scholarly journals Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites

2020 ◽  
Author(s):  
Jacob J. Crow ◽  
Allan R. Albig
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Family Members ◽  
Regulatory Function ◽  
Specific Nature ◽  
Intracellular Domain ◽  
Notch Intracellular Domain ◽  
Highly Sensitive ◽  
Cellular Identity

ABSTRACTNotch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that each family member is capable of dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.

scholarly journals Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0234101
Author(s):  
Jacob J. Crow ◽  
Allan R. Albig
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Family Members ◽  
Regulatory Function ◽  
Specific Nature ◽  
Intracellular Domain ◽  
Notch Intracellular Domain ◽  
Highly Sensitive ◽  
Cellular Identity

Notch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that the Notch 2 and 3 NICDs also exhibit dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Furthermore, our data suggested that Notch4 may also exhibit dimerization-induced signaling, although the amino acids required for Notch4 NICD dimerization appear to be different than those required for Notch 1, 2, and 3 NICD dimerization. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.

scholarly journals Dimerization of the Notch Intracellular Domain Results in Distinct Signaling Activity

2020 ◽  
Author(s):  
Jacob Jeffery Crow
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Notch Signaling Pathway ◽  
Core Component ◽  
High Tunability ◽  
Notch Intracellular Domain ◽  
Signaling Field

The Notch signaling pathway is a core component of multicellularity; enabling cells to directly communicate with both their neighbors and the surrounding microenvironment. These signals are translated directly through the Notch proteins, where a fragment of Notch transitions into the nucleus to act as a co-transcription factor, setting into motion a host of physiological responses. Commonly involved in pathways that define a cell’s identity and fate decisions, what appears to be a simplistic pathway instead exists in a state of high-tunability and strict control. Missteps in this pathway are generally embryonically lethal or lead to a suite of congenital disorders and cancers. Therefore, it’s pertinent to understand the mechanisms of Notch that provide its flexibility and pleiotropic outcomes. One such property is its ability to homodimerize on DNA while within its transcriptional activation complex, resulting in an enhanced transcriptional signal of a select pool of Notch target genes. This dissertation reviews the general mechanics behind Notch signaling, discusses how the field of Notch dimerization came to be and where it stands currently, and finally, details my contributions to the understanding of this regulatory mechanism. Despite Notch’s ubiquitous function in metazoan life, there are still many mysteries behind this signaling pathway. The work detailed here describes my time spent as a basic science researcher, where my findings contribute a couple of puzzle pieces to the expansive Notch signaling field.

scholarly journals Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling

2019 ◽  
Vol 12 (606) ◽  
pp. eaay2369 ◽  
Author(s):  
Sanchez M. Jarrett ◽  
Tom C. M. Seegar ◽  
Mark Andrews ◽  
Guillaume Adelmant ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Negative Feedback ◽  
Transcriptional Activation ◽  
Cultured Cells ◽  
Feedback Inhibition ◽  
Lymphoblastic Leukemia ◽  
Ankyrin Repeat ◽  
Structural Basis ◽  
Intracellular Domain ◽  
Notch Intracellular Domain

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

scholarly journals The Drosophila HP1 family is associated with active gene expression across chromatin contexts

Genetics ◽  
2021 ◽  
Author(s):  
John M Schoelz ◽  
Justina X Feng ◽  
Nicole C Riddle
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Family Members ◽  
Transcription Start ◽  
Heterochromatin Formation ◽  
Transcription Start Sites ◽  
Polymerase Pausing ◽  
Hp1 Proteins

Abstract Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than non-target genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared to non-target genes. Specifically, co-localization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites.

scholarly journals Methylcytosine dioxygenase TET3 interacts with thyroid hormone nuclear receptors and stabilizes their association to chromatin

2017 ◽  
Vol 114 (31) ◽  
pp. 8229-8234 ◽  
Author(s):  
Wenyue Guan ◽  
Romain Guyot ◽  
Jacques Samarut ◽  
Frédéric Flamant ◽  
Jiemin Wong ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Hormone Receptors ◽  
Target Genes ◽  
Nuclear Hormone Receptor ◽  
Regulatory Function ◽  
Interacting Protein ◽  
Differential Ability ◽  
Dominant Activity

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily that act as ligand-dependent transcription factors. Here we identified the ten-eleven translocation protein 3 (TET3) as a TR interacting protein increasing cell sensitivity to T3. The interaction between TET3 and TRs is independent of TET3 catalytic activity and specifically allows the stabilization of TRs on chromatin. We provide evidence that TET3 is required for TR stability, efficient binding of target genes, and transcriptional activation. Interestingly, the differential ability of different TRα1 mutants to interact with TET3 might explain their differential dominant activity in patients carrying TR germline mutations. So this study evidences a mode of action for TET3 as a nonclassical coregulator of TRs, modulating its stability and access to chromatin, rather than its intrinsic transcriptional activity. This regulatory function might be more general toward nuclear receptors. Indeed, TET3 interacts with different members of the superfamily and also enhances their association to chromatin.

scholarly journals The Drosophila HP1 family is associated with active gene expression across chromatin contexts

Genetics ◽  
2021 ◽  
Author(s):  
John M Schoelz ◽  
Justina X Feng ◽  
Nicole C Riddle
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Family Members ◽  
Transcription Start ◽  
Heterochromatin Formation ◽  
Transcription Start Sites ◽  
Polymerase Pausing ◽  
Hp1 Proteins

Abstract Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than non-target genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared to non-target genes. Specifically, co-localization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites.

scholarly journals The Drosophila HP1 family is associated with active gene expression across chromatin contexts

2020 ◽  
Author(s):  
John M. Schoelz ◽  
Justina X. Feng ◽  
Nicole C. Riddle
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Family Members ◽  
Protein Coding ◽  
Heterochromatin Formation ◽  
Transcription Start Sites ◽  
Polymerase Pausing ◽  
Hp1 Proteins

ABSTRACTDrosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed properly. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share a majority of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than non-target genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared to non-target genes. Specifically, co-localization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites (TSSs) to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broadens understanding of the mechanism of transcriptional activation by HP1a and highlights the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at TSSs.

scholarly journals The Non-Canonical Aspects of MicroRNAs: Many Roads to Gene Regulation

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1465 ◽  
Author(s):  
Christiaan J. Stavast ◽  
Stefan J. Erkeland
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Small Rnas ◽  
Target Genes ◽  
Biological Functions ◽  
Rnase Iii ◽  
Mrna Targets ◽  
Argonaute Proteins ◽  
Epigenetic Modifiers ◽  
Encoding Genes

MicroRNAs (miRNAs) are critical regulators of gene expression. As miRNAs are frequently deregulated in many human diseases, including cancer and immunological disorders, it is important to understand their biological functions. Typically, miRNA-encoding genes are transcribed by RNA Polymerase II and generate primary transcripts that are processed by RNase III-endonucleases DROSHA and DICER into small RNAs of approximately 21 nucleotides. All miRNAs are loaded into Argonaute proteins in the RNA-induced silencing complex (RISC) and act as post-transcriptional regulators by binding to the 3′- untranslated region (UTR) of mRNAs. This seed-dependent miRNA binding inhibits the translation and/or promotes the degradation of mRNA targets. Surprisingly, recent data presents evidence for a target-mediated decay mechanism that controls the level of specific miRNAs. In addition, several non-canonical miRNA-containing genes have been recently described and unexpected functions of miRNAs have been identified. For instance, several miRNAs are located in the nucleus, where they are involved in the transcriptional activation or silencing of target genes. These epigenetic modifiers are recruited by RISC and guided by miRNAs to specific loci in the genome. Here, we will review non-canonical aspects of miRNA biology, including novel regulators of miRNA expression and functions of miRNAs in the nucleus.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

scholarly journals Target Gene Specificity of E2F and Pocket Protein Family Members in Living Cells

2000 ◽  
Vol 20 (16) ◽  
pp. 5797-5807 ◽  
Author(s):  
Julie Wells ◽  
Kathryn E. Boyd ◽  
Christopher J. Fry ◽  
Stephanie M. Bartley ◽  
Peggy J. Farnham
Keyword(s):  
Cell Cycle ◽  
Target Gene ◽  
Target Genes ◽  
Protein Complexes ◽  
Current Model ◽  
Family Members ◽  
Living Cells ◽  
Gene Promoters ◽  
Pocket Protein ◽  
Protein Dna Interactions

ABSTRACT E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G0 phase of the cell cycle and release of the pocket protein in late G1, followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G0 and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

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