Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis.

Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. While transcription master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether plasticity is actively suppressed by lineage-specific repressors. Here we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong.

MYT1L haploinsufficiency in human neurons and mice causes autism-associated phenotypes that can be reversed by genetic and pharmacologic intervention.

MYT1L is an autism spectrum disorder (ASD)-associated transcription factor that is expressed in virtually all neurons throughout life. How MYT1L mutations cause neurological phenotypes and whether they can be targeted remains enigmatic. Here, we examine the effects of MYT1L deficiency in human neurons and mice.

Concomitant Activation of OSM and LIF Receptor by a Dual-Specific hlOSM Variant Confers Cardioprotection after Myocardial Infarction in Mice.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) signaling protects the heart after myocardial infarction (MI). In mice, oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) are selectively activated by the respective cognate ligands while OSM activates both the OSMR and LIFR in humans, which prevents efficient translation of mouse data into potential clinical applications. We used an engineered human-like OSM (hlOSM) protein, capable to signal via both OSMR and LIFR, to evaluate beneficial effects on cardiomyocytes and hearts after MI in comparison to selective stimulation of either LIFR or OSMR.

Combining Cell Fate Reprogramming and Protein Engineering to Study Transcription Factor Functions.

Gene expression regulation by transcription factors plays a central role in determining and maintaining cell fate during normal development as well as induced cell fate reprogramming. Induction of cell identity-determining gene regulatory networks by reprogramming factors that act as transcriptional activators is key to induce desired cell fates. Conversely, repression of unwanted genetic programs by transcriptional repressors is equally important to ensure cell fate fidelity.

Isolation and Neuronal Reprogramming of Mouse Embryonic Fibroblasts.

Forced expression of specific neuronal transcription factors in mouse embryonic fibroblasts (MEFs) can lead to their direct conversion into functional neurons. Direct neuronal reprogramming has become a powerful tool to characterize individual factors and molecular mechanisms involved in forced and normal neurogenesis and to generate neuronal cell types for in vitro studies. Here we provide a detailed protocol for the isolation of MEFs devoid of neural tissue and their direct reprogramming into functional neurons by overexpression of neuronal reprogramming factors (Ascl1, Brn2, and Myt1l) using lentiviral vectors.

Pro-neuronal activity of Myod1 due to promiscuous binding to neuronal genes.

The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different.

Identification of Functional Protein Regions Through Chimeric Protein Construction.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay.

The AB loop of oncostatin M (OSM) determines species-specific signaling in humans and mice.

The pleiotropic interleukin-6 (IL-6)-type cytokine oncostatin M (OSM) signals in multiple cell types, affecting processes such as cell differentiation, hematopoiesis, and inflammation. In humans, OSM exerts its effects through activation of either of two different heterodimeric receptor complexes, formed by glycoprotein 130 (gp130) and either OSM receptor (OSMR) or leukemia inhibitory factor receptor (LIFR). In contrast, the mouse OSM orthologue acts mainly through dimers containing OSMR and gp130 and shows limited activity through mouse LIFR.

Reg proteins direct accumulation of functionally distinct macrophage subsets after myocardial infarction.

Aims: Myocardial infarction (MI) causes a massive increase of macrophages in the heart, which serve various non-redundant functions for cardiac repair. The identities of signals controlling recruitment of functionally distinct cardiac macrophages to sites of injury are only partially known. Previous work identified Regenerating islet-derived protein 3 beta (Reg3β) as a novel factor directing macrophages to sites of myocardial injury.

The AB loop and D-helix in binding site III of human Oncostatin M (OSM) are required for OSM receptor activation.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are closely related members of the interleukin-6 (IL-6) cytokine family. Both cytokines share a common origin and structure, and both interact through a specific region, termed binding site III, to activate a dimeric receptor complex formed by glycoprotein 130 (gp130) and LIF receptor (LIFR) in humans. However, only OSM activates the OSM receptor (OSMR)-gp130 complex.

Sirt7 promotes adipogenesis in the mouse by inhibiting autocatalytic activation of Sirt1.

Sirtuins (Sirt1-Sirt7) are NAD-dependent protein deacetylases/ADP ribosyltransferases, which play decisive roles in chromatin silencing, cell cycle regulation, cellular differentiation, and metabolism. Different sirtuins control similar cellular processes, suggesting a coordinated mode of action but information about potential cross-regulatory interactions within the sirtuin family is still limited. Here, we demonstrate that Sirt1 requires autodeacetylation to efficiently deacetylate targets such as p53, H3K9, and H4K16.

Animal Models and "Omics" Technologies for Identification of Novel Biomarkers and Drug Targets to Prevent Heart Failure.

It is now accepted that heart failure (HF) is a complex multifunctional disease rather than simply a hemodynamic dysfunction. Despite its complexity, stressed cardiomyocytes often follow conserved patterns of structural remodelling in order to adapt, survive, and regenerate. When cardiac adaptations cannot cope with mechanical, ischemic, and metabolic loads efficiently or become chronically activated, as, for example, after infection, then the ongoing structural remodelling and dedifferentiation often lead to compromised pump function and patient death.

Myocardial healing requires Reg3β-dependent accumulation of macrophages in the ischemic heart.

Cardiac healing after myocardial ischemia depends on the recruitment and local expansion of myeloid cells, particularly macrophages. Here we identify Reg3β as an essential regulator of macrophage trafficking to the damaged heart. Using mass spectrometry-based secretome analysis, we found that dedifferentiating cardiomyocytes release Reg3β in response to the cytokine OSM, which signals through Jak1 and Stat3.