RESUMO
Pancreatic ductal adenocarcinoma (PDAC) poses a significant threat due to its tendency to evade early detection, frequent metastasis, and the subsequent challenges in devising effective treatments. Processes that govern epithelial-mesenchymal transition (EMT) in PDAC hold promise for advancing novel therapeutic strategies. SAMD1 (SAM domain-containing protein 1) is a CpG island-binding protein that plays a pivotal role in the repression of its target genes. Here, we revealed that SAMD1 acts as a repressor of genes associated with EMT. Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor and negative regulator of SAMD1, which inhibits SAMD1 chromatin-binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.
RESUMO
Acute myeloid leukemia (AML) is a hematological malignancy characterized by abnormal proliferation and accumulation of immature myeloid cells in the bone marrow. Inflammation plays a crucial role in AML progression, but excessive activation of cell-intrinsic inflammatory pathways can also trigger cell death. IRF2BP2 is a chromatin regulator implicated in AML pathogenesis, although its precise role in this disease is not fully understood. In this study, we demonstrate that IRF2BP2 interacts with the AP-1 heterodimer ATF7/JDP2, which is involved in activating inflammatory pathways in AML cells. We show that IRF2BP2 is recruited by the ATF7/JDP2 dimer to chromatin and counteracts its gene-activating function. Loss of IRF2BP2 leads to overactivation of inflammatory pathways, resulting in strongly reduced proliferation. Our research indicates that a precise equilibrium between activating and repressive transcriptional mechanisms creates a pro-oncogenic inflammatory environment in AML cells. The ATF7/JDP2-IRF2BP2 regulatory axis is likely a key regulator of this process and may, therefore, represent a promising therapeutic vulnerability for AML. Thus, our study provides new insights into the molecular mechanisms underlying AML pathogenesis and identifies a potential therapeutic target for AML treatment.
Assuntos
Inflamação , Leucemia Mieloide Aguda , Fator de Transcrição AP-1 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/genética , Inflamação/genética , Inflamação/metabolismo , Linhagem Celular Tumoral , Fatores Ativadores da Transcrição/metabolismo , Fatores Ativadores da Transcrição/genética , Cromatina/metabolismo , Proliferação de Células , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Células HEK293 , Regulação Leucêmica da Expressão Gênica , Multimerização Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNARESUMO
The lysine acetyltransferase KAT6A (MOZ, MYST3) belongs to the MYST family of chromatin regulators, facilitating histone acetylation. Dysregulation of KAT6A has been implicated in developmental syndromes and the onset of acute myeloid leukemia (AML). Previous work suggests that KAT6A is recruited to its genomic targets by a combinatorial function of histone binding PHD fingers, transcription factors and chromatin binding interaction partners. Here, we demonstrate that a winged helix (WH) domain at the very N-terminus of KAT6A specifically interacts with unmethylated CpG motifs. This DNA binding function leads to the association of KAT6A with unmethylated CpG islands (CGIs) genome-wide. Mutation of the essential amino acids for DNA binding completely abrogates the enrichment of KAT6A at CGIs. In contrast, deletion of a second WH domain or the histone tail binding PHD fingers only subtly influences the binding of KAT6A to CGIs. Overexpression of a KAT6A WH1 mutant has a dominant negative effect on H3K9 histone acetylation, which is comparable to the effects upon overexpression of a KAT6A HAT domain mutant. Taken together, our work revealed a previously unrecognized chromatin recruitment mechanism of KAT6A, offering a new perspective on the role of KAT6A in gene regulation and human diseases.
Assuntos
Cromatina , Histona Acetiltransferases , Histonas , Humanos , Cromatina/genética , Ilhas de CpG/genética , DNA , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , AcetilaçãoRESUMO
Gene regulatory networks are pivotal for many biological processes. In mouse embryonic stem cells (mESCs), the transcriptional network can be divided into three functionally distinct modules: Polycomb, Core, and Myc. The Polycomb module represses developmental genes, while the Myc module is associated with proliferative functions, and its mis-regulation is linked to cancer development. Here, we show that, in mESCs, the Polycomb repressive complex 2 (PRC2)-associated protein EPOP (Elongin BC and Polycomb Repressive Complex 2-associated protein; a.k.a. C17orf96, esPRC2p48, and E130012A19Rik) co-localizes at chromatin with members of the Myc and Polycomb module. EPOP interacts with the transcription elongation factor Elongin BC and the H2B deubiquitinase USP7 to modulate transcriptional processes in mESCs similar to MYC. EPOP is commonly upregulated in human cancer, and its loss impairs the proliferation of several human cancer cell lines. Our findings establish EPOP as a transcriptional modulator, which impacts both Polycomb and active gene transcription in mammalian cells.
Assuntos
Cromatina/química , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Complexo Repressor Polycomb 2/genética , Fatores de Transcrição/genética , Proteases Específicas de Ubiquitina/genética , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteínas Cromossômicas não Histona , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Elonguina , Embrião de Mamíferos , Redes Reguladoras de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo , Transcrição Gênica , Peptidase 7 Específica de Ubiquitina , Proteases Específicas de Ubiquitina/metabolismoRESUMO
The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.
Assuntos
Ciclo Celular/genética , Proteínas de Drosophila/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Hemócitos/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular/genética , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Ontologia Genética , Regiões Promotoras Genéticas , Isoformas de Proteínas , Interferência de RNA , RNA-Seq , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Linfócitos T CD8-Positivos , Fibroblastos Associados a Câncer/metabolismo , Interleucina-17/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Proteínas de Homeodomínio , Neoplasias PancreáticasRESUMO
The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.
Assuntos
Ilhas de CpG/genética , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Animais , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas do Grupo Polycomb/química , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
HDGF-related protein 3 (HRP3, also known as HDGFL3) belongs to the family of HDGF-related proteins (HRPs) and plays an essential role in hepatocellular carcinoma pathogenesis. All HRPs have a PWWP domain at the N-terminus that binds both histone and DNA substrates. Despite previous advances in PWWP domains, the molecular basis by which HRP3 interacts with chromatin is unclear. In this study, we solved the crystal structures of the HRP3 PWWP domain in complex with various double-stranded DNAs with/without bound histone peptides. We found that HRP3 PWWP bound to the phosphate backbone of the DNA minor groove and showed a preference for DNA molecules bearing a narrow minor groove width. In addition, HRP3 PWWP preferentially bound to histone peptides bearing the H3K36me3/2 modification. HRP3 PWWP uses two adjacent surfaces to bind both DNA and histone substrates simultaneously, enabling us to generate a model illustrating the recruitment of PWWP to H3K36me3-containing nucleosomes. Cell-based analysis indicated that both DNA and histone binding by the HRP3 PWWP domain is important for HRP3 recruitment to chromatin in vivo. Our work establishes that HRP3 PWWP is a new family of minor groove-specific DNA-binding proteins, which improves our understanding of HRP3 and other PWWP domain-containing proteins.
Assuntos
Cromatina/química , DNA/química , Proteínas Nucleares/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas do Citoesqueleto , Células HEK293 , Células Hep G2 , Histonas/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Nucleossomos/química , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , Frações Subcelulares , Xenopus laevisRESUMO
Increasing evidence shows that many transcription factors execute important biologic functions independent from their DNA-binding capacity. The NF-κB p65 (RELA) subunit is a central regulator of innate immunity. Here, we investigated the relative functional contribution of p65 DNA-binding and dimerization in p65-deficient human and murine cells reconstituted with single amino acid mutants preventing either DNA-binding (p65 E/I) or dimerization (p65 FL/DD). DNA-binding of p65 was required for RelB-dependent stabilization of the NF-κB p100 protein. The antiapoptotic function of p65 and expression of the majority of TNF-α-induced genes were dependent on p65's ability to bind DNA and to dimerize. Chromatin immunoprecipitation with massively parallel DNA sequencing experiments revealed that impaired DNA-binding and dimerization strongly diminish the chromatin association of p65. However, there were also p65-independent TNF-α-inducible genes and a subgroup of p65 binding sites still allowed some residual chromatin association of the mutants. These sites were enriched in activator protein 1 (AP-1) binding motifs and showed increased chromatin accessibility and basal transcription. This suggests a mechanism of assisted p65 chromatin association that can be in part facilitated by chromatin priming and cooperativity with other transcription factors such as AP-1.-Riedlinger, T., Liefke, R., Meier-Soelch, J., Jurida, L., Nist, A., Stiewe, T., Kracht, M., Schmitz, M. L. NF-κB p65 dimerization and DNA-binding is important for inflammatory gene expression.
Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Expressão Gênica/genética , Inflamação/genética , Fator de Transcrição RelA/genética , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Dimerização , Células HeLa , Humanos , Camundongos , Ligação Proteica/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição RelB/genéticaRESUMO
Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. Here we show that the histone demethylase KDM5A is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses.
Assuntos
Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Receptores Notch/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Camundongos , Linfócitos T , Ubiquitina-Proteína LigasesRESUMO
Polycomb-like proteins (PCLs) are a crucial group of proteins associated with the Polycomb repressive complex 2 (PRC2) and are responsible for setting up the PRC2.1 subcomplex. In the vertebrate system, three homologous PCLs exist: PHF1 (PCL1), MTF2 (PCL2), and PHF19 (PCL3). Although the PCLs share a similar domain composition, they differ significantly in their primary sequence. PCLs play a critical role in targeting PRC2.1 to its genomic targets and regulating the functionality of PRC2. However, they also have PRC2-independent functions. In addition to their physiological roles, their dysregulation has been associated with various human cancers. In this review, we summarize the current understanding of the molecular mechanisms of the PCLs and how alterations in their functionality contribute to cancer development. We particularly highlight the nonoverlapping and partially opposing roles of the three PCLs in human cancer. Our review provides important insights into the biological significance of the PCLs and their potential as therapeutic targets for cancer treatment.
Assuntos
Proteínas de Drosophila , Neoplasias , Humanos , Histonas/genética , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Complexo Repressor Polycomb 2/genética , Núcleo Celular/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas de Drosophila/metabolismo , Neoplasias/genéticaRESUMO
SAM domain-containing protein 1 (SAMD1) has been implicated in atherosclerosis, as well as in chromatin and transcriptional regulation, suggesting a versatile and complex biological function. However, its role at an organismal level is currently unknown. Here, we generated SAMD1-/- and SAMD1+/- mice to explore the role of SAMD1 during mouse embryogenesis. Homozygous loss of SAMD1 was embryonic lethal, with no living animals seen after embryonic day 18.5. At embryonic day 14.5, organs were degrading and/or incompletely developed, and no functional blood vessels were observed, suggesting failed blood vessel maturation. Sparse red blood cells were scattered and pooled, primarily near the embryo surface. Some embryos had malformed heads and brains at embryonic day 15.5. In vitro, SAMD1 absence impaired neuronal differentiation processes. Heterozygous SAMD1 knockout mice underwent normal embryogenesis and were born alive. Postnatal genotyping showed a reduced ability of these mice to thrive, possibly due to altered steroidogenesis. In summary, the characterization of SAMD1 knockout mice suggests a critical role of SAMD1 during developmental processes in multiple organs and tissues.
Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Camundongos , Animais , Embrião de Mamíferos/metabolismo , Camundongos Knockout , Heterozigoto , HomozigotoRESUMO
Inhibition of protein-protein interactions (PPIs) via designed peptides is an effective strategy to perturb their biological functions. The Elongin BC heterodimer (ELOB/C) binds to a BC-box motif and is essential for cancer cell growth. Here, we report a peptide that mimics the high-affinity BC-box of the PRC2-associated protein EPOP. This peptide tightly binds to the ELOB/C dimer (kD = 0.46 ± 0.02 nM) and blocks the association of ELOB/C with its interaction partners, both in vitro and in the cellular environment. Cancer cells treated with our peptide inhibitor showed decreased cell viability, increased apoptosis, and perturbed gene expression. Therefore, our work proposes that blocking the BC-box-binding pocket of ELOB/C is a feasible strategy to impair its function and inhibit cancer cell growth. Our peptide inhibitor promises novel mechanistic insights into the biological function of the ELOB/C dimer and offers a starting point for therapeutics linked to ELOB/C dysfunction.
Assuntos
Neoplasias , Fatores de Transcrição , Elonguina/metabolismo , Fatores de Transcrição/metabolismo , Ligação Proteica , Peptídeos/farmacologia , Peptídeos/metabolismo , Apoptose , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias/tratamento farmacológicoRESUMO
The Polycomb repressive complex 2 (PRC2) is an essential chromatin regulatory complex involved in repressing the transcription of diverse developmental genes. PRC2 consists of a core complex; possessing H3K27 methyltransferase activity and various associated factors that are important to modulate its function. During evolution, the composition of PRC2 and the functionality of PRC2 components have changed considerably. Here, we compare the PRC2 complex members of Drosophila and mammals and describe their adaptation to altered biological needs. We also highlight how the PRC2.1 subcomplex has gained multiple novel functions and discuss the implications of these changes for the function of PRC2 in chromatin regulation.
Assuntos
Drosophila , Complexo Repressor Polycomb 2 , Animais , Núcleo Celular , Cromatina/genética , Drosophila/genética , Mamíferos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genéticaRESUMO
The unmethylated CpG island-binding protein SAMD1 is upregulated in many human cancer types, but its cancer-related role has not yet been investigated. Here, we used the hepatocellular carcinoma cell line HepG2 as a cancer model and investigated the cellular and transcriptional roles of SAMD1 using ChIP-Seq and RNA-Seq. SAMD1 targets several thousand gene promoters, where it acts predominantly as a transcriptional repressor. HepG2 cells with SAMD1 deletion showed slightly reduced proliferation, but strongly impaired clonogenicity. This phenotype was accompanied by the decreased expression of pro-proliferative genes, including MYC target genes. Consistently, we observed a decrease in the active H3K4me2 histone mark at most promoters, irrespective of SAMD1 binding. Conversely, we noticed an increase in interferon response pathways and a gain of H3K4me2 at a subset of enhancers that were enriched for IFN-stimulated response elements (ISREs). We identified key transcription factor genes, such as IRF1, STAT2, and FOSL2, that were directly repressed by SAMD1. Moreover, SAMD1 deletion also led to the derepression of the PI3K-inhibitor PIK3IP1, contributing to diminished mTOR signaling and ribosome biogenesis pathways. Our work suggests that SAMD1 is involved in establishing a pro-proliferative setting in hepatocellular carcinoma cells. Inhibiting SAMD1's function in liver cancer cells may therefore lead to a more favorable gene signature.
RESUMO
2-cell-like cells (2CLCs)-which comprise only â¼1% of murine embryonic stem cells (mESCs)-resemble blastomeres of 2-cell-stage embryos and are used to investigate zygotic genome activation (ZGA). Here, we discovered that TRIM66 and DAX1 function together as negative regulators of the 2C-like state in mESCs. Chimeric assays confirmed that mESCs lacking TRIM66 or DAX1 function have bidirectional embryonic and extraembryonic differentiation potential. TRIM66 functions by recruiting the co-repressor DAX1 to the Dux promoter, and TRIM66's repressive effect on Dux is dependent on DAX1. A solved crystal structural shows that TRIM66's PHD finger recognizes H3K4-K9me3, and mutational evidence confirmed that TRIM66's PHD finger is essential for its repression of Dux. Thus, beyond expanding the scope of known 2CLC regulators, our study demonstrates that interventions disrupting TRIM66 or DAX1 function in mESCs yield 2CLCs with expanded bidirectional differentiation potential, opening doors for the practical application of these totipotent-like cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Zigoto , Animais , Células-Tronco Embrionárias , Genoma , Camundongos , Regiões Promotoras GenéticasRESUMO
In recent years, the amount of available literature, data and computational tools has increased exponentially, providing opportunities and challenges to make use of this vast amount of material. Here, we describe how we utilized publicly available information to identify the previously hardly characterized protein SAMD1 (SAM domain-containing protein 1) as a novel unmethylated CpG island-binding protein. This discovery is an example, how the richness of material and tools on the internet can be used to make scientific breakthroughs, but also the hurdles that may occur. Specifically, we discuss how the misrepresentation of SAMD1 in literature and databases may have prevented an earlier characterization of this protein and we address what can be learned from this example.
RESUMO
BACKGROUND: Notch signaling controls cell fate decisions in many contexts during development and adult stem cell homeostasis and, when dysregulated, leads to carcinogenesis. The central transcription factor RBPJ assembles the Notch coactivator complex in the presence of Notch signaling, and represses Notch target gene expression in its absence. RESULTS: We identified L3MBTL2 and additional members of the non-canonical polycomb repressive PRC1.6 complex in DNA-bound RBPJ associated complexes and demonstrate that L3MBTL2 directly interacts with RBPJ. Depletion of RBPJ does not affect occupancy of PRC1.6 components at Notch target genes. Conversely, absence of L3MBTL2 reduces RBPJ occupancy at enhancers of Notch target genes. Since L3MBTL2 and additional members of the PRC1.6 are known to be SUMOylated, we investigated whether RBPJ uses SUMO-moieties as contact points. Indeed, we found that RBPJ binds to SUMO2/3 and that this interaction depends on a defined SUMO-interaction motif. Furthermore, we show that pharmacological inhibition of SUMOylation reduces RBPJ occupancy at Notch target genes. CONCLUSIONS: We propose that the PRC1.6 complex and its conjugated SUMO-modifications provide a favorable environment for binding of RBPJ to Notch target genes.
Assuntos
Proteínas de Drosophila , Fatores de Transcrição , Diferenciação Celular , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Personalized treatment of acute myeloid leukemia (AML) that target individual aberrations strongly improved the survival of AML patients. However, AML is still one of the most lethal cancer diseases of the 21st century, demonstrating the need to find novel drug targets and to explore alternative treatment strategies. Upon investigation of public perturbation data, we identified the transcription factor IRF8 as a novel AML-specific susceptibility gene in humans. IRF8 is upregulated in a subset of AML cells and its deletion leads to impaired proliferation in those cells. Consistently, high IRF8 expression is associated with poorer patients' prognoses. Combining gene expression changes upon IRF8 deletion and the genome-wide localization of IRF8 in the AML cell line MV4-11, we demonstrate that IRF8 directly regulates key signaling molecules, such as the kinases SRC and FAK, the transcription factors RUNX1 and IRF5, and the cell cycle regulator Cyclin D1. IRF8 loss impairs AML-driving signaling pathways, including the WNT, Chemokine, and VEGF signaling pathways. Additionally, many members of the focal adhesion pathway showed reduced expression, providing a putative link between high IRF8 expression and poor prognosis. Thus, this study suggests that IRF8 could serve as a biomarker and potential molecular target in a subset of human AMLs.