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1.
Nucleic Acids Res ; 52(13): 7987-8002, 2024 Jul 22.
Article in English | MEDLINE | ID: mdl-38874471

ABSTRACT

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.


Subject(s)
DNA , Homeodomain Proteins , Protein Binding , DNA/metabolism , DNA/chemistry , Homeodomain Proteins/metabolism , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Animals , Binding Sites , Nucleic Acid Conformation , Models, Molecular , Transcription Factors/metabolism , Transcription Factors/chemistry , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Humans , Thermodynamics
2.
bioRxiv ; 2023 Dec 08.
Article in English | MEDLINE | ID: mdl-38106145

ABSTRACT

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.

3.
Nucleic Acids Res ; 51(12): 6055-6072, 2023 07 07.
Article in English | MEDLINE | ID: mdl-37114997

ABSTRACT

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.


Subject(s)
Homeodomain Proteins , Animals , Binding Sites , DNA/chemistry , Homeodomain Proteins/metabolism , High-Throughput Nucleotide Sequencing
4.
Mol Ther Oncolytics ; 28: 307-320, 2023 Mar 16.
Article in English | MEDLINE | ID: mdl-36938545

ABSTRACT

Notch activation complex kinase (NACK) is a component of the Notch transcriptional machinery critical for the Notch-mediated tumorigenesis. However, the mechanism through which NACK regulates Notch-mediated transcription is not well understood. Here, we demonstrate that NACK binds and hydrolyzes ATP and that only ATP-bound NACK can bind to the Notch ternary complex (NTC). Considering this, we sought to identify inhibitors of this ATP-dependent function and, using computational pipelines, discovered the first small-molecule inhibitor of NACK, Z271-0326, that directly blocks the activity of Notch-mediated transcription and shows potent antineoplastic activity in PDX mouse models. In conclusion, we have discovered the first inhibitor that holds promise for the efficacious treatment of Notch-driven cancers by blocking the Notch activity downstream of the NTC.

5.
Microorganisms ; 11(2)2023 Feb 02.
Article in English | MEDLINE | ID: mdl-36838345

ABSTRACT

Clostridioides difficile, a nosocomial pathogen, is an emerging gut pathobiont causing antibiotic-associated diarrhea. C. difficile infection involves gut colonization and disruption of the gut epithelial barrier, leading to the induction of inflammatory/immune responses. The expression of two major exotoxins, TcdA and TcdB is the major cause of C. difficile pathogenicity. Attachment of bacterial abundant cell wall proteins or surface S-layer proteins (SLPs) such as SlpA with host epithelial cells is critical for virulence. In addition to being toxins, these surface components have been shown to be highly immunogenic. Recent studies indicate that C. difficile SLPs play important roles in the adhesion of the bacteria to the intestinal epithelial cells, disruption of tight junctions, and modulation of the immune response of the host cells. These proteins might serve as new targets for vaccines and new therapeutic agents. This review summarizes our current understanding of the immunological role of SLPs in inducing host immunity and their use in the development of vaccines and novel therapeutics to combat C. difficile infection.

6.
Genes (Basel) ; 14(1)2023 01 13.
Article in English | MEDLINE | ID: mdl-36672946

ABSTRACT

Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). Here, we generate a novel HΔCT allele by genome engineering. Phenotypically, HΔCT behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein-protein interactions, we define the sequences of the CT domain that are involved in Hairless-Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the HΔCT background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations.


Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Repressor Proteins/genetics , Drosophila Proteins/metabolism , Histone Chaperones/genetics , Histone Chaperones/metabolism , Alleles , Receptors, Notch/genetics , Receptors, Notch/metabolism , Drosophila/genetics , Chromatin/metabolism
7.
Nucleic Acids Res ; 50(22): 13083-13099, 2022 12 09.
Article in English | MEDLINE | ID: mdl-36477367

ABSTRACT

The Notch pathway transmits signals between neighboring cells to elicit downstream transcriptional programs. Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The pathway signals through the transcription factor CSL (RBPJ in mammals), which forms an activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified L3MBTL3 as a bona fide RBPJ-binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here, we define the RBPJ-interacting domain of L3MBTL3 and report the 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex. The structure reveals that L3MBTL3 interacts with RBPJ via an unusual binding motif compared to other RBPJ binding partners, which we comprehensively analyze with a series of structure-based mutants. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.


Subject(s)
DNA-Binding Proteins , Gene Expression Regulation , Immunoglobulin J Recombination Signal Sequence-Binding Protein , Animals , Humans , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Histone Demethylases/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Intracellular Signaling Peptides and Proteins/genetics , LIM Domain Proteins/metabolism , Muscle Proteins/genetics , Protein Binding , Receptors, Notch/genetics , Receptors, Notch/metabolism
8.
PLoS Genet ; 18(8): e1010335, 2022 08.
Article in English | MEDLINE | ID: mdl-35951645

ABSTRACT

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.


Subject(s)
Drosophila Proteins , Drosophila , Animals , Co-Repressor Proteins , DNA , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/metabolism , Ectodermal Dysplasia , Humans , Limb Deformities, Congenital , Mammals/genetics , Receptors, Notch/genetics , Receptors, Notch/metabolism , Scalp/metabolism , Scalp Dermatoses/congenital , Skull/metabolism
9.
Sci Transl Med ; 14(635): eabb7695, 2022 03 09.
Article in English | MEDLINE | ID: mdl-35263148

ABSTRACT

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.


Subject(s)
Leukemia, Myeloid, Acute , Ubiquitin-Conjugating Enzymes , Cell Proliferation/genetics , Humans , Leukemia, Myeloid, Acute/metabolism , Oncogenes , Signal Transduction/genetics , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism
10.
PLoS Genet ; 17(11): e1009890, 2021 11.
Article in English | MEDLINE | ID: mdl-34723970

ABSTRACT

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.


Subject(s)
Heart/physiology , Histone Deacetylase 1/metabolism , Myocytes, Cardiac/cytology , Regeneration/physiology , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Cell Proliferation
11.
PLoS Genet ; 17(9): e1009039, 2021 09.
Article in English | MEDLINE | ID: mdl-34559800

ABSTRACT

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.


Subject(s)
Drosophila Proteins/physiology , Enhancer Elements, Genetic , Receptors, Notch/metabolism , Repressor Proteins/physiology , Transcription Factors/physiology , Animals , Binding Sites , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Gene Expression Regulation , Genes, Reporter , Lac Operon , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcriptional Activation
12.
Front Microbiol ; 12: 639362, 2021.
Article in English | MEDLINE | ID: mdl-34220733

ABSTRACT

The life-threatening pandemic concerning multi-drug resistant (MDR) bacteria is an evolving problem involving increased hospitalizations, billions of dollars in medical costs and a remarkably high number of deaths. Bacterial pathogens have demonstrated the capacity for spontaneous or acquired antibiotic resistance and there is virtually no pool of organisms that have not evolved such potentially clinically catastrophic properties. Although many diseases are linked to such organisms, three include cystic fibrosis (CF), burn/blast wounds and urinary tract infections (UTIs), respectively. Thus, there is a critical need to develop novel, effective antimicrobials for the prevention and treatment of such problematic infections. One of the most formidable, naturally MDR bacterial pathogens is Pseudomonas aeruginosa (PA) that is particularly susceptible to nitric oxide (NO), a component of our innate immune response. This susceptibility sets the translational stage for the use of NO-based therapeutics during the aforementioned human infections. First, we discuss how such NO therapeutics may be able to target problematic infections in each of the aforementioned infectious scenarios. Second, we describe a recent discovery based on years of foundational information, a novel drug known as AB569. AB569 is capable of forming a "time release" of NO from S-nitrosothiols (RSNO). AB569, a bactericidal tandem consisting of acidified NaNO2 (A-NO2 -) and Na2-EDTA, is capable of killing all pathogens that are associated with the aforementioned disorders. Third, we described each disease state in brief, the known or predicted effects of AB569 on the viability of PA, its potential toxicity and highly remote possibility for resistance to develop. Finally, we conclude that AB569 can be a viable alternative or addition to conventional antibiotic regimens to treat such highly problematic MDR bacterial infections for civilian and military populations, as well as the economical burden that such organisms pose.

13.
J Biol Chem ; 296: 100593, 2021.
Article in English | MEDLINE | ID: mdl-33775697

ABSTRACT

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.


Subject(s)
Breast Neoplasms/pathology , Carcinogenesis , Proto-Oncogene Proteins c-pim-1/metabolism , Receptor, Notch3/metabolism , Active Transport, Cell Nucleus , Animals , Cell Line, Tumor , Cell Nucleus/metabolism , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Mice , Models, Molecular , Muscle Proteins/metabolism , Phosphorylation , Protein Domains , Receptor, Notch3/chemistry
14.
Adv Exp Med Biol ; 1287: 9-30, 2021.
Article in English | MEDLINE | ID: mdl-33034023

ABSTRACT

The Notch signal transduction cascade requires cell-to-cell contact and results in the proteolytic processing of the Notch receptor and subsequent assembly of a transcriptional coactivator complex containing the Notch intracellular domain (NICD) and transcription factor RBPJ. In the absence of a Notch signal, RBPJ remains at Notch target genes and dampens transcriptional output. Like in other signaling pathways, RBPJ is able to switch from activation to repression by associating with corepressor complexes containing several chromatin-modifying enzymes. Here, we focus on the recent advances concerning RBPJ-corepressor functions, especially in regard to chromatin regulation. We put this into the context of one of the best-studied model systems for Notch, blood cell development. Alterations in the RBPJ-corepressor functions can contribute to the development of leukemia, especially in the case of acute myeloid leukemia (AML). The versatile role of transcription factor RBPJ in regulating pivotal target genes like c-MYC and HES1 may contribute to the better understanding of the development of leukemia.


Subject(s)
Gene Expression Regulation , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Receptors, Notch/metabolism , Chromatin/genetics , Chromatin/metabolism , Humans , Signal Transduction
15.
PLoS Biol ; 18(10): e3000850, 2020 10.
Article in English | MEDLINE | ID: mdl-33017398

ABSTRACT

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.


Subject(s)
B-Lymphocytes/immunology , Gene Dosage , Heart/embryology , Homeostasis , Intestines/pathology , Mite Infestations/immunology , Receptors, Notch/genetics , Stem Cells/pathology , Alleles , Animals , Base Sequence , Cell Proliferation , Chromatin/metabolism , Dextran Sulfate , Heart Ventricles/embryology , Heart Ventricles/pathology , Mice , Mites/physiology , Models, Biological , Protein Multimerization , Receptors, Notch/metabolism , Spleen/immunology , Splenomegaly/immunology , Splenomegaly/parasitology , Stem Cells/metabolism
16.
Elife ; 92020 04 16.
Article in English | MEDLINE | ID: mdl-32297857

ABSTRACT

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.


Subject(s)
Drosophila Proteins/genetics , Enhancer Elements, Genetic/genetics , Haploinsufficiency/genetics , Models, Genetic , Models, Theoretical , Receptors, Notch/genetics , Animals , Drosophila , Phenotype
19.
Dev Cell ; 50(3): 367-380.e7, 2019 08 05.
Article in English | MEDLINE | ID: mdl-31178402

ABSTRACT

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.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Diarrhea/congenital , Malabsorption Syndromes/genetics , Mutation , Nerve Tissue Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Line , Diarrhea/genetics , Human Embryonic Stem Cells/metabolism , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Mice , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Organoids/cytology , Organoids/metabolism , Pancreas/cytology , Pancreas/growth & development , Pancreas/metabolism , Protein Binding , Protein Multimerization , Protein Stability
20.
Cell Rep ; 26(4): 845-854.e6, 2019 01 22.
Article in English | MEDLINE | ID: mdl-30673607

ABSTRACT

Notch is a conserved signaling pathway that is essential for metazoan development and homeostasis; dysregulated signaling underlies the pathophysiology of numerous human diseases. Receptor-ligand interactions result in gene expression changes, which are regulated by the transcription factor RBPJ. RBPJ forms a complex with the intracellular domain of the Notch receptor and the coactivator Mastermind to activate transcription, but it can also function as a repressor by interacting with corepressor proteins. Here, we determine the structure of RBPJ bound to the corepressor SHARP and DNA, revealing its mode of binding to RBPJ. We tested structure-based mutants in biophysical and biochemical-cellular assays to characterize the role of RBPJ as a repressor, clearly demonstrating that RBPJ mutants deficient for SHARP binding are incapable of repressing transcription of genes responsive to Notch signaling in cells. Altogether, our structure-function studies provide significant insights into the repressor function of RBPJ.


Subject(s)
DNA-Binding Proteins , Immunoglobulin J Recombination Signal Sequence-Binding Protein , Multiprotein Complexes , RNA-Binding Proteins , Signal Transduction , Transcription, Genetic , Animals , Binding Sites , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/chemistry , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Structure, Quaternary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Notch/chemistry , Receptors, Notch/genetics , Receptors, Notch/metabolism
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