RESUMO
The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the following question: How do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely 7 bp apart. However, the mechanistic basis for Gsx-DNA binding and cooperativity is poorly understood. Here, we used biochemical, biophysical, structural and modeling approaches to (i) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation, (ii) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions, (iii) solve a high-resolution monomer/DNA structure that reveals that Gsx2 induces a 20° bend in DNA, (iv) identify a Gsx2 protein-protein interface required for cooperative DNA binding and (v) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors.
Assuntos
DNA , Proteínas de Homeodomínio , Ligação Proteica , DNA/metabolismo , DNA/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Animais , Sítios de Ligação , Conformação de Ácido Nucleico , Modelos Moleculares , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , TermodinâmicaRESUMO
The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the question - how do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely seven base pairs apart. However, the mechanistic basis for Gsx DNA binding and cooperativity are poorly understood. Here, we used biochemical, biophysical, structural, and modeling approaches to (1) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation; (2) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions; (3) solve a high-resolution monomer/DNA structure that reveals Gsx2 induces a 20° bend in DNA; (4) identify a Gsx2 protein-protein interface required for cooperative DNA binding; and (5) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors, thereby providing a deeper understanding of HD specificity.
RESUMO
Homeodomain proteins constitute one of the largest families of metazoan transcription factors. Genetic studies have demonstrated that homeodomain proteins regulate many developmental processes. Yet, biochemical data reveal that most bind highly similar DNA sequences. Defining how homeodomain proteins achieve DNA binding specificity has therefore been a long-standing goal. Here, we developed a novel computational approach to predict cooperative dimeric binding of homeodomain proteins using High-Throughput (HT) SELEX data. Importantly, we found that 15 of 88 homeodomain factors form cooperative homodimer complexes on DNA sites with precise spacing requirements. Approximately one third of the paired-like homeodomain proteins cooperatively bind palindromic sequences spaced 3 bp apart, whereas other homeodomain proteins cooperatively bind sites with distinct orientation and spacing requirements. Combining structural models of a paired-like factor with our cooperativity predictions identified key amino acid differences that help differentiate between cooperative and non-cooperative factors. Finally, we confirmed predicted cooperative dimer sites in vivo using available genomic data for a subset of factors. These findings demonstrate how HT-SELEX data can be computationally mined to predict cooperativity. In addition, the binding site spacing requirements of select homeodomain proteins provide a mechanism by which seemingly similar AT-rich DNA sequences can preferentially recruit specific homeodomain factors.
Assuntos
Proteínas de Homeodomínio , Animais , Sítios de Ligação , DNA/química , Proteínas de Homeodomínio/metabolismo , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteínas Correpressoras , DNA , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Displasia Ectodérmica , Humanos , Deformidades Congênitas dos Membros , Mamíferos/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Couro Cabeludo/metabolismo , Dermatoses do Couro Cabeludo/congênito , Crânio/metabolismoRESUMO
Distinct neural stem cells (NSCs) reside in different regions of the subventricular zone (SVZ) and generate multiple olfactory bulb (OB) interneuron subtypes in the adult brain. However, the molecular mechanisms underlying such NSC heterogeneity remain largely unknown. Here, we show that the basic helix-loop-helix transcription factor Olig2 defines a subset of NSCs in the early postnatal and adult SVZ. Olig2-expressing NSCs exist broadly but are most enriched in the ventral SVZ along the dorsoventral axis complementary to dorsally enriched Gsx2-expressing NSCs. Comparisons of Olig2-expressing NSCs from early embryonic to adult stages using single cell transcriptomics reveal stepwise developmental changes in their cell cycle and metabolic properties. Genetic studies further show that cross-repression contributes to the mutually exclusive expression of Olig2 and Gsx2 in NSCs/progenitors during embryogenesis, but that their expression is regulated independently from each other in adult NSCs. Finally, lineage-tracing and conditional inactivation studies demonstrate that Olig2 plays an important role in the specification of OB interneuron subtypes. Altogether, our study demonstrates that Olig2 defines a unique subset of adult NSCs enriched in the ventral aspect of the adult SVZ.
Assuntos
Interneurônios/metabolismo , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/metabolismo , Bulbo Olfatório/crescimento & desenvolvimento , Bulbo Olfatório/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Animais , Ciclo Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Feminino , Técnicas de Inativação de Genes , Ventrículos Laterais/embriologia , Masculino , Camundongos , Camundongos Knockout , Neurogênese/genética , Bulbo Olfatório/embriologia , Fator de Transcrição 2 de Oligodendrócitos/genética , Transdução de Sinais/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: In a large pedigree with an unusual phenotype of spastic paraplegia or dystonia and autosomal dominant inheritance, linkage analysis previously mapped the disease to chromosome 2q24-2q31. OBJECTIVE: The aim of this study is to identify the genetic cause and molecular basis of an unusual autosomal dominant spastic paraplegia and dystonia. METHODS: Whole exome sequencing following linkage analysis was used to identify the genetic cause in a large family. Cosegregation analysis was also performed. An additional 384 individuals with spastic paraplegia or dystonia were screened for pathogenic sequence variants in the adenosine triphosphate (ATP) synthase membrane subunit C locus 3 gene (ATP5MC3). The identified variant was submitted to the "GeneMatcher" program for recruitment of additional subjects. Mitochondrial functions were analyzed in patient-derived fibroblast cell lines. Transgenic Drosophila carrying mutants were studied for movement behavior and mitochondrial function. RESULTS: Exome analysis revealed a variant (c.318C > G; p.Asn106Lys) (NM_001689.4) in ATP5MC3 in a large family with autosomal dominant spastic paraplegia and dystonia that cosegregated with affected individuals. No variants were identified in an additional 384 individuals with spastic paraplegia or dystonia. GeneMatcher identified an individual with the same genetic change, acquired de novo, who manifested upper-limb dystonia. Patient fibroblast studies showed impaired complex V activity, ATP generation, and oxygen consumption. Drosophila carrying orthologous mutations also exhibited impaired mitochondrial function and displayed reduced mobility. CONCLUSION: A unique form of familial spastic paraplegia and dystonia is associated with a heterozygous ATP5MC3 variant that also reduces mitochondrial complex V activity.
Assuntos
Distonia , Distúrbios Distônicos , Paraplegia Espástica Hereditária , Distonia/genética , Distúrbios Distônicos/genética , Humanos , Mutação/genética , Paraplegia/genética , Linhagem , Fenótipo , Paraplegia Espástica Hereditária/genéticaRESUMO
Metazoans differentially express multiple Hox transcription factors to specify diverse cell fates along the developing anterior-posterior axis. Two challenges arise when trying to understand how the Hox transcription factors regulate the required target genes for morphogenesis: First, how does each Hox factor differ from one another to accurately activate and repress target genes required for the formation of distinct segment and regional identities? Second, how can a Hox factor that is broadly expressed in many tissues within a segment impact the development of specific organs by regulating target genes in a cell type-specific manner? In this review, we highlight how recent genomic, interactome, and cis-regulatory studies are providing new insights into answering these two questions. Collectively, these studies suggest that Hox factors may differentially modify the chromatin of gene targets as well as utilize numerous interactions with additional co-activators, co-repressors, and sequence-specific transcription factors to achieve accurate segment and cell type-specific transcriptional outcomes.
RESUMO
Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.
Assuntos
Proteínas de Drosophila/fisiologia , Elementos Facilitadores Genéticos , Receptores Notch/metabolismo , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genes Reporter , Óperon Lac , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The lipogenic enzyme stearoyl CoA desaturase (SCD) plays a key role in tumor lipid metabolism and membrane architecture. SCD is often up-regulated and a therapeutic target in cancer. Here, we report the unexpected finding that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and unintentional monoallelic co-deletion with phosphatase and tensin homolog (PTEN) in a subset of patients. Cell lines from this subset expressed undetectable SCD, yet retained residual SCD enzymatic activity. Unexpectedly, these lines evolved to survive independent of SCD through unknown mechanisms. Cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Last, we identify that SCD-dependent lines acquire resistance through a previously unknown FBJ murine osteosarcoma viral oncogene homolog B (FOSB)-mediated mechanism. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor-bearing mice treated with SCD inhibitor.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias , Estearoil-CoA Dessaturase , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Metabolismo dos Lipídeos , Lipogênese , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. Here, we show that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, we test a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Animais , Proteínas de Drosophila/genética , Genoma/genética , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Camundongos , Ligação Proteica , Telencéfalo/embriologiaRESUMO
Despite a common understanding that Gli TFs are utilized to convey a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. Using the mouse model (Mus musculus), we demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the basic helix-loop-helix TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, 'divergent' Gli-binding motifs (dGBMs). Functional validation revealed these dGBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Desenvolvimento Maxilofacial , Camundongos/genética , Proteínas do Tecido Nervoso/genética , Crânio/crescimento & desenvolvimento , Proteína Gli3 com Dedos de Zinco/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Masculino , Desenvolvimento Maxilofacial/genética , Camundongos/metabolismo , Modelos Animais , Proteínas do Tecido Nervoso/metabolismo , Crânio/metabolismo , Proteína Gli3 com Dedos de Zinco/metabolismoRESUMO
Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.
Assuntos
Linfócitos B/imunologia , Dosagem de Genes , Coração/embriologia , Homeostase , Intestinos/patologia , Infestações por Ácaros/imunologia , Receptores Notch/genética , Células-Tronco/patologia , Alelos , Animais , Sequência de Bases , Proliferação de Células , Cromatina/metabolismo , Sulfato de Dextrana , Ventrículos do Coração/embriologia , Ventrículos do Coração/patologia , Camundongos , Ácaros/fisiologia , Modelos Biológicos , Multimerização Proteica , Receptores Notch/metabolismo , Baço/imunologia , Esplenomegalia/imunologia , Esplenomegalia/parasitologia , Células-Tronco/metabolismoRESUMO
Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.
Assuntos
Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos/genética , Haploinsuficiência/genética , Modelos Genéticos , Modelos Teóricos , Receptores Notch/genética , Animais , Drosophila , FenótipoRESUMO
The Gsx2 homeodomain transcription factor promotes neural progenitor identity in the lateral ganglionic eminence (LGE), despite upregulating the neurogenic factor Ascl1. How this balance in maturation is maintained is unclear. Here, we show that Gsx2 and Ascl1 are co-expressed in subapical progenitors that have unique transcriptional signatures in LGE ventricular zone (VZ) cells. Moreover, whereas Ascl1 misexpression promotes neurogenesis in dorsal telencephalic progenitors, the co-expression of Gsx2 with Ascl1 inhibits neurogenesis. Using luciferase assays, we found that Gsx2 reduces the ability of Ascl1 to activate gene expression in a dose-dependent and DNA binding-independent manner. Furthermore, Gsx2 physically interacts with the basic helix-loop-helix (bHLH) domain of Ascl1, and DNA-binding assays demonstrated that this interaction interferes with the ability of Ascl1 to bind DNA. Finally, we modified a proximity ligation assay for tissue sections and found that Ascl1-Gsx2 interactions are enriched within LGE VZ progenitors, whereas Ascl1-Tcf3 (E-protein) interactions predominate in the subventricular zone. Thus, Gsx2 contributes to the balance between progenitor maintenance and neurogenesis by physically interacting with Ascl1, interfering with its DNA binding and limiting neurogenesis within LGE progenitors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/embriologia , Proliferação de Células , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/metabolismo , Proliferação de Células/genética , Células Cultivadas , Drosophila , Embrião de Mamíferos , Feminino , Gânglios/citologia , Gânglios/embriologia , Proteínas de Homeodomínio/genética , Homeostase/genética , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica , Telencéfalo/citologia , Telencéfalo/embriologiaRESUMO
Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells, whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues, whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability, DNA binding, and gene transcription. Moreover, NEUROG3 was highly unstable in the intestinal epithelium, explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diarreia/congênito , Síndromes de Malabsorção/genética , Mutação , Proteínas do Tecido Nervoso/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Diarreia/genética , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Organoides/citologia , Organoides/metabolismo , Pâncreas/citologia , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade ProteicaRESUMO
During development diverse transcription factor inputs are integrated by cis-regulatory modules (CRMs) to yield cell-specific gene expression. Defining how CRMs recruit the appropriate combinations of factors to either activate or repress gene expression remains a challenge. In this study, we compare and contrast the ability of two CRMs within the Drosophila embryo to recruit functional Hox transcription factor complexes. The DCRE CRM recruits Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox complexes that include the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the Distal-less leg selector gene, whereas the RhoA CRM selectively recruits Abd-A/Exd/Hth complexes to activate rhomboid and stimulate Epidermal Growth Factor secretion in sensory cell precursors. By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE. We further show that the orientation and spacing of Hox sites relative to additional binding sites within the RhoA and DCRE is critical to mediate cell- and segment-specific output. These results indicate that the configuration of Exd, Hth, and Hox site within RhoA is neither Abd-A specific nor activation specific. Instead Hox specific output is largely dependent upon the presence of appropriately spaced and oriented binding sites for additional TF inputs. Taken together, these studies provide insight into the cis-regulatory logic used to generate cell-specific outputs via recruiting Hox transcription factor complexes.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Proteínas de Homeodomínio/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs.
Assuntos
Proteínas de Drosophila/química , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Proteínas Nucleares/química , Fator de Transcrição PAX2/química , Fatores de Transcrição/química , Animais , Animais Geneticamente Modificados/genética , Sítios de Ligação/genética , Ligação Competitiva , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Fator de Transcrição PAX2/genética , Fatores de Transcrição/genéticaRESUMO
Superior manual dexterity in higher primates emerged together with the appearance of cortico-motoneuronal (CM) connections during the evolution of the mammalian corticospinal (CS) system. Previously thought to be specific to higher primates, we identified transient CM connections in early postnatal mice, which are eventually eliminated by Sema6D-PlexA1 signaling. PlexA1 mutant mice maintain CM connections into adulthood and exhibit superior manual dexterity as compared with that of controls. Last, differing PlexA1 expression in layer 5 of the motor cortex, which is strong in wild-type mice but weak in humans, may be explained by FEZF2-mediated cis-regulatory elements that are found only in higher primates. Thus, species-dependent regulation of PlexA1 expression may have been crucial in the evolution of mammalian CS systems that improved fine motor control in higher primates.
Assuntos
Lateralidade Funcional/genética , Regulação da Expressão Gênica , Córtex Motor/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tratos Piramidais/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Semaforinas/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Hox transcription factors specify distinct cell types along the anterior-posterior axis of metazoans by regulating target genes that modulate signaling pathways. A well-established example is the induction of Epidermal Growth Factor (EGF) signaling by an Abdominal-A (Abd-A) Hox complex during the specification of Drosophila hepatocyte-like cells (oenocytes). Previous studies revealed that Abd-A is non-cell autonomously required to promote oenocyte fate by directly activating a gene (rhomboid) that triggers EGF secretion from sensory organ precursor (SOP) cells. Neighboring cells that receive the EGF signal initiate a largely unknown pathway to promote oenocyte fate. Here, we show that Abd-A also plays a cell autonomous role in inducing oenocyte fate by activating the expression of the Pointed-P1 (PntP1) ETS transcription factor downstream of EGF signaling. Genetic studies demonstrate that both PntP1 and PntP2 are required for oenocyte specification. Moreover, we found that PntP1 contains a conserved enhancer (PntP1OE) that is activated in oenocyte precursor cells by EGF signaling via direct regulation by the Pnt transcription factors as well as a transcription factor complex consisting of Abd-A, Extradenticle, and Homothorax. Our findings demonstrate that the same Abd-A Hox complex required for sending the EGF signal from SOP cells, enhances the competency of receiving cells to select oenocyte cell fate by up-regulating PntP1. Since PntP1 is a downstream effector of EGF signaling, these findings provide insight into how a Hox factor can both trigger and potentiate the EGF signal to promote an essential cell fate along the body plan.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Fator de Crescimento Epidérmico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Drosophila/enzimologia , Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/citologia , Hepatócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Órgãos dos Sentidos/crescimento & desenvolvimento , Fatores de Transcrição/genéticaRESUMO
The Notch signaling pathway relies on a proteolytic cascade to release its transcriptionally active intracellular domain, on force to unfold a protective domain and permit proteolysis, on extracellular domain glycosylation to tune the forces exerted by endocytosed ligands, and on a motley crew of nuclear proteins, chromatin modifiers, ubiquitin ligases, and a few kinases to regulate activity and half-life. Herein we provide a review of recent molecular insights into how Notch signals are triggered and how cell shape affects these events, and we use the new insights to illuminate a few perplexing observations.