scholarly journals Dynamics of Notch-dependent transcriptional bursting in its native context

2018 ◽  
Author(s):  
ChangHwan Lee ◽  
Heaji Shin ◽  
Judith Kimble
Keyword(s):  
Transcriptional Activation ◽  
Burst Size ◽  
Notch Receptor ◽  
Regulation Of Transcription ◽  
Germline Stem Cells ◽  
C Elegans ◽  
Burst Intensity ◽  
Native Context ◽  
Transcriptional Bursting ◽  
Nascent Transcripts

Transcription is well known to be inherently stochastic and episodic, but the regulation of transcriptional dynamics is not well understood. Here we analyze how Notch signaling modulates transcriptional bursting during animal development. Our focus is Notch regulation of transcription in germline stem cells of the nematode C. elegans. Using the MS2 system to visualize nascent transcripts and live imaging to record dynamics, we analyze bursting as a function of position within the intact animal. We find that Notch-dependent transcriptional activation is indeed bursty; that wild-type Notch modulates burst duration (ON-time) rather than duration of pauses between bursts (OFF-time) or mean burst intensity; and that a mutant Notch receptor, which is compromised for assembly into the Notch transcription factor complex, primarily modifies burst size (duration x intensity). To our knowledge, this work is the first to visualize regulation of metazoan transcriptional bursting by a canonical signaling pathway in its native context.

scholarly journals C. elegans GLP-1/Notch activates transcription in a probability gradient across the germline stem cell pool

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
ChangHwan Lee ◽  
Erika B Sorensen ◽  
Tina R Lynch ◽  
Judith Kimble
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcriptional Response ◽  
Germline Stem Cells ◽  
C Elegans ◽  
Cell Pool ◽  
Transcription Sites ◽  
Active Transcription ◽  
Nascent Transcripts ◽  
Stem Cell Pool

C. elegans Notch signaling maintains a pool of germline stem cells within their single-celled mesenchymal niche. Here we investigate the Notch transcriptional response in germline stem cells using single-molecule fluorescence in situ hybridization coupled with automated, high-throughput quantitation. This approach allows us to distinguish Notch-dependent nascent transcripts in the nucleus from mature mRNAs in the cytoplasm. We find that Notch-dependent active transcription sites occur in a probabilistic fashion and, unexpectedly, do so in a steep gradient across the stem cell pool. Yet these graded nuclear sites create a nearly uniform field of mRNAs that extends beyond the region of transcriptional activation. Therefore, active transcription sites provide a precise view of where the Notch-dependent transcriptional complex is productively engaged. Our findings offer a new window into the Notch transcriptional response and demonstrate the importance of assaying nascent transcripts at active transcription sites as a readout for canonical signaling.

scholarly journals A sensitized genetic screen to identify regulators of C. elegans germline stem cells

2021 ◽  
Author(s):  
Sarah Robinson-Thiewes ◽  
Aaron M Kershner ◽  
Heaji Shin ◽  
Kimberly A Haupt ◽  
Peggy Kroll-Connor ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Polymerase ◽  
Transcriptional Activation ◽  
Regulatory Network ◽  
Genetic Screen ◽  
Germline Stem Cells ◽  
Meiotic Cell ◽  
Genetic Screens ◽  
C Elegans ◽  
Forward Genetic Screens

Abstract GLP-1/Notch signaling and a downstream RNA regulatory network maintain germline stem cells (GSCs) in Caenorhabditis elegans. In mutants lacking the GLP-1 receptor, all GSCs enter the meiotic cell cycle precociously and differentiate into sperm. This dramatic GSC defect is called the “Glp” phenotype. The lst-1 and sygl-1 genes are direct targets of Notch transcriptional activation and functionally redundant. Whereas single lst-1 and sygl-1 mutants are fertile, lst-1 sygl-1 double mutants are sterile with a Glp phenotype. We set out to identify genes that function redundantly with either lst-1 or sygl-1 to maintain GSCs. To this end, we conducted forward genetic screens for mutants with a Glp phenotype in genetic backgrounds lacking functional copies of either lst-1 or sygl-1. The screens generated nine glp-1 alleles, two lst-1 alleles, and one allele of pole-1, which encodes the catalytic subunit of DNA polymerase ε. Three glp-1 alleles reside in Ankyrin (ANK) repeats not previously mutated. pole-1 single mutants have a low penetrance Glp phenotype that is enhanced by loss of sygl-1. Thus, the screen uncovered one locus that interacts genetically with sygl-1 and generated useful mutations for further studies of GSC regulation.

scholarly journals Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal

2021 ◽  
Author(s):  
Tina R. Lynch ◽  
Mingyu Xue ◽  
Cazza W. Czerniak ◽  
ChangHwan Lee ◽  
Judith Kimble
Keyword(s):  
Stem Cell ◽  
Regulatory Elements ◽  
Single Element ◽  
Developmental Context ◽  
C Elegans ◽  
Stem Cell Maintenance ◽  
Nascent Transcripts ◽  
Functional Analyses ◽  
Dose Dependent

A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. The use of reporter constructs, cell culture and computational modeling has made enormous contributions to understanding this fundamental question, but analysis of regulatory elements in their natural developmental context is an essential but rarely used complement. Here, we edited Notch-dependent cis-regulatory elements in the endogenous C. elegans sygl-1 gene, which encodes a key stem cell regulator. We then analyzed the in vivo consequences of those mutations – on both gene expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of a single element in a three-element homotypic cluster reduced expression as well as stem cell pool size by about half, while mutation of two elements essentially abolished them. We find that LBS number and LBS neighborhood are both important to activity: elements on separate chromosomes function additively, while elements in the same cluster act synergistically. Our approach of precise CRISPR/Cas9 gene editing coupled with quantitation of both molecular and biological readouts establishes a powerful model for in vivo functional analyses of DNA cis-regulatory elements.

scholarly journals Transcriptional bursting explains the noise-versus-mean relationship in mRNA and protein levels

2016 ◽  
Author(s):  
Roy D. Dar ◽  
Sydney M. Schaffer ◽  
Siddarth S. Dey ◽  
Jonathan E. Foley ◽  
Abhyudai Singh ◽  
...  
Keyword(s):  
Conflict Of Interest ◽  
Burst Size ◽  
Inverse Correlation ◽  
Recent Analysis ◽  
Mrna Levels ◽  
Burst Frequency ◽  
Expression Variability ◽  
Protein Levels ◽  
Transcriptional Bursting ◽  
Cell Expression

Recent analysis (Dey et al, 2015), demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.Conflict of InterestThe authors declare that they have no conflict of interest.

scholarly journals Interaction between Acetylated MyoD and the Bromodomain of CBP and/or p300

2001 ◽  
Vol 21 (16) ◽  
pp. 5312-5320 ◽  
Author(s):  
Anna Polesskaya ◽  
Irina Naguibneva ◽  
Arnaud Duquet ◽  
Eyal Bengal ◽  
Philippe Robin ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Protein Function ◽  
Live Cells ◽  
Regulation Of Transcription ◽  
Muscle Creatine Kinase ◽  
Nonhistone Protein ◽  
Myogenic Factor ◽  
First Time

ABSTRACT Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.

scholarly journals Long-term experimental evolution reveals purifying selection on piRNA-mediated control of transposable element expression

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Ulfar Bergthorsson ◽  
Caroline J. Sheeba ◽  
Anke Konrad ◽  
Tony Belicard ◽  
Toni Beltran ◽  
...  
Keyword(s):  
Natural Selection ◽  
Transcriptional Activation ◽  
Small Rnas ◽  
Active Regions ◽  
Purifying Selection ◽  
Ancestral Population ◽  
Sequence Context ◽  
C Elegans ◽  
Population Sizes

Abstract Background Transposable elements (TEs) are an almost universal constituent of eukaryotic genomes. In animals, Piwi-interacting small RNAs (piRNAs) and repressive chromatin often play crucial roles in preventing TE transcription and thus restricting TE activity. Nevertheless, TE content varies widely across eukaryotes and the dynamics of TE activity and TE silencing across evolutionary time is poorly understood. Results Here, we used experimentally evolved populations of C. elegans to study the dynamics of TE expression over 409 generations. The experimental populations were evolved at population sizes of 1, 10 and 100 individuals to manipulate the efficiency of natural selection versus genetic drift. We demonstrate increased TE expression relative to the ancestral population, with the largest increases occurring in the smallest populations. We show that the transcriptional activation of TEs within active regions of the genome is associated with failure of piRNA-mediated silencing, whilst desilenced TEs in repressed chromatin domains retain small RNAs. Additionally, we find that the sequence context of the surrounding region influences the propensity of TEs to lose silencing through failure of small RNA-mediated silencing. Conclusions Our results show that natural selection in C. elegans is responsible for maintaining low levels of TE expression, and provide new insights into the epigenomic features responsible.

scholarly journals Stem Cells: Muscle Cells Enwrap Escaped Germline Stem Cells in C. elegans

2019 ◽  
Vol 29 (5) ◽  
pp. R150-R152
Author(s):  
Charlotte A. Kelley ◽  
Erin J. Cram
Keyword(s):  
Stem Cells ◽  
Muscle Cells ◽  
Germline Stem Cells ◽  
C Elegans

scholarly journals Signaling Mechanism of Transcriptional Bursting: A Technical Resolution-Independent Study

Biology ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 339
Author(s):  
Yaolai Wang ◽  
Jiaming Qi ◽  
Jie Shao ◽  
Xu-Qing Tang
Keyword(s):  
Frequency Modulation ◽  
Transcription Initiation ◽  
Burst Size ◽  
Independent Study ◽  
Transcriptional Activators ◽  
Signaling Mechanism ◽  
Theoretical Predictions ◽  
Support Size ◽  
Analytical Research ◽  
Transcriptional Bursting

Gene transcription has been uncovered to occur in sporadic bursts. However, due to technical difficulties in differentiating individual transcription initiation events, it remains debated as to whether the burst size, frequency, or both are subject to modulation by transcriptional activators. Here, to bypass technical constraints, we addressed this issue by introducing two independent theoretical methods including analytical research based on the classic two-model and information entropy research based on the architecture of transcription apparatus. Both methods connect the signaling mechanism of transcriptional bursting to the characteristics of transcriptional uncertainty (i.e., the differences in transcriptional levels of the same genes that are equally activated). By comparing the theoretical predictions with abundant experimental data collected from published papers, the results exclusively support frequency modulation. To further validate this conclusion, we showed that the data that appeared to support size modulation essentially supported frequency modulation taking into account the existence of burst clusters. This work provides a unified scheme that reconciles the debate on burst signaling.

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