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Embryonic development and cell specification have been viewed as an epigenetically rigid process. Through accumulation of irreversible epigenetic marks, the differentiation process has been considered unidirectional, and once completed cell specification would be permanent and stable. However, somatic cell nuclear transfer that involved the implantation of a somatic nucleus into a previously enucleated oocyte accomplished in amphibians in the 1950s and in mammals in the late 1990s-resulting in the birth of "Dolly the sheep"-clearly showed that "terminal" differentiation is reversible. In parallel, work on lineage-determining factors like MyoD revealed surprising potential to modulate lineage identity in somatic cells. This work culminated in the discovery that a set of four defined factors can reprogram fibroblasts into induced pluripotent stem (iPS) cells, which were shown to be molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells, thus essentially showing that defined factors can induce authentic reprogramming without the need of oocytes. This concept was further extended when it was shown that fibroblasts can be directly converted into neurons, showing induced lineage conversion is possible even between cells representing two different germ layers. These findings suggest that "everything is possible" (i.e., once key lineage reprogramming factors are identified, cells should be able to convert into any desired lineage).

Gene regulation is a tightly controlled process in Eukaryotes. Coding and non-coding regions work together to ensure proper spatiotemporal gene expression through 3D chromatin organization, nucleosome positioning, histone tail post-translational modifications, epigenetic DNA modifications, and non-canonical nucleic acid structures such as RNA:DNA triplexes. In this thesis, I investigate two of these mechanisms, nucleosomes and RNA:DNA triplexes. In the first chapter, I focus on characterizing nucleosomal properties, such as stability and accessibility, and explore how these properties change to regulate gene expression. I show that Eukaryotic organisms can modulate nucleosome stability and change the RNA polymerase pausing rate, which in turn regulates gene expression. Intriguingly, I find a distinct group of un-stable nucleosomes enriched at the TSS of promoters marked with motif one, an M1BP TF-specific motif, in D. melanogaster. Modulation of stability may ensure a fast response to environmental cues and proper spatiotemporal expression of developmental genes. Furthermore, I developed a bioinformatic pipeline called nucMACC, with which scientists can study nucleosomal properties and positioning in their projects. I show the nucMACC pipeline is consistent and robust and provide recommendations for minimum sequencing depth, MNase titrations, and spike-in use. In summary, the nucMACC pipeline provides high-resolution nucleosome positions and an automated way of calling non-canonical nucleosomes, un-stable nucleosomes, hyper-accessible nucleosomes, hypo-accessible nucleosomes, and stable canonical nucleosomes. In the second chapter, I study the triplex binding code and explore how the code changes based on the triplex motif (Purine, pyrimidine, and mixed), sequence Guanine content, length, and the nucleic acid (RNA or DNA). Triplexes are non-canonical DNA/RNA structures consisting of three nucleotide strands, most commonly a double-stranded DNA molecule and a single-stranded RNA molecule in its major groove. I show that major differences exist between the triplex motifs and between RNA:DNA and DNA:DNA triplexes. Interestingly, I discovered that the mixed RNA:DNA triplex motif permits triplex formation only at very narrow Guanine contents, while DNA:DNA mixed motif triplexes are able to form at almost any Guanine content. Moreover, I confirm the newly defined triplex code by testing published triplex pairs, of which half are unable to form a triplex under physiological conditions. Furthermore, I developed a high throughput method to investigate the triplex binding code in the context of molecular crowding and competition. I find that certain mismatches in the motif stabilize triplex formation and are preferentially selected for triplex formation over the complementary Hoogsten base-pairing motif. Moreover, I investigate how the location of a mismatch modifies triplex stability and show that the middle section of the triplex is more sensitive to mismatches than flanking regions. In summary, I add to the existing triplex code by delineating the differences in the binding code between triplex motifs and RNA:DNA and DNA:DNA triplexes. I show how the binding code changes in the context of molecular crowding and competition and moreover, show the differential effect of different mismatch locations., Die Genregulation ist ein genau kontrollierter Prozess in Eukaryonten. Kodierende und nicht-kodierende Regionen arbeiten zusammen, um eine korrekte räumlich-zeitliche Genexpression durch 3D-Chromatinorganisation, Nukleosomenpositionierung, posttranslationale Histonschwanzmodifikationen, epigenetische DNA-Modifikationen und nicht-kanonische Nukleinsäurestrukturen wie RNA:DNA-Triplexe sicherzustellen. In dieser Arbeit untersuche ich zwei dieser Mechanismen, Nukleosomen und RNA:DNA-Triplexe. Im ersten Kapitel konzentriere ich mich auf die Charakterisierung der Eigenschaften von Nukleosomen, wie Stabilität und Zugänglichkeit, und untersuche, wie sich diese Eigenschaften ändern, um die Genexpression zu regulieren. Ich zeige, dass eukaryotische Organismen die Nukleosomenstabilität modulieren und die Pausenrate der RNA-Polymerase verändern können, was wiederum die Genexpression reguliert. Interessanterweise finde ich in D. melanogaster eine bestimmte Gruppe instabiler Nukleosomen, die an der TSS von Promotoren mit dem „Motif one“ , einem M1BP TF-spezifischen Motiv, angereichert sind. Eine Modulation der Stabilität könnte eine schnelle Reaktion auf Umweltreize und eine angemessene räumlich-zeitliche Expression von Entwicklungsgenen gewährleisten. Darüber hinaus habe ich eine bioinformatische Pipeline namens nucMACC entwickelt, mit der Wissenschaftler die Eigenschaften und Positionierung von Nukleosomen in ihren Projekten untersuchen können. Ich zeige, dass die nucMACC-Pipeline konsistent und robust ist, und gebe Empfehlungen für die Mindestsequenzierungstiefe, MNase-Titrationen und die Verwendung von Spike-in DNA. Zusammenfassend erlaubt die nucMACC-Pipeline sowohl die Bestimmung von hochauflösenden Nukleosomenpositionen als auch eine automatische Kategorisierung von nicht-kanonischen, instabilen, hyper- und hypo-zugänglichen und stabilen kanonischen Nukleosomen. Im zweiten Kapitel erforsche ich den Triplex-Bindungscode und untersuche, wie sich der Code je nach Triplex-Motiv (Purin, Pyrimidin und gemischt), Sequenz, Guanin-Gehalt, Länge und Nukleinsäure (RNA oder DNA) verhält. Triplexe sind nicht-kanonische DNA/RNA-Strukturen, die aus drei Nukleotidsträngen bestehen, meist einem doppelsträngigen DNA-Molekül und einem einzelsträngigen RNA-Molekül in seiner großen Furche . Ich zeige, dass es große Unterschiede zwischen den Triplex-Motiven und zwischen RNA:DNA- und DNA:DNA-Triplexen gibt. Interessanterweise entdeckte ich, dass das gemischte RNA:DNA-Triplex-Motiv die Triplex-Bildung nur bei einem sehr geringen Guanin-Gehalt ermöglicht, während Triplexe mit gemischtem DNA:DNA-Motiv bei fast jedem Guanin-Gehalt gebildet werden können. Darüber hinaus bestätige ich den neu definierten Triplex-Code, indem ich schon veröffentlichte Triplex-Paare teste und zeige außerdem, dass von diesen Paaren die Hälfte unter physiologischen Bedingungen keine Triplex bildet. Zusätzlich habe ich eine Hochdurchsatzmethode entwickelt, um den Triplex-Bindungscode im Kontext von molekularem Gedränge und Kompetetion zu untersuchen. Ich stelle fest, dass bestimmte Fehlpaarungen im Motiv die Triplexbildung stabilisieren und gegenüber dem komplementären Hoogsten-Basenpaarungsmotiv bevorzugt für die Triplexbildung ausgewählt werden. Außerdem untersuche ich, wie die Lage einer Fehlpaarung die Triplex-Stabilität verändert, und zeige, dass der mittlere Abschnitt des Triplex empfindlicher auf Fehlpaarungen reagiert als flankierende Regionen. Zusammenfassend lässt sich sagen, dass ich den bestehenden Triplex-Code ergänze, indem ich die Unterschiede im Bindungscode zwischen Triplex-Motiven und RNA:DNA- und DNA:DNA-Triplexen beschreibe. Ich zeige, wie sich der Bindungscode im Kontext von molekularem Gedränge und Kompetetion verändert, und zeige darüber hinaus die unterschiedliche Wirkung verschiedener Mismatch-Positionen.

Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

Objective. The insulin tolerance test (ITT) is the gold standard endocrine test used to assess the integrity of the growth hormone (GH) and cortisol axes. The ITT has potential risks, and severe hypoglycaemia may necessitate intravenous glucose rescue. There is no clear consensus as to the optimal insulin dose for the ITT. Therefore, we sought to compare the standard dose (0.15 U/kg) and a low-dose ITT (0.1 U/kg). Design. Single-centre audit of ITT data (2012–2021). Patients and Measurements. Patients who underwent an ITT to assess possible GH deficiency/adrenal insufficiency were included. Glucose, GH, and cortisol were measured at baseline and 30, 45, 60, 90, and 120 minutes following I.V. insulin bolus (0.15 U/kg or 0.10 U/kg). Results. Of the ITTs performed, only 3/177 (1.7%) did not achieve adequate hypoglycaemia (≤2.2 mmol/L) with a single insulin dose. In total, 174 patients (43.5 ± 12.1 yrs, mean ± standard deviation) were included for analysis (0.15 U/kg: n = 113, 0.10 U/kg: n = 61). All 174 subjects had adequate hypoglycaemia regardless of baseline fasting blood glucose level or insulin dose. Neither nadir glucose nor glucose delta (i.e., baseline minus nadir) differed between insulin doses. Trends in both cortisol and GH responses over time were similar between groups, and a greater proportion of patients receiving the standard dose had an adequate cortisol response (77/106 (72.6%) vs. 32/60 (53.3%), p = 0.01 ). The rates of glucose rescue did not differ in a subset of 79 patients, with on-demand glucose rescue in 4/35 (11%) for the standard dose and 2/44 (5%) for the low dose ( p = 0.25 ). Conclusions. Our results suggest that the low-dose ITT produces comparable glucose, cortisol, and GH responses to the higher dose. Given the risks associated with hypoglycaemia, the low dose appears to be preferable to the standard dose ITT in most circumstances.

Introduction Phaeochromocytomas/paraganglioma (PPGL) surgery was historically associated with significant risks of perioperative complications. The decreased mortality (Methods A retrospective study using data from our institutional database was conducted. All patients undergoing adrenalectomy for PPGL from October 2011 to September 2020 were included. All patients were routinely alpha-blocked. Intraoperative cardiovascular instability (ICI) was assessed through number of systolic blood pressure (SBP) episodes >160mmHg, SBP Results A total of 100 consecutive patients undergoing surgery were identified of whom 53 patients had complete anaesthetic records available for analysis. Thirty-two patients (60%) had at least one episode with an SBP >160mmHg. Nine (17%) cases had no intraoperative hypotensive episodes, while 3 (6%) patients had >10 intraoperative episodes of an SBP Conclusions Cardiac instability remained significant in PPGL surgery despite optimal alpha- and beta-blockade. While omitting blockade would appear empirically questionable, a randomised controlled trial (RCT) of surgery with and without alpha-blockade will provide an answer.

PDF file - 190K, JAK2-inhibitor trial (proliferation assay, colony assay)

PDF file - 626K, Leukemia phenotype (FACS, immunohistochemistry)

PDF file - 339K, LSC sorting strategy

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PDF file - 58K, Colony assay with and without cytokines

SummaryThe role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer’s disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-β inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.

SummaryMultiple sclerosis (MS) is an autoimmune disease associated with inflammatory demyelination in the central nervous system (CNS). Autologous hematopoietic cell transplantation (HCT) is under investigation as a promising therapy for treatment-refractory MS. Here we identify a reactive myeloid state in chronic experimental autoimmune encephalitis (EAE) mice and MS patients that is surprisingly associated with neuroprotection and immune suppression. HCT in EAE mice leads to an enhancement of this myeloid state, as well as clinical improvement, reduction of demyelinated lesions, suppression of cytotoxic T cells, and amelioration of reactive astrogliosis reflected in reduced expression of EAE- associated gene signatures in oligodendrocytes and astrocytes. Further enhancement of myeloid cell incorporation into the CNS following a modified HCT protocol results in an even more consistent therapeutic effect corroborated by additional amplification of HCT-induced transcriptional changes, underlining myeloid-derived beneficial effects in the chronic phase of EAE. Replacement or manipulation of CNS myeloid cells thus represents an intriguing therapeutic direction for inflammatory demyelinating disease.

SummaryChromatin-associated long non-coding RNAs (lncRNAs) have been shown to define chromatin density, regulate gene expression, and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA modulation, little data is showing the direct effects of lncRNA on regulatory elements (REs) that drive gene expression. Here we explored the molecular mechanism of the chromatin-interacting lncRNA, MALAT1, through RNA- and ATAC-seq, using HeLa cells as a model system. Time-resolved MALAT1 knock-down assays revealed its direct regulation of a limited number of protein-coding genes. Loss of MALAT1 resulted in a substantial loss of chromatin accessibility downstream of theNR4A1gene, associated with its down-regulation. CRISPR-i assays revealed that this region corresponds to a new downstream RE. Next, using TCGA data, we identified a direct correlation between the expression of NR4A1 and the accessibility of the downstream RE in breast cancer. The molecular mechanism was validated on estrogen receptor (ER) positive breast cancer cells (MCF7) and Pancreatic Duct Epithelioid Carcinoma (PANC1) cells, not showing this effect according to TCGA data. Indeed, MALAT1 regulates the expression of NR4A1 in a cell type-specific manner by changing the accessibility of the downstream RE. MALAT1 exhibits a molecular mechanism that fine-tunes the expression of cancer drivers, like NR4A1, in ER-positive breast cancer cells, but not in other cell types.

SummaryBackgroundGene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.MethodsWe developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar- free,COL7A1corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing.FindingsiPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT- treated mice.InterpretationDEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

COVID-19 remains a global pandemic of an unprecedented magnitude with millions of people now developing “COVID lung fibrosis.” Single-cell transcriptomics of lungs of patients with long COVID revealed a unique immune signature demonstrating the upregulation of key proinflammatory and innate immune effector genes CD47, IL-6, and JUN. We modeled the transition to lung fibrosis after COVID and profiled the immune response with single-cell mass cytometry in JUN mice. These studies revealed that COVID mediated chronic immune activation reminiscent to long COVID in humans. It was characterized by increased CD47, IL-6, and phospho-JUN (pJUN) expression which correlated with disease severity and pathogenic fibroblast populations. When we subsequently treated a humanized COVID lung fibrosis model by combined blockade of inflammation and fibrosis, we not only ameliorated fibrosis but also restored innate immune equilibrium indicating possible implications for clinical management of COVID lung fibrosis in patients.

Funder: National Multiple Sclerosis Society; Id: http://dx.doi.org/10.13039/100000890, Funder: Association Européenne contre les Leucodystrophies; Id: http://dx.doi.org/10.13039/501100008731, Funder: New York Stem Cell Foundation; Id: http://dx.doi.org/10.13039/100003194, Funder: University of California, San Francisco; Id: http://dx.doi.org/10.13039/100008069, Funder: Sandler Foundation; Id: http://dx.doi.org/10.13039/100007100, Funder: Action Medical Research; Id: http://dx.doi.org/10.13039/501100000317, Funder: Dr. Miriam and Sheldon G. Adelson Medical Research Foundation; Id: http://dx.doi.org/10.13039/100005984, Funder: National Institute for Health Research; Id: http://dx.doi.org/10.13039/501100000272, Funder: Stanford University; Id: http://dx.doi.org/10.13039/100005492, Funder: Foundation Optic Atrophy 1, Oligodendrocytes, the myelinating cells of the central nervous system, possess great potential for disease modeling and cell transplantation-based therapies for leukodystrophies. However, caveats to oligodendrocyte differentiation protocols ( Ehrlich et al., 2017; Wang et al., 2013; Douvaras and Fossati, 2015) from human embryonic stem and induced pluripotent stem cells (iPSCs), which include slow and inefficient differentiation, and tumorigenic potential of contaminating undifferentiated pluripotent cells, are major bottlenecks towards their translational utility. Here, we report the rapid generation of human oligodendrocytes by direct lineage conversion of human dermal fibroblasts (HDFs). We show that the combination of the four transcription factors OLIG2, SOX10, ASCL1 and NKX2.2 is sufficient to convert HDFs to induced oligodendrocyte precursor cells (iOPCs). iOPCs resemble human primary and iPSC-derived OPCs based on morphology and transcriptomic analysis. Importantly, iOPCs can differentiate into mature myelinating oligodendrocytes in vitro and in vivo. Finally, iOPCs derived from patients with Pelizaeus Merzbacher disease, a hypomyelinating leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene, showed increased cell death compared with iOPCs from healthy donors. Thus, human iOPCs generated by direct lineage conversion represent an attractive new source for human cell-based disease models and potentially myelinating cell grafts.

Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen α3 VI (COL6A3) C-terminal domain (CTD). TheseCol6a3CTTmice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner.Col6a3CTTmice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid (eCB) signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance inCol6a3CTTmice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control.SIGNIFICANCE STATEMENTDystonia is a movement disorder characterized by involuntary movements. We previously identified genetic variants affecting a specific domain of the COL6A3 protein as a cause of dystonia. Here, we created mice lacking the affected domain and observed an analogous movement disorder. Using a protein interaction screen, we found that the affected COL6A3 domain mediates an interaction with the cannabinoid receptor 1 (CB1R). Concordantly, our COL6A3-deficient mice showed a deficit in synaptic plasticity linked to a deficit in cannabinoid signaling. Pharmacological cannabinoid augmentation rescued the motor impairment of the mice. Thus, cannabinoid augmentation could be a promising avenue for treating dystonia, and we have identified a possible molecular mechanism mediating this.

Patients with severe COVID-19 often suffer from lymphopenia, which is linked to T cell sequestration, cytokine storm and mortality. However, it remains largely unknown how SARS-CoV-2 induces lymphopenia. Here, we studied the transcriptomic profile and epigenomic alterations involved in cytokine production by SARS-CoV-2-infected cells. We adopted a reverse time-order gene coexpression network (TO-GCN) approach to analyze time-series RNA-sequencing data, revealing epigenetic modifications at the late stage of viral egress. Furthermore, we identified SARS-CoV-2-activated NF-κB and IRF1 pathways contributing to viral infection and COVID-19 severity through epigenetic analysis of H3K4me3 ChIP-sequencing. Cross-referencing our transcriptomic and epigenomic datasets revealed that coupling NF-κB and IRF1 pathways mediate PD-L1 immunosuppressive programs. Interestingly, we observed higher PD-L1 expression in Omicron-infected cells than SARS-CoV-2 infected cells. Blocking PD-L1 at an early stage of virally-infected AAV-hACE2 mice significantly recovered lymphocyte counts and lowered inflammatory cytokine levels. Our findings indicate that targeting the SARS-CoV-2-mediated NF-κB and IRF1-PD-L1 axis may represent an alternative strategy to reduce COVID-19 severity. This article is protected by copyright. All rights reserved.

Micrococcal nuclease sequencing (MNase-seq) is the state-of-the-art method for determining the chromatin structure and nucleosome positioning. Analysis of MNase-seq data is complex due to variable degrees of sample quality, sequence bias of the endonuclease, and high sequencing depth requirement. Therefore, we developed an optimized analysis pipeline written in nextflow and publicly available on GitHub. The nucMACC pipeline provides high-resolution nucleosome positions and associated properties such as nucleosome accessibility and stability. Hyper-/hypo-accessible nucleosomes or non-canonical and unstable nucleosome positions in the genome are called automatically. We provide recommendations for the minimum sequencing depth and show robust results using two differential MNase conditions.Using the nucMACC pipeline, we characterized the nucleosomal properties in D. melanogaster and explored the correlation between these properties and gene expression. Intriguingly, we found a distinct group of unstable nucleosomes enriched in promoters marked with the M1BP motif. The expression rate of the genes harboring an unstable nucleosome in the promoter was not altered compared to genes with a nucleosome-depleted promoter region, whereas the RNA polymerase pausing rate was affected.

The chromodomain helicase DNA-binding protein CHD8 is among the most frequently found de-novo mutations in autism (1–3). Unlike most other autism-risk genes, CHD8 mutations appear to be fully penetrant (4). Despite its prominent disease involvement, little known about its molecular function. Based on sequence homology, CHD8 is believed to be a chromatin regulator, but mechanisms for its genomic targeting and its role on chromatin are unclear. Here, we developed a human cell model carrying conditional CHD8 loss-of-function alleles. Full knockout CHD8 was required for the viability of undifferentiated human embryonic stem (ES) cells, whereas postmitotic neurons survived following CHD8 depletion. However, chromatin accessibility maps and transcriptional profiling revealed that CHD8 is a potent general chromatin activator, enhancing transcription of its direct target genes, including a large group of autism genes. CHD8’s genomic binding sites in human neurons were significantly enriched for ELK1 (ETS) motifs. Moreover, positive CHD8-dependent chromatin remodeling was enhanced at ELK1 motif-containing CHD8 binding sites. ELK1 was the most prominent ETS factor expressed in human neurons and was necessary for CHD8 to target the sites that contained the ELK1 motif, demonstrating a cooperative interaction between ELK1 and CHD8 on chromatin. We also observed potential role of CHD8 in ELK1 localization on nuclear compartments in a transcription-stage-dependent manner. Finally, inhibition of ELK1 activity or ELK1 knockdown that enhances the neurogenesis from embryonic stem cells (ES) was dependent on the presence of CHD8. In summary, our results establish that CHD8 is a strong activator of chromatin accessibility and transcription in neurons and reveals a role in regulating many high-risk autism genes. Additionally, we show there is molecular and functional interdependence of CHD8 and ELK1 in chromatin binding of CHD8, nuclear interaction of ELK1, and neurogenesis enhancement. These data imply the involvement of the MAPK/ERK pathway effector ELK1 in pathogenesis of autism caused by CHD8 mutations (5).

The SARS-CoV-2 Omicron variant and its sublineages show pronounced viral escape from neutralizing antibodies elicited by vaccination or prior SARS-CoV-2 variant infection owing to over 30-amino acid alterations within the spike (S) glycoprotein. Breakthrough infection of vaccinated individuals with Omicron sublineages BA.1 and BA.2 is associated with distinct patterns of cross-neutralizing activity against SARS-CoV-2 variants of concern (VOCs). In continuation of our previous work, we characterized the effect of Omicron BA.4/BA.5 S glycoprotein exposure on the neutralizing antibody response upon breakthrough infection in vaccinated individuals and upon variant-adapted booster vaccination in mice. We found that immune sera from triple mRNA-vaccinated individuals with subsequent breakthrough infection during the Omicron BA.4/BA.5 wave showed cross-neutralizing activity against previous Omicron variants BA.1, BA.2, BA.2.12.1, and BA.4/BA.5 itself. Administration of a prototypic BA.4/BA.5-adapted mRNA booster vaccine to mice after SARS-CoV-2 wild-type strain-based primary immunization is associated with broader cross-neutralizing activity than a BA.1-adapted booster. Whereas the Omicron BA.1-adapted mRNA vaccine in a bivalent format (wild-type + BA.1) broadens cross-neutralizing activity relative to the BA.1 monovalent booster, cross-neutralization of BA.2 and descendants is more effective in mice boosted with a bivalent wild-type + BA.4/BA.5 vaccine. In naïve mice, primary immunization with the bivalent wild-type + Omicron BA.4/BA.5 vaccine induces strong cross-neutralizing activity against Omicron VOCs and previous variants. These findings suggest that, when administered as boosters, mono- and bivalent Omicron BA.4/BA.5-adapted vaccines enhance neutralization breadth and that the bivalent version also has the potential to confer protection to individuals with no preexisting immunity against SARS-CoV-2.

Based on the recent interest in oxy-carbide materials in catalysis, we employ a thin film model concept to highlight that variation of key reaction parameters in the reactive magnetron sputtering of zirconium carbide films (sputtering power, template temperature or reactive plasma environment) under realistic preparation and application conditions often results in zirconium oxy-carbide films of varying stoichiometry. The composition of the films grown on silicon wafers and in vacuo - cleaved NaCl (001) single crystal facets was confirmed by depth profiling X-ray photoelectron spectroscopy and electron microscopy analysis. A correlation between methane-to-argon ratio, excess carbon and template temperature with elemental composition emphasizes the exclusive presence of oxygen-containing zirconium carbides. To generalize the approach, we also show that embedding of highly ordered Cu particles with uniform sizes in zirconium oxy-carbide matrices yields well-defined metal / oxy-carbide interfaces. As the presence of an oxy-carbide and its reactivity has been inextricably linked to enhanced activity and selectivity in a variety of processes, including hydrogenation, oxidation or reduction reactions, our model thin film approach provides the necessary well-defined catalysts to derive mechanistic details and to study the decomposition/re-carburization cycles of oxy-carbides. We have exemplified the concept for zirconium oxy-carbide, but deliberate extension to similar systems is easily possible.

Mutations in β-amyloid (Aβ) precursor protein (APP) cause familial Alzheimer’s disease (AD) probably by enhancing Aβ peptides production from APP. An antibody targeting Aβ (aducanumab) was approved as an AD treatment; however, some Aβ antibodies have been reported to accelerate, instead of ameliorating, cognitive decline in individuals with AD. Using conditional APP mutations in human neurons for perfect isogenic controls and translational relevance, we found that the APP-Swedish mutation in familial AD increased synapse numbers and synaptic transmission, whereas the APP deletion decreased synapse numbers and synaptic transmission. Inhibition of BACE1, the protease that initiates Aβ production from APP, lowered synapse numbers, suppressed synaptic transmission in wild-type neurons, and occluded the phenotype of APP-Swedish–mutant neurons. Modest elevations of Aβ, conversely, elevated synapse numbers and synaptic transmission. Thus, the familial AD-linked APP-Swedish mutation under physiologically relevant conditions increased synaptic connectivity in human neurons via a modestly enhanced production of Aβ. These data are consistent with the relative inefficacy of BACE1 and anti-Aβ treatments in AD and the chronic nature of AD pathogenesis, suggesting that AD pathogenesis is not simply caused by overproduction of toxic Aβ but rather by a long-term effect of elevated Aβ concentrations.

Mutations in β-amyloid (Aβ) precursor protein ( APP ) cause familial Alzheimer’s disease (AD) probably by enhancing Aβ peptides production from APP. An antibody targeting Aβ (aducanumab) was approved as an AD treatment; however, some Aβ antibodies have been reported to accelerate, instead of ameliorating, cognitive decline in individuals with AD. Using conditional APP mutations in human neurons for perfect isogenic controls and translational relevance, we found that the APP -Swedish mutation in familial AD increased synapse numbers and synaptic transmission, whereas the APP deletion decreased synapse numbers and synaptic transmission. Inhibition of BACE1, the protease that initiates Aβ production from APP, lowered synapse numbers, suppressed synaptic transmission in wild-type neurons, and occluded the phenotype of APP -Swedish–mutant neurons. Modest elevations of Aβ, conversely, elevated synapse numbers and synaptic transmission. Thus, the familial AD-linked APP -Swedish mutation under physiologically relevant conditions increased synaptic connectivity in human neurons via a modestly enhanced production of Aβ. These data are consistent with the relative inefficacy of BACE1 and anti-Aβ treatments in AD and the chronic nature of AD pathogenesis, suggesting that AD pathogenesis is not simply caused by overproduction of toxic Aβ but rather by a long-term effect of elevated Aβ concentrations.

The SARS-CoV-2 Omicron variant and its sublineages show pronounced viral escape from neutralizing antibodies elicited by vaccination or prior SARS-CoV-2 variant infection owing to over 30 amino acid alterations within the spike (S) glycoprotein. We and others have recently reported that breakthrough infection of vaccinated individuals with Omicron sublineages BA.1 and BA.2 are associated with distinct patterns of cross-neutralizing activity against SARS-CoV-2 variants of concern (VOCs). BA.2 breakthrough infection mediated overall stronger cross-neutralization of BA.2 and its descendants (BA.2.12.1, BA.4, and BA.5) compared to BA.1 breakthrough infection. Here we characterized the effect of Omicron BA.4/BA.5 S glycoprotein exposure on the magnitude and breadth of the neutralizing antibody response upon breakthrough infection in vaccinated individuals and in mice upon booster vaccination. We show that immune sera from triple mRNA-vaccinated individuals with subsequent Omicron BA.4/BA.5 breakthrough infection display broad and robust neutralizing activity against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5. Administration of a prototypic BA.4/BA.5-adapted mRNA booster vaccine to mice following SARS-CoV-2 wild-type strain-based primary immunization is associated with similarly broad neutralizing activity. Immunization of naïve mice with a bivalent mRNA vaccine (wild-type + Omicron BA.4/BA.5) induces strong and broad neutralizing activity against Omicron VOCs and previous variants. These findings suggest that when administered as boosters, mono- and bivalent Omicron BA.4/BA.5-adapted vaccines may enhance neutralization breadth, and in a bivalent format may also have the potential to confer protection to individuals with no pre-existing immunity against SARS-CoV-2.

Summary The differentiation of pluripotent stem cells can be accomplished by sequential activation of signaling pathways or through transcription factor programming. Multistep differentiation imitates embryonic development to obtain authentic cell types, but it suffers from asynchronous differentiation with variable efficiency. Transcription factor programming induces synchronous and efficient differentiation with higher reproducibility but may not always yield authentic cell types. We systematically explored the generation of dopaminergic induced neuronal cells from mouse and human pluripotent stem cells. We found that the proneural factor Ascl1 in combination with mesencephalic factors Lmx1a and Nurr1 induce peripheral dopaminergic neurons. Co-delivery of additional midbrain transcription factors En1, FoxA2, and Pitx3 resulted in facile and robust generation of functional dopaminergic neurons of midbrain character. Our results suggest that more complex combinations of transcription factors may be needed for proper regional specification of induced neuronal cells generated by direct lineage induction., Graphical abstract, Highlights • Ascl1 alone can induce tyrosine hydroxylase (TH)-positive ES-iN cells • Ascl1 alone, or with Nurr1 and Lmx1a, induce peripheral TH-positive cells • WNT1 and neurotrophic factors increase TH-positive iN cells in culture • A 6-factor combination induces TH-positive dopamine iN cells of central identity, Dopaminergic neurons can be induced from pluripotent cells using overexpression of transcription factors. Existing transcription factor combinations result in cells of mixed regional identity. Here, Wernig and colleagues report that with extracellular signals, a six-factor combination delivered via piggyBac transposons is capable of inducing dopamine neurons of central identity from mouse and human embryonic stem cells.

The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.

Compound materials, such as transition-metal (TM) carbides, are anticipated to be effective electrocatalysts for the carbon dioxide reduction reaction (CO2RR) to useful chemicals. This expectation is nurtured by density functional theory (DFT) predictions of a break of key adsorption energy scaling relations that limit CO2RR at parent TMs. Here, we evaluate these prospects for hexagonal Mo2C in aqueous electrolytes in a multimethod experiment and theory approach. We find that surface oxide formation completely suppresses the CO2 activation. The oxides are stable down to potentials as low as −1.9 V versus the standard hydrogen electrode, and solely the hydrogen evolution reaction (HER) is found to be active. This generally points to the absolute imperative of recognizing the true interface establishing under operando conditions in computational screening of catalyst materials. When protected from ambient air and used in nonaqueous electrolyte, Mo2C indeed shows CO2RR activity.

Introduction Adrenocortical carcinomas (ACCs) are rare and aggressive malignant tumors often with an unfavorable prognosis accounting for 0. 05-0.2% of all cancers. ACCs can be separated into 2 groups: Functioning (hormone-producing) and non-functioning tumors. About 60% of ACCs synthesize hormones. Hormone-secreting ACCs most commonly cause virilization or Cushing's syndrome. On very rare occasions, ACCs can lead to feminization in male subjects. Clinical manifestations are gynecomastia and/or hypogonadism. Here, we present a case of an estradiol-secreting adrenal cortical carcinoma. Case presentation A 58-year-old male presented with patchy hair loss, change in hair colour to white, loss of body hair and eyebrows, and breast tenderness. Hormone testing revealed significantly elevated estradiol came 582 pmol/L [NR Conclusion We describe a rare case of an estradiol-secreting ACC presenting with feminization and Marie-Antoinette syndrome as well as possible paraneoplastic aortitis. Targeted molecular tumor analysis may guide further treatment options and enrolment in clinical trials should be considered. Presentation: No date and time listed

Meta je termin u kulturi video igara koji opisuje najpopularniji način igranja u jednoj igri, a nastaje od strane igrača koji igraju igru i izvođača koji utječu na mehanizme u igri. Video igre koje će služiti kao primjer da analiziramo metu i njezin utjecaj na drugačije aktere i njihov međusobni odnos su Apex Legends i Dead by Daylight. Ovo Istraživanje opisuje što je meta, kako nastaje i kako se mijena te koji faktori najviše utječu na njezino promjenu. Također, opisujem igre Apex Legends i Dead by Daylight kao primjer utjecaja mete na odnose između aktera ali i aktore poput ekonomije i politike na koje taj pojam može utjecati svojim postojanjem. Zaključno, zašto bi taj pojam bio u interesu studija antropologije i etnologije te kako digitalni pojam utječe na stvarni svijet. Meta is a term used in video game culture which is used to describe the most popular way of playing a game, and the term is developed by the players who play the game and the developers who influence and change the in game mechanics. Video games which will be used as examples to analyze the meta and its influence on the different actors and the relationship between them are the game Apex Legends and Dead by Daylight. This research describes what is meta, how it is developed, how does it change and lastly which factors have the most influence on the term changing. Also I will be describe the games Apex Legends and Dead by Daylight as examples of meta's influence and also other actors such as economy and politics which in turn can also be influenced by its existence. In conclusion, why would this term be in the interesting to the sciences of anthropology and ethnology while also seeing how a digital term influences the real world.

Endogenous Cushing’s syndrome (CS) poses considerable diagnostic challenges. Although late-night salivary cortisol (LNSC) is recommended as a first-line screening investigation, it remains the least widely used test in many countries. The combined measurement of LNSC and late-night salivary cortisone (LNS cortisone) has shown to further improve diagnostic accuracy. We present a retrospective study in a tertiary referral centre comparing LNSC, LNS cortisone, overnight dexamethasone suppression test, low-dose dexamethasone suppression test and 24-h urinary free cortisol results of patients investigated for CS. Patients were categorised into those who had CS (21 patients) and those who did not (33 patients). LNSC had a sensitivity of 95% and a specificity of 91%. LNS cortisone had a specificity of 100% and a sensitivity of 86%. With an optimal cut-off for LNS cortisone of >14.5 nmol/L the sensitivity was 95.2%, and the specificity was 100% with an area under the curve of 0.997, for diagnosing CS. Saliva collection is non-invasive and can be carried out at home. We therefore advocate simultaneous measurement of LNSC and LNS cortisone as the first-line screening test to evaluate patients with suspected CS.

Background The zinc finger domain containing transcription factor Myt1l is tightly associated with neuronal identity and is the only transcription factor known that is both neuron-specific and expressed in all neuronal subtypes. We identified Myt1l as a powerful reprogramming factor that, in combination with the proneural bHLH factor Ascl1, could induce neuronal fate in fibroblasts. Molecularly, we found it to repress many non-neuronal gene programs, explaining its supportive role to induce and safeguard neuronal identity in combination with proneural bHLH transcriptional activators. Moreover, human genetics studies found MYT1L mutations to cause intellectual disability and autism spectrum disorder often coupled with obesity. Methods Here, we generated and characterized Myt1l-deficient mice. A comprehensive, longitudinal behavioral phenotyping approach was applied. Results Myt1l was necessary for survival beyond 24 h but not for overall histological brain organization. Myt1l heterozygous mice became increasingly overweight and exhibited multifaceted behavioral alterations. In mouse pups, Myt1l haploinsufficiency caused mild alterations in early socio-affective communication through ultrasonic vocalizations. In adulthood, Myt1l heterozygous mice displayed hyperactivity due to impaired habituation learning. Motor performance was reduced in Myt1l heterozygous mice despite intact motor learning, possibly due to muscular hypotonia. While anxiety-related behavior was reduced, acoustic startle reactivity was enhanced, in line with higher sensitivity to loud sound. Finally, Myt1l haploinsufficiency had a negative impact on contextual fear memory retrieval, while cued fear memory retrieval appeared to be intact. Limitations In future studies, additional phenotypes might be identified and a detailed characterization of direct reciprocal social interaction behavior might help to reveal effects of Myt1l haploinsufficiency on social behavior in juvenile and adult mice. Conclusions Behavioral alterations in Myt1l haploinsufficient mice recapitulate several clinical phenotypes observed in humans carrying heterozygous MYT1L mutations and thus serve as an informative model of the human MYT1L syndrome.

Retinal ganglion cell (RGC) replacement therapy could restore vision in glaucoma and other optic neuropathies. We developed a rapid protocol for directly induced RGC (iRGC) differentiation from human stem cells, leveraging overexpression of NGN2. Neuronal morphology and neurite growth were observed within 1 week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated γ-aminobutyric acid (GABA)-induced excitation characteristic of immature RGCs. Single-cell RNA sequencing showed more similarities between iRGCs and early-stage fetal human RGCs than retinal organoid-derived RGCs. Intravitreally transplanted iRGCs survived and migrated into host retinas independent of prior optic nerve trauma, but iRGCs protected host RGCs from neurodegeneration. These data demonstrate rapid iRGC generation in vitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplantation and neuroprotective function after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.

The development of low-temperature fuel cells for clean energy production is an appealing alternative to fossil-fuel technologies. CO is a key intermediate in the electro-oxidation of energy carrying fuels and, due to its strong interaction with state-of-the-art Pt electrodes, it is known to act as a poison. Here we demonstrate the ability of Earth-abundant Cu to electro-oxidize CO efficiently in alkaline media, reaching high current densities of ≥0.35 mA cm−2 on single-crystal Cu(111) model catalysts. Strong and continuous surface structural changes are observed under reaction conditions. Supported by first-principles microkinetic modelling, we show that the concomitant presence of high-energy undercoordinated Cu structures at the surface is a prerequisite for the high activity. Similar CO-induced self-activation has been reported for gas–surface reactions at coinage metals, demonstrating the strong parallels between heterogeneous thermal catalysis and heterogeneous electrocatalysis. CO is a key intermediate in the electro-oxidation of energy carrying fuels which typically acts as a poison. Here, the authors demonstrate that Cu is an efficient CO electro-oxidation catalyst in alkaline electrolyte due to the continuous formation of undercoordinated active Cu adatom sites in the presence of CO and OH.

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients., Abdominal adhesions are a common cause of bowel obstruction, but knowledge regarding adhesion biology and anti-adhesion therapies remains limited. Here the authors report a systematic analysis of mouse and human adhesion tissues demonstrating that visceral fibroblast JUN and associated PDGFRA expression promote adhesions, and JUN suppression can prevent adhesion formation.

Objectives: Primary aldosteronism is one of the most common causes of secondary hypertension. Distinguishing unilateral from bilateral disease is essential as those with unilateral adrenal disease will benefit from adrenalectomy. This is best achieved by adrenal vein sampling (AVS) which may be a difficult procedure with significant radiation to both patient and operator. This study aims to measure the radiation dose during AVS before and after the installation of a new angiography platform. Materials and Methods: The dose area product (DAP), air kerma, and fluoroscopy time were collected retrospectively together with demographic data for the first ten patients who underwent AVS between April and September 2018 following the installation of the Philips Azurion 7 M20 interventional platform. These results were compared with those from ten patients who underwent AVS before the installation of the new machine using the Philips Allura Xper FD20 platform. Mann–Whitney test was used to compare DAP, air kerma, and fluoroscopy time. P < 0.05 was considered statistically significant. Results: Successful bilateral adrenal vein catheterization was achieved in all cases in both groups. There was no significant difference in fluoroscopy time: 5.5 (3.3–10.8) min (new) versus 5.3 (4.5–8.4) min (old) (P = 0.9502). The DAP and air kerma were both significantly lower on the new machine: 11.3 (5.2–26.7) Gy.cm2 versus 45.5 (17.2–56.5) Gy.cm2 (P = 0.0089) and 201.5 (88.1–464.0) mGy versus 682.5 (300.5–998.5) mGy (P = 0.0115), respectively. Conclusion: The installation of the Philips Azurion 7 M20 interventional platform has allowed a significant reduction in radiation dose during AVS.

Most fungal fatty acid synthases assemble from two multidomain subunits, α and β, into a heterododecameric FAS complex. It has been recently shown that the complex assembly occurs in a cotranslational manner and is initiated by an interaction between the termini of α and β subunits. This initial engagement of subunits may be the rate-limiting phase of the assembly and subject to cellular regulation. Therefore, we hypothesized that bypassing this step by genetically fusing the subunits could be beneficial for biotechnological production of fatty acids. To test the concept, we expressed fused FAS subunits engineered for production of octanoic acid in Saccharomyces cerevisiae. Collectively, our data indicate that FAS activity is a limiting factor of fatty acid production and that FAS fusion proteins show a superior performance compared to their split counterparts. This strategy is likely a generalizable approach to optimize the production of fatty acids and derived compounds in microbial chassis organisms.

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. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein–DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity. Lee, Mall et al. explore why the sequence-related transcription factors Myod1 and Ascl1 lead to different reprogramming outcomes when expressed in fibroblasts, despite binding similar targets.