RESUMO
Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.
Assuntos
Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Repetições de Microssatélites , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Priônicas/metabolismo , Domínios Proteicos , Sarcoma de Ewing/patologiaRESUMO
Eukaryotic DNA is packaged into chromatin in the nucleus, restricting the binding of transcription factors (TFs) to their target DNA sites. FOXA1 functions as a pioneer TF to bind condensed chromatin and initiate the opening of local chromatin for gene expression. However, the principles of FOXA1 recruitment and how it subsequently unpacks the condensed chromatin remain elusive. Here, we revealed that FOXA1 intrinsically forms submicron-sized condensates through its N- and C-terminal intrinsically disordered regions (IDRs). Notably, both IDRs enable FOXA1 to dissolve the condensed chromatin. In addition, the DNA-binding capacity of FOXA1 contributes to its ability to both form condensates and dissolve condensed chromatin. Further genome-wide investigation showed that IDRs enable FOXA1 to bind and unpack the condensed chromatin to regulate the proliferation and migration of breast cancer cells. This work provides a principle of how pioneer TFs function to initiate competent chromatin states using their IDRs.
Assuntos
Condensados Biomoleculares , Cromatina , Fator 3-alfa Nuclear de Hepatócito , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Heterocromatina , HumanosRESUMO
Despite the unique ability of pioneer factors (PFs) to target nucleosomal sites in closed chromatin, they only bind a small fraction of their genomic motifs. The underlying mechanism of this selectivity is not well understood. Here, we design a high-throughput assay called chromatin immunoprecipitation with integrated synthetic oligonucleotides (ChIP-ISO) to systematically dissect sequence features affecting the binding specificity of a classic PF, FOXA1, in human A549 cells. Combining ChIP-ISO with in vitro and neural network analyses, we find that (1) FOXA1 binding is strongly affected by co-binding transcription factors (TFs) AP-1 and CEBPB; (2) FOXA1 and AP-1 show binding cooperativity in vitro; (3) FOXA1's binding is determined more by local sequences than chromatin context, including eu-/heterochromatin; and (4) AP-1 is partially responsible for differential binding of FOXA1 in different cell types. Our study presents a framework for elucidating genetic rules underlying PF binding specificity and reveals a mechanism for context-specific regulation of its binding.
Assuntos
Fator 3-alfa Nuclear de Hepatócito , Ligação Proteica , Fator de Transcrição AP-1 , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/genética , Sítios de Ligação , Células A549 , Cromatina/metabolismo , Cromatina/genética , Imunoprecipitação da Cromatina , Oligonucleotídeos/metabolismo , Oligonucleotídeos/genéticaRESUMO
Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA sequences. Two use their POUS domains while the other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap â¼25 base pair DNA. Our analysis of previous genomic data and determination of the ESRRB-nucleosome-OCT4 structure confirmed the generality of these structural features. Moreover, biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Thus, our study suggests a mechanism of how OCT4 can target the nucleosome and open closed chromatin.
Assuntos
Cromatina , Nucleossomos , Fator 3 de Transcrição de Octâmero , Proteínas de Ligação a RNA , Humanos , Sequência de Bases , Reprogramação Celular , Cromatina/genética , DNA/metabolismo , Nucleossomos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.
Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regiões Promotoras Genéticas , Regulação da Expressão Gênica , Sequência de Bases , Cromatina/genéticaRESUMO
The influence of genome organization on transcription is central to our understanding of cell type specification. Higher-order genome organization is established through short- and long-range DNA interactions. Coordination of these interactions, from single atoms to entire chromosomes, plays a fundamental role in transcriptional control of gene expression. Loss of this coupling can result in disease. Analysis of transcriptional regulation typically involves disparate experimental approaches, from structural studies that define angstrom-level interactions to cell-biological and genomic approaches that assess mesoscale relationships. Thus, to fully understand the mechanisms that regulate gene expression, it is critical to integrate the findings gained across these distinct size scales. In this review, I illustrate fundamental ways in which cells regulate transcription in the context of genome organization.
Assuntos
Pareamento de Bases/genética , Cromossomos/genética , Transcrição Gênica/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Elementos Reguladores de Transcrição/genéticaRESUMO
Pioneer transcription factors have the intrinsic biochemical ability to scan partial DNA sequence motifs that are exposed on the surface of a nucleosome and thus access silent genes that are inaccessible to other transcription factors. Pioneer factors subsequently enable other transcription factors, nucleosome remodeling complexes, and histone modifiers to engage chromatin, thereby initiating the formation of an activating or repressive regulatory sequence. Thus, pioneer factors endow the competence for fate changes in embryonic development, are essential for cellular reprogramming, and rewire gene networks in cancer cells. Recent studies with reconstituted nucleosomes in vitro and chromatin binding in vivo reveal that pioneer factors can directly perturb nucleosome structure and chromatin accessibility in different ways. This review focuses on our current understanding of the mechanisms by which pioneer factors initiate gene network changes and will ultimately contribute to our ability to control cell fates at will.
Assuntos
Redes Reguladoras de Genes/genética , Fatores de Transcrição/genética , Animais , Reprogramação Celular/genética , Cromatina/genética , Desenvolvimento Embrionário/genética , Histonas/genética , Humanos , Nucleossomos/genéticaRESUMO
Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.
Assuntos
Fator 3-gama Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/citologia , Fígado/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas , Reprogramação Celular/fisiologia , Cromatina/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Camundongos Endogâmicos C57BL , Domínios Proteicos , Sítio de Iniciação de TranscriçãoRESUMO
The FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex regulatory network thought to become progressively resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for maintaining this regulatory network, we ablated all FoxA genes in the adult mouse liver. Remarkably, loss of FoxA caused rapid and massive reduction in the expression of critical liver genes. Activity of these genes was reduced back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining enhancer activity, chromatin accessibility, nucleosome positioning, and binding of HNF4α. Thus, the FoxA factors act continuously, guarding hepatic enhancer activity throughout adult life.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Redes Reguladoras de Genes , Fígado/metabolismo , Animais , Sítios de Ligação , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/patologia , Falência Hepática/etiologia , Falência Hepática/patologia , Masculino , Camundongos , NucleossomosRESUMO
Pioneer factors are transcriptional regulators with the capacity to bind inactive regions of chromatin and induce changes in accessibility that underpin cell fate decisions. The FOXA family of transcription factors is well understood to have pioneer capacity. Indeed, researchers have uncovered numerous examples of FOXA-dependent epigenomic modulation in developmental and disease processes. Despite the presence of FOXA being essential for correct epigenetic patterning, the need for continued FOXA presence postchromatin modulation has been debated. In a recent study in this issue of Genes & Development, Reizel and colleagues (pp. 1039-1050) show that the tissue-specific ablation of FOXA1/2/3 in the adult mouse liver results in the collapse of the epigenetic profile that maintains the hepatic gene expression profile. Thus, FOXA functions as a key, opening regions of chromatin during development, and as a doorstep, maintaining the established euchromatic structure in adult tissue.
Assuntos
Cromatina , Fator 3-alfa Nuclear de Hepatócito , Animais , Diferenciação Celular , Fator 3-alfa Nuclear de Hepatócito/genética , Fígado , Camundongos , OrganogêneseRESUMO
To induce cell type-specific forms of gene regulation, pioneer factors open tightly packed, inaccessible chromatin sites, enabling the molecular machinery to act on functionally significant information encoded in DNA. While previous studies of pioneer factors have revealed their functions in transcriptional regulation, pioneer factors that open chromatin for other physiological events remain undetermined. In this issue of Genes & Development, Spruce and colleagues (pp. 398-412) report the functional significance of a "pioneer complex" in mouse meiotic recombination. This complex, comprised of the zinc finger DNA-binding protein PRDM9 and the SNF2 family chromatin remodeler HELLS, exposes nucleosomal DNA to designate the sites of DNA double-strand breaks that initiate meiotic recombination. Both HELLS and PRDM9 are required for the determination of these recombination hot spots. Through the identification of a pioneer complex for meiotic recombination, this study broadens the conceptual scope of pioneer factors, indicating their functional significance in biological processes beyond transcriptional regulation.
Assuntos
Meiose/fisiologia , Recombinação Genética/fisiologia , Animais , DNA Helicases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismoRESUMO
Chromatin barriers prevent spurious interactions between regulatory elements and DNA-binding proteins. One such barrier, whose mechanism for overcoming is poorly understood, is access to recombination hot spots during meiosis. Here we show that the chromatin remodeler HELLS and DNA-binding protein PRDM9 function together to open chromatin at hot spots and provide access for the DNA double-strand break (DSB) machinery. Recombination hot spots are decorated by a unique combination of histone modifications not found at other regulatory elements. HELLS is recruited to hot spots by PRDM9 and is necessary for both histone modifications and DNA accessibility at hot spots. In male mice lacking HELLS, DSBs are retargeted to other sites of open chromatin, leading to germ cell death and sterility. Together, these data provide a model for hot spot activation in which HELLS and PRDM9 form a pioneer complex to create a unique epigenomic environment of open chromatin, permitting correct placement and repair of DSBs.
Assuntos
DNA Helicases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga/genética , Meiose/fisiologia , Animais , Morte Celular/genética , Quebras de DNA de Cadeia Dupla , Células Germinativas/patologia , Código das Histonas/genética , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Substâncias Macromoleculares/metabolismo , Masculino , Meiose/genética , CamundongosRESUMO
Pioneer factors are a subclass of transcription factors that can bind and initiate opening of silent chromatin regions. Pioneer factors subsequently regulate lineage-specific genes and enhancers and, thus, activate the zygotic genome after fertilization, guide cell fate transitions during development, and promote various forms of human cancers. As such, pioneer factors are useful in directed cell reprogramming. In this review, we define the structural and functional characteristics of pioneer factors, how they bind and initiate opening of closed chromatin regions, and the consequences for chromatin dynamics and gene expression during cell differentiation. We also discuss emerging mechanisms that modulate pioneer factors during development.
Assuntos
Cromatina , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Diferenciação Celular/genética , Reprogramação Celular , ZigotoRESUMO
Development is regulated by coordinated changes in gene expression. Control of these changes in expression is largely governed by the binding of transcription factors to specific regulatory elements. However, the packaging of DNA into chromatin prevents the binding of many transcription factors. Pioneer factors overcome this barrier owing to unique properties that enable them to bind closed chromatin, promote accessibility and, in so doing, mediate binding of additional factors that activate gene expression. Because of these properties, pioneer factors act at the top of gene-regulatory networks and drive developmental transitions. Despite the ability to bind target motifs in closed chromatin, pioneer factors have cell type-specific chromatin occupancy and activity. Thus, developmental context clearly shapes pioneer-factor function. Here, we discuss this reciprocal interplay between pioneer factors and development: how pioneer factors control changes in cell fate and how cellular environment influences pioneer-factor binding and activity.
Assuntos
Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromatina/metabolismo , Humanos , Redes Reguladoras de Genes , Ligação ProteicaRESUMO
Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, we developed an optogenetic strategy to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, we demonstrate that continued Zelda activity is required throughout genome activation. We show that Zelda binds DNA in the context of nucleosomes and suggest that this allows Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.
Assuntos
Reprogramação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , DNA/genética , DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Optogenética , Ligação Proteica , Fase SRESUMO
Chromatin is densely packed with nucleosomes, which limits the accessibility of many chromatin-associated proteins. Pioneer factors (PFs) are usually viewed as a special group of sequence-specific transcription factors (TFs) that can recognize nucleosome-embedded motifs, invade compact chromatin, and generate open chromatin regions. Through this process, PFs initiate a cascade of events that play key roles in gene regulation and cell differentiation. A current debate in the field is if PFs belong to a unique subset of TFs with intrinsic "pioneering activity", or if all TFs have the potential to function as PFs within certain cellular contexts. There are also different views regarding the key feature(s) that define pioneering activity. In this review, we present evidence from the literature related to these alternative views and discuss how to potentially reconcile them. It is possible that both intrinsic properties, like tight nucleosome binding and structural compatibility, and cellular conditions, like concentration and co-factor availability, are important for PF function.
RESUMO
Nucleosomes present a barrier for the binding of most transcription factors (TFs). However, special TFs known as nucleosome-displacing factors (NDFs) can access embedded sites and cause the depletion of the local nucleosomes as well as repositioning of the neighboring nucleosomes. Here, we developed a novel high-throughput method in yeast to identify NDFs among 104 TFs and systematically characterized the impact of orientation, affinity, location, and copy number of their binding motifs on the nucleosome occupancy. Using this assay, we identified 29 NDF motifs and divided the nuclear TFs into three groups with strong, weak, and no nucleosome-displacing activities. Further studies revealed that tight DNA binding is the key property that underlies NDF activity, and the NDFs may partially rely on the DNA replication to compete with nucleosome. Overall, our study presents a framework to functionally characterize NDFs and elucidate the mechanism of nucleosome invasion.
Assuntos
Nucleossomos/metabolismo , Saccharomycetales/metabolismo , Cromatina/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica , Saccharomycetales/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The transcription factor SOX2 is a vital regulator of stem cell activity in various developing and adult tissues. Mounting evidence has demonstrated the importance of SOX2 in regulating the induction and maintenance of stemness as well as in controlling cell proliferation, lineage decisions and differentiation. Recent studies have revealed that the ability of SOX2 to regulate these stem cell features involves its function as a pioneer factor, with the capacity to target nucleosomal DNA, modulate chromatin accessibility and prepare silent genes for subsequent activation. Moreover, although SOX2 binds to similar DNA motifs in different stem cells, its multifaceted and cell type-specific functions are reliant on context-dependent features. These cell type-specific properties include variations in partner factor availability and SOX2 protein expression levels. In this Primer, we discuss recent findings that have increased our understanding of how SOX2 executes its versatile functions as a master regulator of stem cell activities.
Assuntos
Nucleossomos , Fatores de Transcrição SOXB1 , Diferenciação Celular/genética , Proliferação de Células/genética , Cromatina , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
MYB is a master regulator and pioneer factor highly expressed in hematopoietic progenitor cells (HPCs) where it contributes to the reprogramming processes operating during hematopoietic development. MYB plays a complex role being involved in several lineages of the hematopoietic system. At the molecular level, the MYB gene is subject to intricate regulation at many levels through several enhancer and promoter elements, through transcriptional elongation control, as well as post-transcriptional regulation. The protein is modulated by post-translational modifications (PTMs) such as SUMOylation restricting the expression of its downstream targets. Together with a range of interaction partners, cooperating transcription factors (TFs) and epigenetic regulators, MYB orchestrates a fine-tuned symphony of genes expressed during various stages of haematopoiesis. At the same time, the complex MYB system is vulnerable, being a target for unbalanced control and cancer development.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Proteínas Proto-Oncogênicas c-myb , Humanos , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Animais , Processamento de Proteína Pós-Traducional , Epigênese Genética , Regulação da Expressão GênicaRESUMO
The dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor Grainy head (Grh). Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. We demonstrated that Grh remains bound to mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking maternal and/or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage.