem cell maintenance and oncogenesis. While several Notch inhibitors have been developed, effective small molecule activators are scarce. Here, we identify a small molecule that robustly activates Notch signaling, leading to significant upregulation of Notch target genes in several human cell lines and in Danio rerio embryos. Mechanistically, this compound induces the expression of activated NOTCH3 protein via a cryptic internal promoter within the NOTCH3 locus. Notch induction is markedly diminished upon genetic depletion of NOTCH3 or the canonical Notch transcription factor RBPJ. Furthermore, we demonstrate that the compound inhibits complex I of the respiratory chain. This is accompanied by a raise of cytosolic Ca2+ concentration, which is pivotal for the induction of NOTCH3. Functionally, the Notch inducer enforces cell-cycle arrest and promotes terminal differentiation in acute myeloid leukemia (AML) cells. These findings suggest that this small molecule serves as a potent tool for modulating Notch signaling and holds therapeutic potential for Notch-responsive malignancies.

Histone replacement systems are valuable tools for studying histone modifications, but in vertebrates this is so far only possible by labor-intensive CRISPR base editing of each single histone gene. To facilitate such studies, we developed an alternative method and conducted proof-of-principle experiments using histone 2B (H2B) as an example. This method relies on shRNA targeted degradation of endogenous histone mRNA and simultaneous re-expression of a replacement histone. Due to their limited histone gene number, chicken cells proved suitable as a tetrapod model system for this approach. In the first-generation system we developed an shRNA design tool that identified shRNAs leading to efficient downregulation of each of the canonical histones using a single doxycycline (Dox)-inducible shRNA. As H2B knockdown cells are not viable, they were rescued by simultaneous Dox-inducible re-expression of H2B. Since the comparability between wild-type and mutated histone variants is limited due to random chromosomal integration, we developed a second-generation "inducible knockdown-re-expression" system. This new version employs genomic landing pads to facilitate Bxb1 recombinase-mediated cassette exchange with mutated histones. The system successfully rescued reconstituted cells from histone depletion-induced cell death, but requires optimization to align histone expression levels with endogenous levels. Overall, this system offers a promising new method for studying specific histone modifications in chicken cells.

The accurate determination of protein-protein interactions (PPIs) within living cells is essential for our understanding of cellular processes. Molecular interactions can be determined through complementation systems, in which the spatial proximity of two molecular fragments reconstitutes their activity. Among these, NanoLuc Binary Technology (NanoBiT®) utilizes the functional complementation of a large (LgBiT) and a small (SmBiT) luciferase fragment, but its reliability and biological interpretability depends critically on balanced expression of the interacting proteins. To ensure a highly reproducible and finely controlled expression of the interaction partners, we developed a novel plasmid system allowing stable and biallelic integration of NanoLuc plasmids into the safe harbor locus on human chromosome 19. To avoid overexpression artifacts the expression of both interaction partners is precisely regulated by the Tet-On system. We used this inducible binary technology (iBiT) system to investigate a largely unexplored interaction between the full-length isoform 1 and the N-terminally truncated isoform 2 of p62/Sequestosome 1. These experiments demonstrate that cell lines stably expressing iBiT constructs enable highly reproducible, longitudinal, and time-resolved analyses of protein-protein interactions, providing a robust platform for assessing both protein-protein interaction dynamics and their pharmacological modulation in intact cells.

The protein interactome of p65/RELA, the most active subunit of the transcription factor (TF) NF-κB, has not been previously determined in living cells. Using p65-miniTurbo fusion proteins and biotin tagging, we identify >350 RELA interactors from untreated and IL-1α-stimulated cells, including many TFs (47% of all interactors) and >50 epigenetic regulators belonging to different classes of chromatin remodeling complexes. A comparison with the interactomes of two point mutants of p65 reveals that the interactions primarily require intact dimerization rather than DNA-binding properties. A targeted RNAi screen for 38 interactors and subsequent functional transcriptome and bioinformatics studies identify gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3/TFEB, or S100A8/A9. The large, dynamic and versatile high-resolution interactome of RELA and its gene regulatory logics provides a rich resource and a new framework for explaining how RELA cooperativity determines gene expression patterns.

The well-studied NF-κB signaling system is a key mediator of the inflammatory response. Large-scale sequencing studies in humans now allow initial insights into non-essential human genes in which both alleles carry mutations that prevent protein expression or function. Here, we compiled the non-essential genes identified in various sequencing studies and analyzed the occurrence of knockouts in the human NF-κB signaling system. This revealed a lower knockout frequency in the NF-κB system compared to the entire genome. Since drugs inhibiting NF-κB pathway components were unsuccessful in clinical trials so far, the naturally occurring knockouts of NF-κB and its upstream regulators could provide new candidates for therapeutic intervention. To investigate the potential functional importance of posttranslational modifications (PTMs) occurring on NF-κB components, we analyzed not only their evolutionary conservation but also, as a second criterion, their genetic constraint in the sequenced individuals. This approach revealed the absence of missense mutations at key modification sites involved in NF-κB activation and identified additional candidate sites for future studies.

As cancer cell specific rewiring of metabolic networks creates potential therapeutic opportunities, we conducted a synthetic lethal screen utilizing inhibitors of metabolic pathways. Simultaneous administration of (R)-GNE-140 and BMS-986205 (Linrodostat) preferentially halted proliferation of ovarian cancer cells, but not of their non-oncogenically transformed progenitor cells. While (R)-GNE-140 inhibits lactate dehydrogenase (LDH)A/B and thus effective glycolysis, BMS-986205, in addition to its known inhibitory activity on Indoleamine 2,3-dioxygenase (IDO1), also restricts oxidative phosphorylation (OXPHOS), as revealed here. BMS-986205, which is being tested in multiple Phase III clinical trials, inhibits the ubiquinone reduction site of respiratory complex I and thus compromises mitochondrial ATP production. The energetic catastrophe caused by simultaneous interference with glycolysis and OXPHOS resulted in either cell death or the induction of senescence in tumor cells, with the latter being eliminated by senolytics. The frequent synergy observed with combined inhibitor treatment was comprehensively confirmed through testing on tumor cell lines from the DepMap panel and on human colorectal cancer organoids. These experiments revealed highly synergistic activity of the compounds in a third of the tested tumor cell lines, correlating with alterations in genes with known roles in metabolic regulation and demonstrating the therapeutic potential of metabolic intervention.

The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.

The family of hypoxia-inducible transcription factors (HIF) is typically activated by low oxygen concentrations. We observed a transient increase of the HIF-1α protein levels during the G1 phase of the cell cycle, designated as G1-HIF. This cell cycle-regulated form of HIF ensures cell proliferation and survival under conditions of metabolic stress by increasing the levels of key metabolites during the cell cycle. The generation of G1-HIF depends on an AMPK-mediated signaling pathway.

The protein interactome of p65 / RELA, the most active subunit of the transcription factor (TF) NF-κB, has not been previously determined in living cells. Using p65-miniTurbo fusion proteins, we identified by biotin tagging > 350 RELA interactors from untreated and IL-1α-stimulated cells, including many TFs (47 % of all interactors) and > 50 epigenetic regulators belonging to different classes of chromatin remodeling complexes. According to point mutants of p65, the interactions primarily require intact dimerization rather than DNA binding properties. A targeted RNAi screen for 38 interactors and subsequent functional transcriptome and bioinformatics studies identified gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3 / TFEB or S100A8 / A9. The remarkably large, dynamic and versatile high resolution interactome of RELA and its gene-regulatory logics provides a rich resource and a new framework for explaining how RELA cooperativity determines gene expression patterns.Highlights ### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACT The large group of negative-strand RNA viruses (NSVs) comprises many important pathogens. To identify conserved patterns in host responses, we systematically compared changes in the cellular RNA levels after infection of human hepatoma cells with nine different NSVs of different virulence degrees. RNA sequencing experiments indicated that the amount of viral RNA in host cells correlates with the number of differentially expressed host cell transcripts. Time-resolved differential gene expression analysis revealed a common set of 178 RNAs that are regulated by all NSVs analyzed. A newly developed open access web application allows downloads and visualizations of all gene expression comparisons for individual viruses over time or between several viruses. Most of the genes included in the core set of commonly differentially expressed genes (DEGs) encode proteins that serve as membrane receptors, signaling proteins and regulators of transcription. They mainly function in signal transduction and control immunity, metabolism, and cell survival. One hundred sixty-five of the DEGs encode host proteins from which 47 have already been linked to the regulation of viral infections in previous studies and 89 proteins form a complex interaction network that may function as a core hub to control NSV infections. IMPORTANCE The infection of cells with negative-strand RNA viruses leads to the differential expression of many host cell RNAs. The differential spectrum of virus-regulated RNAs reflects a large variety of events including anti-viral responses, cell remodeling, and cell damage. Here, these virus-specific differences and similarities in the regulated RNAs were measured in a highly standardized model. A newly developed app allows interested scientists a wide range of comparisons and visualizations.

The role of hypoxia-inducible factor (HIF)-1α in the control of proliferation under non-hypoxic conditions has been investigated in numerous studies, but does not yield a coherent picture. Therefore, we conducted this meta-analysis of existing literature to systematically evaluate the role of HIF-1α, based on a number of inclusion and exclusion criteria. Studies analyzing non-transformed, primary cells showed a largely heterogeneous distribution of pro-proliferative, anti-proliferative or absent functions for HIF-1α, which are co-determined by several parameters, including the type and age of the cell and its localization in tissues and organs. In contrast, the analyses of tumor cells showed a predominantly pro-proliferative role of HIF-1α by cell-intrinsic and cell-extrinsic molecular mechanism not yet understood.

Of the seven in absentia homologue (SIAH) family, three members have been identified in the human genome. In contrast to the E3 ubiquitin ligase encoding SIAH1 and SIAH2, little is known on the regulation and function of SIAH3 in tumorigenesis. In this study, we reveal that SIAH3 is frequently epigenetically silenced in different cancer entities, including cutaneous melanoma, lung adenocarcinoma and head and neck cancer. Low SIAH3 levels correlate with an impaired survival of cancer patients. Additionally, induced expression of SIAH3 reduces cell proliferation and induces cell death. Functionally, SIAH3 negatively affects cellular metabolism by shifting cells form aerobic oxidative phosphorylation to glycolysis. SIAH3 is localized in the mitochondrion and interacts with proteins involved in mitochondrial ribosome biogenesis and translation. We also report that SIAH3 interacts with ubiquitin ligases, including SIAH1 or SIAH2, and is degraded by them. These results suggest that SIAH3 acts as an epigenetically controlled tumor suppressor by regulating cellular metabolism through the inhibition of oxidative phosphorylation.

The right ventricle (RV) differs developmentally, anatomically and functionally from the left ventricle (LV). Therefore, characteristics of LV adaptation to chronic pressure overload cannot easily be extrapolated to the RV. Mitochondrial abnormalities are considered a crucial contributor in heart failure (HF), but have never been compared directly between RV and LV tissues and cardiomyocytes. To identify ventricle-specific mitochondrial molecular and functional signatures, we established rat models with two slowly developing disease stages (compensated and decompensated) in response to pulmonary artery banding (PAB) or ascending aortic banding (AOB). Genome-wide transcriptomic and proteomic analyses were used to identify differentially expressed mitochondrial genes and proteins and were accompanied by a detailed characterization of mitochondrial function and morphology. Two clearly distinguishable disease stages, which culminated in a comparable systolic impairment of the respective ventricle, were observed. Mitochondrial respiration was similarly impaired at the decompensated stage, while respiratory chain activity or mitochondrial biogenesis were more severely deteriorated in the failing LV. Bioinformatics analyses of the RNA-seq. and proteomic data sets identified specifically deregulated mitochondrial components and pathways. Although the top regulated mitochondrial genes and proteins differed between the RV and LV, the overall changes in tissue and cardiomyocyte gene expression were highly similar. In conclusion, mitochondrial dysfuntion contributes to disease progression in right and left heart failure. Ventricle-specific differences in mitochondrial gene and protein expression are mostly related to the extent of observed changes, suggesting that despite developmental, anatomical and functional differences mitochondrial adaptations to chronic pressure overload are comparable in both ventricles.

Influenza viruses pose a major threat to human health and cause annual epidemics and occasional pandemics. Many virus-encoded proteins exert various functions in different subcellular compartments, as exemplified by the M1 protein, but the molecular mechanisms endowing the multiplicity of functions remain incompletely understood.

Signaling networks function as highly intertwined regulatory hubs rather than linear cascades with a single endpoint [...]

A variety of stress signals leads to activation of the inducible transcription factor NF-κB, one of the master regulators of the innate immune response. Despite a wealth of information available on the NF-κB core components and its control by different activation pathways and negative feedback loops, several levels of complexity hamper our understanding of the system. This has also contributed to the limited success of NF-κB inhibitors in the clinic and explains some of their unexpected effects. Here we consider the molecular and cellular events generating this complexity at all levels and point to a number of unresolved questions in the field. We also discuss potential future experimental and computational strategies to provide a deeper understanding of NF-κB and its coregulatory signaling networks.

The influenza A virus (IAV) polymerase is a multifunctional machine that can adopt alternative configurations to perform transcription and replication of the viral RNA genome in a temporally ordered manner. Although the structure of polymerase is well understood, our knowledge of its regulation by phosphorylation is still incomplete. The heterotrimeric polymerase can be regulated by posttranslational modifications, but the endogenously occurring phosphorylations at the PA and PB2 subunits of the IAV polymerase have not been studied. Mutation of phosphosites in PB2 and PA subunits revealed that PA mutants resembling constitutive phosphorylation have a partial (S395) or complete (Y393) defect in the ability to synthesize mRNA and cRNA. As PA phosphorylation at Y393 prevents binding of the 5′ promoter of the genomic RNA, recombinant viruses harboring such a mutation could not be rescued. These data show the functional relevance of PA phosphorylations to control the activity of viral polymerase during the influenza infectious cycle.

In the central nervous system, thrombin-mediated activation of protease-activated receptors (PARs) results in neuroinflammation and increased vascular permeability. These events have been linked to cancer and neurodegeneration. Endothelial cells (ECs) isolated from sporadic cerebral cavernous malformation (CCM) specimens showed dysregulation of genes involved in “thrombin-mediated PAR-1 activation” signaling. CCM is a vascular disease involving brain capillaries. In CCM, ECs show defective cell junctions. Oxidative stress and neuroinflammation play a key role in disease onset and progression. In order to confirm the possible role of thrombin pathway in sporadic CCM pathogenesis, we evaluated PARs expression in CCM-ECs. We found that sporadic CCM-ECs overexpress PAR1, PAR3 and PAR4, together with other coagulation factor encoding genes. Moreover, we investigated about expression of the three familial CCM genes (KRIT1, CCM2 and PDCD10) in human cerebral microvascular ECs, following thrombin exposure, as well as protein level. Thrombin exposure affects EC viability and results in dysregulation of CCM gene expression and, then, in decreased protein level. Our results confirm amplification of PAR pathway in CCM suggesting, for the first time, the possible role of PAR1-mediated thrombin signaling in sporadic CCM. Thrombin-mediated PARs over activation results in increased blood-brain barrier permeability due to loss of cell junction integrity and, in this context, also the three familial CCM genes may be involved.

Background: Atrial fibrillation (AF) is accompanied with structural remodeling. Profibrotic signals lead to interstitial fibrosis, which is considered a structural basis for the development, maintenance, and progression of AF. The development and maintenance of interstitial fibrosis involves expressional changes in cardiac microRNAs (miRNAs).

The family of hypoxia-inducible transcription factors (HIF) is activated to adapt cells to low oxygen conditions, but is also known to regulate some biological processes under normoxic conditions. Here we show that HIF-1α protein levels transiently increase during the G1 phase of the cell cycle (designated as G1-HIF) in an AMP-activated protein kinase (AMPK)-dependent manner. The transient elimination of G1-HIF by a degron system revealed its contribution to cell survival under unfavorable metabolic conditions. Indeed, G1-HIF plays a key role in the cell cycle-dependent expression of genes encoding metabolic regulators and the maintenance of mTOR activity under conditions of nutrient deprivation. Accordingly, transient elimination of G1-HIF led to a significant reduction in the concentration of key proteinogenic amino acids and carbohydrates. These data indicate that G1-HIF acts as a cell cycle-dependent surveillance factor that prevents the onset of starvation-induced apoptosis.