RESUMO
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 (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus, the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin. Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence-specific manner. Here, we reveal the structural dynamics of nucleosomes that mediate Oct4 binding from molecular dynamics simulations. Nucleosome flexibility and the amplitude of nucleosome motions such as breathing and twisting are enhanced in nucleosomes with multiple TF binding sites. Moreover, the regions around the binding sites display higher local structural flexibility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations in which Oct4 recognizes partial binding sites display stable TF-DNA interactions similar to those observed in complexes with free DNA and compatible with the DNA curvature and DNA-histone interactions. Therefore, we propose a structural basis for nucleosome recognition by a pioneer TF that is essential for understanding how chromatin is unraveled during cell fate conversions.
Assuntos
DNA , Nucleossomos , Sítios de Ligação , Cromatina , Histonas/metabolismoRESUMO
The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA-binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re-analyzing ChIP-Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type-specific POU factor function is determined by select residues that affect DNA-dependent dimerization.
Assuntos
Transdiferenciação Celular/genética , Reprogramação Celular/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Fatores do Domínio POU/química , Fatores do Domínio POU/metabolismo , Multimerização Proteica , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos , Epigênese Genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Moleculares , Motivos de Nucleotídeos , Fatores de Transcrição de Octâmero/química , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Fatores do Domínio POU/genética , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , TranscriptomaRESUMO
Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Camundongos , Ratos , Animais , Suínos , Macaca fascicularis/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reprogramação Celular , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular , Mamíferos/metabolismoRESUMO
Oct4 collaborates primarily with other transcriptional factors or coregulators to maintain pluripotency. However, how Oct4 exerts its function is still unclear. Here, we show that the Oct4 linker interface mediates competing yet balanced Oct4 protein interactions that are crucial for maintaining pluripotency. Oct4 linker mutant embryonic stem cells (ESCs) show decreased expression of self-renewal genes and increased expression of differentiation genes, resulting in impaired ESC self-renewal and early embryonic development. The linker mutation interrupts the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 is decreased. In contrast, interactions between Oct4 and Cbx1, Ctr9, and Cdc73 are increased, disrupting the epigenetic state of ESCs. Control of the expression level of Klf5, Cbx1, or Cdc73 rebalances the Oct4 interactome and rescues the pluripotency of linker mutant ESCs, indicating that such factors interact with Oct4 competitively. Thus, we provide previously unidentified molecular insights into how Oct4 maintains pluripotency.
RESUMO
The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2.
Assuntos
Cromatina , Células-Tronco Pluripotentes , Animais , Cromatina/metabolismo , Redes Reguladoras de Genes , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transcrição GênicaRESUMO
Caveolin-1 (CAV1) has been implicated in the regulation of several signaling pathways and in oncogenesis. Previously, we identified CAV1 as a key determinant of the oncogenic phenotype and tumorigenic activity of cells from tumors of the Ewing's Sarcoma Family (ESFT). However, the possible CAV1 involvement in the chemotherapy resistance commonly presented by an ESFT subset has not been established to date. This report shows that CAV1 expression determines the sensitivity of ESFT cells to clinically relevant chemotherapeutic agents. Analyses of endogenous CAV1 levels in several ESFT cells and ectopic CAV1 expression into ESFT cells expressing low endogenous CAV1 showed that the higher the CAV1 levels, the greater their resistance to drug treatment. Moreover, results from antisense- and shRNA-mediated gene expression knockdown and protein re-expression experiments demonstrated that CAV1 increases the resistance of ESFT cells to doxorubicin (Dox)- and cisplatin (Cp)-induced apoptosis by a mechanism involving the activating phosphorylation of PKCalpha. CAV1 knockdown in ESFT cells led to decreased phospho(Thr(638))-PKCalpha levels and a concomitant sensitization to apoptosis, which were reversed by CAV1 re-expression. These results were recapitulated by PKCalpha knockdown and re-expression in ESFT cells in which CAV1 was previously knocked down, thus demonstrating that phospho(Thr(638))-PKCalpha acts downstream of CAV1 to determine the sensitivity of ESFT cells to chemotherapeutic drugs. These data, along with the finding that CAV1 and phospho(Thr(638))-PKCalpha are co-expressed in approximately 45% of ESFT specimens tested, imply that targeting CAV1 and/or PKCalpha may allow the development of new molecular therapeutic strategies to improve the treatment outcome for patients with ESFT.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caveolina 1/metabolismo , Proteína Quinase C-alfa/metabolismo , Western Blotting , Carbazóis/farmacologia , Caveolina 1/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Treonina/metabolismoRESUMO
OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.
RESUMO
Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here, we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network, even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs and thereby bear major implications for further development and application of iPSC technology.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Fator 3 de Transcrição de Octâmero/genética , Reação em Cadeia da Polimerase , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de RNA , Tetraploidia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Simultaneous expression of Oct4, Klf4, Sox2, and cMyc induces pluripotency in somatic cells (iPSCs). Replacing Oct4 with the neuro-specific factor Brn4 leads to transdifferentiation of fibroblasts into induced neural stem cells (iNSCs). However, Brn4 was recently found to induce transient acquisition of pluripotency before establishing the neural fate. We employed genetic lineage tracing and found that induction of iNSCs with individual vectors leads to direct lineage conversion. In contrast, polycistronic expression produces a Brn4-Klf4 fusion protein that enables induction of pluripotency. Our study demonstrates that a combination of pluripotency and tissue-specific factors allows direct somatic cell transdifferentiation, bypassing the acquisition of a pluripotent state. This result has major implications for lineage conversion technologies, which hold potential for providing a safer alternative to iPSCs for clinical application both in vitro and in vivo.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Transdiferenciação Celular/genética , Reprogramação Celular/genética , Células Híbridas/fisiologia , Fatores de Transcrição/genética , Animais , Fusão Celular , Células Cultivadas , Diploide , Embrião de Mamíferos , Feminino , Células-Tronco Pluripotentes Induzidas/fisiologia , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Neurais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Expression of the ENTPD5/mt-PCPH onco-protein and overexpression of the normal ENTPD5/PCPH protein contribute to the malignant transformation of diverse mammalian cell types, and PCPH is mutated and/or deregulated in various human tumor types. Expression of PCPH or mt-PCPH caused similar phenotypes, yet the effects promoted by mt-PCPH expression were consistently and substantially greater. ATP depletion and increased stressresistance are phenotypes commonly associated with PCPH and mt-PCPH expression. It was suggested that the intrinsic nucleoside triphosphate diphosphohydrolase (NTPDase) activity of PCPH and mt-PCPH may be responsible for these phenotypes, but direct supporting evidence remains to be established. Results from experiments designed to test such hypothesis demonstrate that, as expected, mt-PCPH expression in human colorectal carcinoma (CRC) cells decreased their ATP levels and conferred resistance to oxaliplatin, a colorectal cancer-relevant chemotherapeutic agent. Using a combination of site-directed mutagenesis, immunoprecipitation methods, in vitro enzyme activity assays and in situ enzyme activity determinations in live cells, this report also demonstrates that the mt-PCPH oncoprotein lacks detectable NTPDase activity, indicating that direct ATP cleavage by mt-PCPH did not cause the ATP depletion observed in mt-PCPH-expressing CRC cells. These results strongly suggest that the mt-PCPH oncoprotein may regulate the cellular energy levels and subsequent chemoresistance by an NTPDase-independent mechanism. Understanding possible alternative mechanisms will be essential to devise strategies for the successful treatment of predictably therapeutically resistant tumors expressing either increased PCPH levels or, particularly, the mt-PCPH oncoprotein.
Assuntos
Antígenos CD/genética , Apirase/genética , Neoplasias Colorretais/genética , Proteínas Oncogênicas/metabolismo , Pirofosfatases/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Carcinogênese , Catálise , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Pirofosfatases/químicaRESUMO
Previous reports showed that PCPH is mutated or deregulated in some human tumors, suggesting its participation in malignant progression. Immunohistochemical analyses showed that PCPH is not expressed in normal prostate, but its expression increases along cancer progression stages, being detectable in benign prostatic hyperplasia, highly expressed in prostatic intraepithelial neoplasia, and remaining at high levels in prostate carcinoma. Experiments designed to investigate the contribution of PCPH to the malignant phenotype of prostate cancer cells showed that PCPH overexpression in PC-3 cells, which express nearly undetectable PCPH levels, increased collagen I expression and enhanced invasiveness, whereas shRNA-mediated PCPH knockdown in LNCaP cells, which express high PCPH levels, down-regulated collagen I expression and decreased invasiveness. PCPH regulated invasiveness and collagen I expression by a mechanism involving protein kinase C delta (PKC delta): (a) PCPH knockdown in LNCaP cells decreased PKC delta levels relative to control cells; (b) PKC delta knockdown in LNCaP cells recapitulated all changes caused by PCPH knockdown; and (c) forced expression of PKC delta in cells with knocked down PCPH reverted all changes provoked by PCPH down-regulation and rescued the original phenotype of LNCaP cells. These results strongly suggested that the expression level and/or mutational status of PCPH contributes to determine the invasiveness of prostate cancer cells through a mechanism involving PKC delta. Data from immunohistochemical analyses in serial sections of normal, premalignant, and malignant prostate specimens underscored the clinical significance of our findings by showing remarkably similar patterns of expression for PCPH and PKC delta, thus strongly suggesting their likely coregulation in human tumors.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Oncogênicas/metabolismo , Neoplasias da Próstata/metabolismo , Proteína Quinase C-delta/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Colágeno Tipo I/biossíntese , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Técnicas Genéticas , Humanos , Imuno-Histoquímica/métodos , Masculino , Invasividade Neoplásica , Metástase Neoplásica , PirofosfatasesRESUMO
It is generally accepted that the functional activity of biological macromolecules requires tightly packed three-dimensional structures. Recent theoretical and experimental evidence indicates, however, the importance of molecular flexibility for the proper functioning of some proteins. We examined high resolution structures of proteins in various functional categories with respect to the secondary structure assessment. The latter was considered as a characteristic of the inherent flexibility of a polypeptide chain. We found that the proteins in functionally competent conformational states might be comprised of 20-70% flexible residues. For instance, proteins involved in gene regulation, e.g. transcription factors, are on average largely disordered molecules with over 60% of amino acids residing in "coiled" configurations. In contrast, oxygen transporters constitute a class of relatively rigid molecules with only 30% of residues being locally flexible. Phylogenic comparison of a large number of protein families with respect to the propagation of secondary structure illuminates the growing role of the local flexibility in organisms of greater complexity. Furthermore the local flexibility in protein molecules appears to be dependent on the molecular confinement and is essentially larger in extracellular proteins.