RESUMO
Oct4 is essential to maintain pluripotency and has a pivotal role in establishing the germline. Its DNA-binding POU domain was recently found to bind motifs with methylated CpG elements normally associated with epigenetic silencing. However, the mode of binding and the consequences of this capability has remained unclear. Here, we show that Oct4 binds to a compact palindromic DNA element with a methylated CpG core (CpGpal) in alternative states of pluripotency and during cellular reprogramming towards induced pluripotent stem cells (iPSCs). During cellular reprogramming, typical Oct4 bound enhancers are uniformly demethylated, with the prominent exception of the CpGpal sites where DNA methylation is often maintained. We demonstrate that Oct4 cooperatively binds the CpGpal element as a homodimer, which contrasts with the ectoderm-expressed POU factor Brn2. Indeed, binding to CpGpal is Oct4-specific as other POU factors expressed in somatic cells avoid this element. Binding assays combined with structural analyses and molecular dynamic simulations show that dimeric Oct4-binding to CpGpal is driven by the POU-homeodomain whilst the POU-specific domain is detached from DNA. Collectively, we report that Oct4 exerts parts of its regulatory function in the context of methylated DNA through a DNA recognition mechanism that solely relies on its homeodomain.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Fator 3 de Transcrição de Octâmero , Diferenciação Celular/genética , DNA/metabolismo , Metilação de DNA , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Humanos , Animais , CamundongosRESUMO
An engineered SOX17 variant with point mutations within its DNA binding domain termed SOX17FNV is a more potent pluripotency inducer than SOX2, yet the underlying mechanism remains unclear. Although wild-type SOX17 was incapable of inducing pluripotency, SOX17FNV outperformed SOX2 in mouse and human pluripotency reprogramming. In embryonic stem cells, SOX17FNV could replace SOX2 to maintain pluripotency despite considerable sequence differences and upregulated genes expressed in cleavage-stage embryos. Mechanistically, SOX17FNV co-bound OCT4 more cooperatively than SOX2 in the context of the canonical SoxOct DNA element. SOX2, SOX17, and SOX17FNV were all able to bind nucleosome core particles in vitro, which is a prerequisite for pioneer transcription factors. Experiments using purified proteins and in cellular contexts showed that SOX17 variants phase-separated more efficiently than SOX2, suggesting an enhanced ability to self-organise. Systematic deletion analyses showed that the N-terminus of SOX17FNV was dispensable for its reprogramming activity. However, the C-terminus encodes essential domains indicating multivalent interactions that drive transactivation and reprogramming. We defined a minimal SOX17FNV (miniSOX) that can support reprogramming with high activity, reducing the payload of reprogramming cassettes. This study uncovers the mechanisms behind SOX17FNV-induced pluripotency and establishes engineered SOX factors as powerful cell engineering tools.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias/metabolismo , DNA/metabolismo , Mutação Puntual , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismoRESUMO
Pioneer transcription factors are proteins that induce cellular identity transitions by binding to inaccessible regions of DNA in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA elements. The structural features and dynamics modulating their interaction with nucleosomes are still unresolved. From a combination of experiments and molecular simulations, we reveal here how the pioneer factor and master regulator of pluripotency, Oct4, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4's impact on nucleosome dynamics depends on the binding site position and the mobility of the unstructured tails of nucleosomal histone proteins. Oct4 uses both its DNA binding domains to propagate and stabilize open nucleosome conformations, one for specific sequence recognition and the other for nonspecific interactions with nearby regions of DNA. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics.
Assuntos
Nucleossomos , Fator 3 de Transcrição de Octâmero/metabolismo , Cromatina/genética , Histonas/metabolismo , Nucleossomos/genética , Fatores de Transcrição/metabolismoRESUMO
Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.
Assuntos
Técnicas de Reprogramação Celular , Evolução Molecular Direcionada , Fatores do Domínio POU/genética , Animais , Fator 4 Semelhante a Kruppel , Camundongos , Engenharia de ProteínasRESUMO
SOX17 is a transcription factor directing the specification and development of the primitive endoderm, primitive germ cells, definitive endoderm and, subsequently, is involved in the cardiovascular system and several endoderm-derived organs. The analysis of cancer genome sequencing data classified SOX17 as mutated cancer driver gene in endometrial cancer. These studies identified hotspot missense mutations within its DNA binding and transactivation domains. In somatic cell reprogramming, structure-based protein re-engineering showed a single missense mutation in SOX17 can change the DNA dependent heterodimer formation with OCT4 and enables the replacement of SOX2 with SOX17 mutants to induce pluripotency. This reveals the profound impact of specific missense mutations on gene function and regulatory activity. Here, we review the roles of SOX17 in cancer and discuss its cross-talk with the WNT/ß-catenin pathway, potentially reconciling its activity as re-engineered reprogramming factor and mutated cancer driver gene.
Assuntos
Diferenciação Celular , Reprogramação Celular , Neoplasias/patologia , Fatores de Transcrição SOXF/metabolismo , Via de Sinalização Wnt , Animais , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Transcrição SOXF/genética , Transdução de SinaisRESUMO
PURPOSE: XY individuals with disorders/differences of sex development (DSD) are characterized by reduced androgenization caused, in some children, by gonadal dysgenesis or testis regression during fetal development. The genetic etiology for most patients with 46,XY gonadal dysgenesis and for all patients with testicular regression syndrome (TRS) is unknown. METHODS: We performed exome and/or Sanger sequencing in 145 individuals with 46,XY DSD of unknown etiology including gonadal dysgenesis and TRS. RESULTS: Thirteen children carried heterozygous missense pathogenic variants involving the RNA helicase DHX37, which is essential for ribosome biogenesis. Enrichment of rare/novel DHX37 missense variants in 46,XY DSD is highly significant compared with controls (P value = 5.8 × 10-10). Five variants are de novo (P value = 1.5 × 10-5). Twelve variants are clustered in two highly conserved functional domains and were specifically associated with gonadal dysgenesis and TRS. Consistent with a role in early testis development, DHX37 is expressed specifically in somatic cells of the developing human and mouse testis. CONCLUSION: DHX37 pathogenic variants are a new cause of an autosomal dominant form of 46,XY DSD, including gonadal dysgenesis and TRS, showing that these conditions are part of a clinical spectrum. This raises the possibility that some forms of DSD may be a ribosomopathy.
Assuntos
Disgenesia Gonadal 46 XY/genética , Mutação de Sentido Incorreto , RNA Helicases/genética , Análise de Sequência de DNA/métodos , Testículo/crescimento & desenvolvimento , Adolescente , Animais , Pré-Escolar , Feminino , Predisposição Genética para Doença , Heterozigoto , Humanos , Recém-Nascido , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Taxa de Mutação , Domínios Proteicos , RNA Helicases/química , Testículo/metabolismo , Adulto JovemRESUMO
Advanced strategies to interconvert cell types provide promising avenues to model cellular pathologies and to develop therapies for neurological disorders. Yet, methods to directly transdifferentiate somatic cells into multipotent induced neural stem cells (iNSCs) are slow and inefficient, and it is unclear whether cells pass through a pluripotent state with full epigenetic reset. We report iNSC reprogramming from embryonic and aged mouse fibroblasts as well as from human blood using an engineered Sox17 (eSox17FNV). eSox17FNV efficiently drives iNSC reprogramming while Sox2 or Sox17 fail. eSox17FNV acquires the capacity to bind different protein partners on regulatory DNA to scan the genome more efficiently and has a more potent transactivation domain than Sox2. Lineage tracing and time-resolved transcriptomics show that emerging iNSCs do not transit through a pluripotent state. Our work distinguishes lineage from pluripotency reprogramming with the potential to generate more authentic cell models for aging-associated neurodegenerative diseases.
Assuntos
Células-Tronco Neurais , Humanos , Animais , Camundongos , Envelhecimento , Epigenômica , Perfilação da Expressão Gênica , Proteínas HMGB , Fatores de Transcrição SOXF/genéticaRESUMO
The functional consequences of cancer-associated missense mutations are unclear for the majority of proteins. We have previously demonstrated that the activity of SOX and Pit-Oct-Unc (POU) family factors during pluripotency reprogramming can be switched and enhanced with rationally placed point mutations. Here, we interrogated cancer mutation databases and identified recurrently mutated positions at critical structural interfaces of the DNA-binding domains of paralogous SOX and POU family transcription factors. Using the conversion of mouse embryonic fibroblasts to induced pluripotent stem cells as functional readout, we identified several gain-of-function mutations that enhance pluripotency reprogramming by SOX2 and OCT4. Wild-type SOX17 cannot support reprogramming but the recurrent missense mutation SOX17-V118M is capable of inducing pluripotency. Furthermore, SOX17-V118M promotes oncogenic transformation, enhances thermostability and elevates cellular protein levels of SOX17. We conclude that the mutational profile of SOX and POU family factors in cancer can guide the design of high-performance reprogramming factors. Furthermore, we propose cellular reprogramming as a suitable assay to study the functional impact of cancer-associated mutations.