RESUMO
Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição SOXB1 , Super Intensificadores , Transcrição Gênica , DNA/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição SOXB1/genética , Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Microscopia/métodosRESUMO
The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.
Assuntos
Neoplasias da Mama/genética , Metástase Neoplásica/genética , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteína Homeobox Nanog/metabolismo , Metástase Neoplásica/fisiopatologia , Células Neoplásicas Circulantes/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.
Assuntos
Reprogramação Celular , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Fibroblastos/metabolismo , Código das Histonas , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elementos Reguladores de Transcrição , Fatores de Transcrição SOXB1/metabolismo , Elementos Silenciadores TranscricionaisRESUMO
Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.
Assuntos
Fator de Ligação a CCCTC , Elementos Facilitadores Genéticos , Fator de Crescimento Insulin-Like II , RNA Longo não Codificante , Fatores de Transcrição SOXB1 , Animais , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Região de Controle de Locus Gênico/genética , Impressão Genômica , Genômica/métodosRESUMO
Metastasis frequently develops years after the removal of a primary tumor, from a minority of disseminated cancer cells that survived as latent entities through unknown mechanisms. We isolated latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines and defined the mechanisms that suppress outgrowth, support long-term survival, and maintain tumor-initiating potential in these cells during the latent metastasis stage. LCC cells show stem-cell-like characteristics and express SOX2 and SOX9 transcription factors, which are essential for their survival in host organs under immune surveillance and for metastatic outgrowth under permissive conditions. Through expression of the WNT inhibitor DKK1, LCC cells self-impose a slow-cycling state with broad downregulation of ULBP ligands for NK cells and evasion of NK-cell-mediated clearance. By expressing a Sox-dependent stem-like state and actively silencing WNT signaling, LCC cells can enter quiescence and evade innate immunity to remain latent for extended periods.
Assuntos
Comunicação Autócrina , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metástase Neoplásica/imunologia , Metástase Neoplásica/patologia , Evasão Tumoral , Via de Sinalização Wnt , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXB2/metabolismo , Animais , Blastocisto/metabolismo , Fator de Transcrição CDX2 , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Análise de Célula Única , Fatores de Transcrição/genéticaRESUMO
Transcription factor (TF) binding to DNA is fundamental for gene regulation. However, it remains unknown how the dynamics of TF-DNA interactions change during cell-fate determination in vivo. Here, we use photo-activatable FCS to quantify TF-DNA binding in single cells of developing mouse embryos. In blastocysts, the TFs Oct4 and Sox2, which control pluripotency, bind DNA more stably in pluripotent than in extraembryonic cells. By contrast, in the four-cell embryo, Sox2 engages in more long-lived interactions than does Oct4. Sox2 long-lived binding varies between blastomeres and is regulated by H3R26 methylation. Live-cell tracking demonstrates that those blastomeres with more long-lived binding contribute more pluripotent progeny, and reducing H3R26 methylation decreases long-lived binding, Sox2 target expression, and pluripotent cell numbers. Therefore, Sox2-DNA binding predicts mammalian cell fate as early as the four-cell stage. More generally, we reveal the dynamic repartitioning of TFs between DNA sites driven by physiological epigenetic changes. VIDEO ABSTRACT.
Assuntos
Fatores de Transcrição SOXB1/metabolismo , Animais , Blastocisto/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , DNA/metabolismo , Difusão , Regulação para Baixo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/análise , Histonas/metabolismo , Cinética , Metilação , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Espectrometria de FluorescênciaRESUMO
Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci.
Assuntos
Regulação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico , Animais , Camundongos , Elementos Facilitadores Genéticos , Genômica , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution.
Assuntos
Metilação de DNA , Análise de Célula Única , Animais , Ilhas de CpG , Metilases de Modificação do DNA/metabolismo , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos , Humanos , Camundongos , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/metabolismoRESUMO
How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.
Assuntos
Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Animais , Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Transcription factors are defined by their sequence-specific binding to DNA and by their selective impacts on gene expression, depending on specific binding sites. The factor binding motifs in the DNA should thus represent a blueprint of regulatory logic, suggesting that transcription factor binding patterns on the genome (e.g., measured by ChIP-seq) should indicate which target genes the factors are directly controlling. However, although genetic data confirm high impacts of transcription factor perturbation in embryology, transcription factors bind to far more sites than the number of genes they dynamically regulate, when measured by direct perturbation in a given cell type. Also, deletion of carefully chosen transcription factor binding sites often gives disappointingly weak results. In a new study in the previous issue of Genes & Development, Lo and colleagues (pp. 1079-1095) reconcile these contradictions by using an elegant experimental system to directly compare the roles of transcription factor-binding site interaction in gene regulation maintenance with roles of the same factor-site interactions in gene regulation through developmental change. They examine Oct4:Sox2 shared target genes under maintained versus reinduced pluripotency conditions within the same cell clone. The results show that the same factor-site interaction impacts can appear modest in assays in developmental steady-state but are far more important as regulatory catalysts of developmental change.
Assuntos
Células-Tronco Embrionárias , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Sítios de Ligação , Fator 3 de Transcrição de Octâmero/metabolismo , DNA/metabolismo , Fatores de Transcrição SOXB1/genética , Diferenciação Celular/genéticaRESUMO
Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
Assuntos
DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Análise de Célula Única , Animais , Imunoprecipitação da Cromatina , Epigênese Genética , Estudo de Associação Genômica Ampla , Cinética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2-4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.
Assuntos
Epigênese Genética , Código das Histonas , Histonas , Nucleossomos , Fator 3 de Transcrição de Octâmero , Fatores de Transcrição SOXB1 , Humanos , Microscopia Crioeletrônica , DNA/química , DNA/genética , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Histonas/ultraestrutura , Nucleossomos/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/ultraestrutura , Processamento de Proteína Pós-Traducional , Fatores de Transcrição SOXB1/metabolismo , Regulação Alostérica , Proteínas de Ligação a RNA/genética , Proteínas Matrilinas/genética , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Diferenciação Celular/genética , Domínios ProteicosRESUMO
Recent exon sequencing studies have revealed that over 20% of human tumors have mutations in subunits of mSWI/SNF (BAF) complexes. To investigate the underlying mechanism, we studied human synovial sarcoma (SS), in which transformation results from the translocation of exactly 78 amino acids of SSX to the SS18 subunit of BAF complexes. We demonstrate that the SS18-SSX fusion protein competes for assembly with wild-type SS18, forming an altered complex lacking the tumor suppressor BAF47 (hSNF5). The altered complex binds the Sox2 locus and reverses polycomb-mediated repression, resulting in Sox2 activation. Sox2 is uniformly expressed in SS tumors and is essential for proliferation. Increasing the concentration of wild-type SS18 leads to reassembly of wild-type complexes retargeted away from the Sox2 locus, polycomb-mediated repression of Sox2, and cessation of proliferation. This mechanism of transformation depends on only two amino acids of SSX, providing a potential foundation for therapeutic intervention.
Assuntos
Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/patologia , Proliferação de Células , Transformação Celular Neoplásica , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Repressoras/química , Proteína SMARCB1 , Fatores de Transcrição SOXB1/genética , Sarcoma Sinovial/genética , Fatores de Transcrição/metabolismoRESUMO
Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of â¼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.
Assuntos
Cromatina/química , Cromatina/genética , Metilação de DNA , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Hibridização in Situ Fluorescente , Lisina/genética , Lisina/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição SOXB1/genética , Proteína de Homoeobox de Baixa Estatura/genética , Fatores de Transcrição/genéticaRESUMO
Genome-wide transcriptional activity involves the binding of many transcription factors (TFs) to thousands of sites in the genome. Pioneer TFs are a class of TFs that maintain open chromatin and allow non-pioneer TFs access to their target sites. Determining which TF binding sites directly drive transcription remains a challenge. Here, we use acute protein depletion of the pioneer TF SOX2 to establish its functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of the pioneer factor. To understand the relationship with transcription, we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to all other SOX2 binding sites. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted mainly at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.
Assuntos
Cromatina , Fatores de Transcrição SOXB1 , Fatores de Transcrição , Sítios de Ligação , Cromatina/genética , Regulação da Expressão Gênica , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , CamundongosRESUMO
SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Crista Neural , Placa Neural , Fatores de Transcrição SOXB1 , Proteínas de Xenopus , Xenopus laevis , Animais , Placa Neural/metabolismo , Placa Neural/embriologia , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Crista Neural/metabolismo , Crista Neural/citologia , Blástula/metabolismo , Embrião não Mamífero/metabolismoRESUMO
Neutrophils express Toll-like receptors (TLRs) for the recognition of conserved bacterial elements to initiate antimicrobial responses. However, whether other cytosolic DNA sensors are expressed by neutrophils remains elusive. Here we found constitutive expression of the transcription factor Sox2 in the cytoplasm of mouse and human neutrophils. Neutrophil-specific Sox2 deficiency exacerbated bacterial infection. Sox2 directly recognized microbial DNA through its high-mobility-group (HMG) domain. Upon challenge with bacterial DNA, Sox2 dimerization was needed to activate a complex of the kinase TAK1 and its binding partner TAB2, which led to activation of the transcription factors NF-κB and AP-1 in neutrophils. Deficiency in TAK1 or TAB2 impaired Sox2-mediated antibacterial immunity. Overall, we reveal a previously unrecognized role for Sox2 as a cytosolic sequence-specific DNA sensor in neutrophils, which might provide potential therapeutic strategies for the treatment of infectious diseases.
Assuntos
DNA Bacteriano/imunologia , Imunidade Inata , Listeriose/imunologia , Neutrófilos/imunologia , Fatores de Transcrição SOXB1/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Citoplasma/imunologia , Citoplasma/microbiologia , Regulação da Expressão Gênica , Humanos , Listeria monocytogenes/imunologia , Listeriose/genética , Listeriose/microbiologia , Listeriose/mortalidade , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , Neutrófilos/microbiologia , Multimerização Proteica , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Análise de Sobrevida , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologiaRESUMO
The factors that shape the distinctive tumor-immune landscapes of various types and subtypes of cancer remain poorly understood. In this issue of Immunity, Mollaoglu et al. (2018) reveal a mechanistic link between the function of lineage specifiers SOX2 and NKX2-1 and the presence of neutrophils in the tumor-immune microenvironment of lung cancer.
Assuntos
Neoplasias Pulmonares , Diferenciação Celular , Humanos , Fatores de Transcrição SOXB1 , Microambiente TumoralRESUMO
The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.