scholarly journals E2F4’s cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6

2021 ◽  
pp. mbc.E21-01-0039
Author(s):  
Renin Hazan ◽  
Munemasa Mori ◽  
Paul S. Danielian ◽  
Vincent J. Guen ◽  
Seth M. Rubin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Reproductive Organs ◽  
Transcriptional Program ◽  
Replication Machinery ◽  
Terminal Domain ◽  
Multiciliated Cells ◽  
N Terminus ◽  
Shared Roles

Multiciliated cells play critical roles in the airway, reproductive organs and brain. Generation of multiple cilia requires both activation of a specialized transcriptional program and subsequent massive amplification of centrioles within the cytoplasm. The E2F4 transcription factor is required for both roles, and consequently for multiciliogenesis. Here, we establish that E2F4 associates with two distinct components of the centriole replication machinery, Deup1 and SAS6, targeting non-homologous domains in these proteins. We map Deup1 and SAS6 binding to E2F4’s N-terminus, and show that this domain is sufficient to mediate E2F4’s cytoplasmic role in multiciliogenesis. This sequence is highly conserved across the E2F family, but the ability to bind Deup1 and SAS6 is specific to E2F4 and E2F5, consistent with their shared roles in multiciliogenesis. By generating E2F4/E2F1 chimeras, we identify a six-residue motif that is critical for Deup1 and SAS6 binding. We propose that the ability of E2F4 and E2F5 to recruit Deup1 and/or SAS6, and enable centriole replication, contributes to their cytoplasmic roles in multiciliogenesis.

scholarly journals Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Munemasa Mori ◽  
Renin Hazan ◽  
Paul S. Danielian ◽  
John E. Mahoney ◽  
Huijun Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Large Scale ◽  
Abnormal Development ◽  
Basal Bodies ◽  
Transcriptional Program ◽  
Mutant Proteins ◽  
Multiprotein Complexes ◽  
Airway Diseases ◽  
Multiciliated Cells ◽  
Cytoplasmic Structures

Abstract Abnormal development of multiciliated cells is a hallmark of a variety of human conditions associated with chronic airway diseases, hydrocephalus and infertility. Multiciliogenesis requires both activation of a specialized transcriptional program and assembly of cytoplasmic structures for large-scale centriole amplification that generates basal bodies. It remains unclear, however, what mechanism initiates formation of these multiprotein complexes in epithelial progenitors. Here we show that this is triggered by nucleocytoplasmic translocation of the transcription factor E2f4. After inducing a transcriptional program of centriole biogenesis, E2f4 forms apical cytoplasmic organizing centres for assembly and nucleation of deuterosomes. Using genetically altered mice and E2F4 mutant proteins we demonstrate that centriole amplification is crucially dependent on these organizing centres and that, without cytoplasmic E2f4, deuterosomes are not assembled, halting multiciliogenesis. Thus, E2f4 integrates nuclear and previously unsuspected cytoplasmic events of centriole amplification, providing new perspectives for the understanding of normal ciliogenesis, ciliopathies and cancer.

scholarly journals The signal response of IkappaB alpha is regulated by transferable N- and C-terminal domains.

1997 ◽  
Vol 17 (6) ◽  
pp. 3021-3027 ◽  
Author(s):  
K Brown ◽  
G Franzoso ◽  
L Baldi ◽  
L Carlson ◽  
L Mills ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Chimeric Proteins ◽  
Glutathione S Transferase ◽  
Terminal Domain ◽  
N Terminus ◽  
Signal Response ◽  
Nf Kappab ◽  
Phosphopeptide Mapping ◽  
Terminal Domains

IkappaB alpha retains the transcription factor NF-kappaB in the cytoplasm, thus inhibiting its function. Various stimuli inactivate IkappaB alpha by triggering phosphorylation of the N-terminal residues Ser32 and Ser36. Phosphorylation of both serines is demonstrated directly by phosphopeptide mapping utilizing calpain protease, which cuts approximately 60 residues from the N terminus, and by analysis of mutants lacking one or both serine residues. Phosphorylation is followed by rapid proteolysis, and the liberated NF-kappaB translocates to the nucleus, where it activates transcription of its target genes. Transfer of the N-terminal domain of IkappaB alpha to the ankyrin domain of the related oncoprotein Bcl-3 or to the unrelated protein glutathione S-transferase confers signal-induced phosphorylation on the resulting chimeric proteins. If the C-terminal domain of IkappaB alpha is transferred as well, the resulting chimeras exhibit both signal-induced phosphorylation and rapid proteolysis. Thus, the signal response of IkappaB alpha is controlled by transferable N-terminal and C-terminal domains.

scholarly journals Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis

Blood ◽  
2019 ◽  
Vol 134 (19) ◽  
pp. 1619-1631 ◽  
Author(s):  
Te Ling ◽  
Yehudit Birger ◽  
Monika J. Stankiewicz ◽  
Nissim Ben-Haim ◽  
Tomer Kalisky ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Differentiation ◽  
Full Length ◽  
Red Cell ◽  
Terminal Domain ◽  
N Terminus ◽  
Epigenetic Analysis

GATA1 has a foundational role in erythropoiesis. The investigators compare the function of 2 forms (the full-length protein and a shorter form) of the transcription factor GATA1 and show that the N-terminal domain of GATA1 is critical to red cell differentiation.

scholarly journals Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Seungwoo Cha ◽  
Chang Pyo Hong ◽  
Hyun Ah Kang ◽  
Ji-Sook Hahn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Target Genes ◽  
Gene Encoding ◽  
Metabolic Switch ◽  
Differential Activation ◽  
N Terminus ◽  
Activation Mechanisms ◽  
In Trans ◽  
Separate Activation

Abstract Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.

scholarly journals Histone demethylase JMJD2B/KDM4B regulates transcriptional program via distinctive epigenetic targets and protein interactors for the maintenance of trophoblast stem cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kylie Hin-Man Mak ◽  
Yuk Man Lam ◽  
Ray Kit Ng
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Cell Fate ◽  
Histone Demethylase ◽  
Gene Promoters ◽  
Transcriptional Program ◽  
Binding Motifs ◽  
Trophoblast Stem Cells ◽  
Trophoblast Stem ◽  
Trophoblast Stem Cell

AbstractTrophoblast stem cell (TSC) is crucial to the formation of placenta in mammals. Histone demethylase JMJD2 (also known as KDM4) family proteins have been previously shown to support self-renewal and differentiation of stem cells. However, their roles in the context of the trophoblast lineage remain unclear. Here, we find that knockdown of Jmjd2b resulted in differentiation of TSCs, suggesting an indispensable role of JMJD2B/KDM4B in maintaining the stemness. Through the integration of transcriptome and ChIP-seq profiling data, we show that JMJD2B is associated with a loss of H3K36me3 in a subset of embryonic lineage genes which are marked by H3K9me3 for stable repression. By characterizing the JMJD2B binding motifs and other transcription factor binding datasets, we discover that JMJD2B forms a protein complex with AP-2 family transcription factor TFAP2C and histone demethylase LSD1. The JMJD2B–TFAP2C–LSD1 complex predominantly occupies active gene promoters, whereas the TFAP2C–LSD1 complex is located at putative enhancers, suggesting that these proteins mediate enhancer–promoter interaction for gene regulation. We conclude that JMJD2B is vital to the TSC transcriptional program and safeguards the trophoblast cell fate via distinctive protein interactors and epigenetic targets.

On mechanisms of colicin import: the outer membrane quandary

2018 ◽  
Vol 475 (23) ◽  
pp. 3903-3915 ◽  
Author(s):  
William A. Cramer ◽  
Onkar Sharma ◽  
S.D. Zakharov
Keyword(s):  
Outer Membrane ◽  
Catalytic Domain ◽  
Efflux Pump ◽  
Crystal Structure Analysis ◽  
Multidrug Efflux Pump ◽  
Terminal Domain ◽  
Colicin E1 ◽  
N Terminus ◽  
T Domain

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10−9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

scholarly journals Salmonella SipA mimics a cognate SNARE for host Syntaxin8 to promote fusion with early endosomes

2018 ◽  
Vol 217 (12) ◽  
pp. 4199-4214 ◽  
Author(s):  
Pawan Kishor Singh ◽  
Anjali Kapoor ◽  
Richa Madan Lomash ◽  
Kamal Kumar ◽  
Sukrut C. Kamerkar ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Enteric Fever ◽  
Caspase 3 ◽  
Arginine Residue ◽  
Terminal Domain ◽  
Trafficking Pathway ◽  
Early Endosomes ◽  
N Terminus ◽  
Snare Motif ◽  
Salmonella Survival

SipA is a major effector of Salmonella, which causes gastroenteritis and enteric fever. Caspase-3 cleaves SipA into two domains: the C-terminal domain regulates actin polymerization, whereas the function of the N terminus is unknown. We show that the cleaved SipA N terminus binds and recruits host Syntaxin8 (Syn8) to Salmonella-containing vacuoles (SCVs). The SipA N terminus contains a SNARE motif with a conserved arginine residue like mammalian R-SNAREs. SipAR204Q and SipA1–435R204Q do not bind Syn8, demonstrating that SipA mimics a cognate R-SNARE for Syn8. Consequently, Salmonella lacking SipA or that express the SipA1–435R204Q SNARE mutant are unable to recruit Syn8 to SCVs. Finally, we show that SipA mimicking an R-SNARE recruits Syn8, Syn13, and Syn7 to the SCV and promotes its fusion with early endosomes to potentially arrest its maturation. Our results reveal that SipA functionally substitutes endogenous SNAREs in order to hijack the host trafficking pathway and promote Salmonella survival.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

scholarly journals The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

2019 ◽  
Author(s):  
Ana M. Oliveira Paiva ◽  
Leen de Jong ◽  
Annemieke H. Friggen ◽  
Wiep Klaas Smits ◽  
Jeroen Corver
Keyword(s):  
Sigma Factor ◽  
Negative Regulator ◽  
Toxin Gene ◽  
Cytoplasmic Localization ◽  
Terminal Domain ◽  
Clostridioides Difficile ◽  
C Terminus ◽  
N Terminus ◽  
Ob Fold ◽  
Toxin Gene Expression

AbstractClostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold.In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors.ImportanceTranscription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain.Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.

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