Tineke L Lenstra

Tineke L Lenstra
Netherlands Cancer Institute · Division of Gene Regulation

PhD

About

57
Publications
3,498
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
1,244
Citations
Introduction
Skills and Expertise
Additional affiliations
January 2013 - present
National Institutes of Health
Position
  • PostDoc Position
September 2008 - September 2012
University Medical Center Utrecht
Position
  • PhD

Publications

Publications (57)
Preprint
Transcription occurs in stochastic bursts, i.e., transcription events are temporally clustered. The clustering does not ensue from environmental fluctuations but springs from the intrinsically stochastic behavior of the regulatory process that controls transcription. Based on microscopic observations of transcription at a single gene copy of yeast,...
Preprint
DNA supercoiling has emerged as a major contributor to gene regulation in bacteria. The impact of DNA supercoiling on transcription dynamics in eukaryotes is less clear. Here, using single-molecule dual-color RNA imaging in budding yeast, we show that transcriptional bursting of the divergent and tandem GAL genes is coupled. Upon topoisomerase degr...
Article
Transcription, the process of copying genetic information from DNA to messenger RNA, is regulated by sequence-specific DNA binding proteins known as transcription factors (TFs). Recent advances in single-molecule tracking (SMT) technologies have enabled visualization of individual TF molecules as they diffuse and interact with the DNA in the contex...
Article
Full-text available
The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed important mechanisms for minimizing these conflicts and their consequences, little is known regarding how the replication and transcription machinery are coordinated in real-time. Here...
Preprint
Full-text available
Transcriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in budding yeast to measure how transcriptional bursting changes upon single and double perturbations of chr...
Preprint
Full-text available
Transcriptional rates are often estimated by fitting the distribution of mature mRNA numbers measured using smFISH (single molecule fluorescence in situ hybridization) with the distribution predicted by the telegraph model of gene expression, which defines two promoter states of activity and inactivity. However, fluctuations in mature mRNA numbers...
Article
Full-text available
Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non-coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription...
Article
Full-text available
Single-molecule RNA fluorescence in situ hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell, revealing both past and instantaneous transcript...
Preprint
Full-text available
Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA Polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA, and a divergent non-coding (DNC) transcript with an unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcr...
Article
Full-text available
This protocol describes how to image fluorescently tagged proteins, RNA, or DNA inside living Saccharomyces cerevisiae cells at the single-molecule level. Imaging inside living cells, as opposed to fixed materials, gives access to real-time kinetic information. Although various single-molecule imaging applications are discussed, we focus on imaging...
Preprint
Full-text available
RNA Polymerase II contains a disordered C-terminal domain (CTD) whose length enigmatically correlates with genome size. The CTD is crucial to eukaryotic transcription, yet the functional and evolutionary relevance of this variation remains unclear. Here, we use smFISH, live imaging, and RNA-seq to investigate how CTD length and disorder influence t...
Article
RNA Polymerase II contains a disordered C-terminal domain (CTD) whose length enigmatically correlates with genome size. The CTD is crucial to eukaryotic transcription, yet the functional and evolutionary relevance of this variation remains unclear. Here, we use smFISH, live imaging, and RNA-seq to investigate how CTD length and disorder influence t...
Article
Full-text available
Visualization of transcription in living cells has taught us that genes are often transcribed in bursts, with periods of gene activity interspersed by periods of inactivity. Recently, technological advances in live-cell imaging have provided a more detailed picture of the characteristics of transcriptional bursts, and have allowed direct visualizat...
Article
Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bu...
Preprint
Full-text available
Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the transcription factor Gal4 with the transcriptional bu...
Article
The transcription cycle can be roughly divided into three stages: initiation, elongation, and termination. Understanding the molecular events that regulate all these stages requires a dynamic view of the underlying processes. The development of techniques to visualize and quantify transcription in single living cells has been essential in revealing...
Article
Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub-second temporal resolution and nanometer spatial resolution. Following transcription in single cells indicates that gene activity is heterogeneous between cells and also...
Article
Full-text available
Background: Genetic interactions, or non-additive effects between genes, play a crucial role in many cellular processes and disease. Which mechanisms underlie these genetic interactions has hardly been characterized. Understanding the molecular basis of genetic interactions is crucial in deciphering pathway organization and understanding the relat...
Article
Eukaryotic transcription is pervasive, and many of the resulting RNAs are non-coding. It is unknown whether ubiquitous transcription is functional or simply reflects stochastic transcriptional noise. By single-molecule visualization of the dynamic interplay between coding and non-coding transcription at the GAL locus in living yeast cells, we show...
Article
The eukaryotic genome is pervasively transcribed, giving rise to various sorts of non-coding RNAs whose mechanisms of action are for the most part not understood. Recent technological advances now allow direct visualization of the synthesis of nascent transcripts from individual genes over time by decorating RNAs with fluorescent proteins. Using th...
Article
Full-text available
Growth condition perturbation or gene function disruption are commonly used strategies to study cellular systems. Although it is widely appreciated that such experiments may involve indirect effects, these frequently remain uncharacterized. Here, analysis of functionally unrelated Saccharyomyces cerevisiae deletion strains reveals a common gene exp...
Article
To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect man...
Article
Full-text available
Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (...
Article
Full-text available
Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify th...
Article
Full-text available
Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed triple-mutant analysis (TMA). We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histon...
Article
Full-text available
Transcription and mRNA export are linked processes. However, the molecular mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we investigate the coordinated action of SAGA an...
Article
Full-text available
Enhanced signaling by the small guanosine triphosphatase Ras is common in T cell acute lymphoblastic leukemia/lymphoma (T-ALL), but the underlying mechanisms are unclear. We identified the guanine nucleotide exchange factor RasGRP1 (Rasgrp1 in mice) as a Ras activator that contributes to leukemogenesis. We found increased RasGRP1 expression in many...
Article
Full-text available
The influence of chromatin on many cellular processes is well appreciated. Much has been learned by studying the role of chromatin remodeling and modifying complexes on individual genes. The seemingly straightforward models that inevitably arise from such studies are challenged by genome-wide analyses. Two recent studies in Saccharomyces cerevisiae...
Data
Old and new histone H3 binding to Hat1 and Asf1. (A) As explained in Figure 7, following a RITE epitope-tag switch (H3-HAH3-T7 and H3-T7H3-HA) tap-tagged Hat1 and Asf1 were immunoprecipitated from cells expressing a mix of old and new histone H3 proteins. Bound histone proteins were analyzed by immunoblots against the C-terminus of histone H3. H3-H...
Data
Growth of mutants of the NuB4 complex. (A) Wild-type (BY4742) and NuB4 mutant strains (all derived from BY4742) were grown under the conditions indicated after spotting on agar plates in 10-fold dilution series. Photos were taken after incubating the plates for 2–3 days. (B) Analysis of cell cycle profiles by staining for DNA content and analysis b...
Data
Primers used for deep sequencing. (DOC)
Data
Scheme showing PCR amplification strategy of barcoded regions around the KANMX selectable marker gene. A first round of amplification introduces an index sequence to barcoded regions of each experimental condition. A second round of amplification introduces the sequences required for Illumina sequencing. All mutants were grown individually to starv...
Data
Immunoblot and ChIP analysis of new histone H3-T7 in starved cells. (A) Immunoblot analysis of new histone H3-T7 and old histone H3-HA before and after induction of the tag switch in starved cells. The H3-HAT7 switch (strain NKI2215) was performed in duplicate. Quantification is shown in Figure 2C. (B) The amount of new H3-T7/input was determined f...
Data
Recombination defect in hap2Δ mutant. Upon deletion of HAP2, the efficiency of recombination (percent of cells that had lost the Hygromycin resistance gene) was impaired, leading to more background recombination before and less recombination after induction of the switch. (TIF)
Data
Role of H4K5 and K12 in histone turnover. The amount of histone turnover at the promoter region of four genes was determined by dividing the ChIP signal of H3-T7 over H3-HA (new/old) and plotted relative to WT. The standard error shows the spread of biological duplicates. Histone turnover was measured in histone H4 mutants carrying mutated lysines...
Data
Role of NuB4 in histone turnover in replicating cells. Histone turnover (ChIP new/old H3) was determined in replicating cells by inducing the tag switch in cells that had been growing in log phase for at least 16 hours and by taking samples two population doublings after induction of Cre recombinase. During this time-period the population of cells...
Data
Effects of histone H3 tags on mRNA expression levels. Microarray analysis of mRNA expression of target genes in RITE strains in mid-log expressing 100% HA-tagged histone H3 or 100% T7-tagged histone H3 (changes vs isogenic RITE strain expressing untagged H3; strains NKI2176/NKI2300/NKI2301). HHT2 and HHF2 represent the genes encoding histone H3 and...
Data
Expression of genes encoding HATs in NuB4 mutants. Microarray analysis of mRNA expression changes in genes encoding (putative) HATs in NuB4 and H4K5,12 mutant strains (fold change vs isogenic wild-type RITE strain in G0 t = 3d). Strains: NKI2148/NKI2191/NKI2192/NKI2187/NKI4169/NKI4170/NKI2193/NKI2194. (TIF)
Data
Yeast strains in Epi-ID histone turnover screen. (DOC)
Data
Recombination efficiencies in tag switch experiments. (DOC)
Article
Full-text available
Author Summary Packaging of eukaryotic genomes by the histone proteins influences many processes that use the DNA, such as transcription, repair, and replication. One well-known mechanism of regulation of histone function is the covalent modification of histone proteins. Replacement of modified histones by new histones has recently emerged as an ad...
Article
Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. T...
Article
Full-text available
The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific pro...
Article
Full-text available
Methylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. Tar...
Data
Chromatin immunoprecipitation of Sir2 and Sir3 normalized to an actively transcribed gene. Chromatin immunoprecipitation (ChIP) using specific antibodies against Sir2 and Sir3 [24] was followed by quantitative PCR to determine binding to telomeric URA3 and ACT1 upon targeting of Dot1 or Dot1G401R (NKI5128). Average ChIP signals were normalized to i...
Data
Gcn5 is required for relocalization of ARS1413 to the nuclear interior by Gcn4. Subnuclear positioning of ARS1413 was monitored in a strain harboring Lac operators next to the origin of replication. The locus was visualized by binding of a GFP-LacI fusion protein to the Lac operators (indicated by green boxes). Subnuclear position was scored relati...
Data
Characterization of the role of Gcn5 in derepression by Dot1. (A) URA3 silencing was determined in strains with a different genomic context at telomere VIIL. The indicated numbers refer to the distance (kb) from the URA3 promoter. Strains from top to bottom are NKI1084, NKI1087, NKI5376 and NKI1088. Gene cassettes inserted on the centromeric side o...
Data
Human Dot1 has derepressor activity in yeast. (A) Human DOT1L (1537 residues) consists of a N-terminal methyltransferase domain homologous to the yeast methyltransferase domain (hDot11-340), followed by a lysine-rich region that shows weak homology to the N-terminal domain of yDot1 (hDot1318-430), and a large domain of unknown function [52,98]. The...
Article
To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic...
Article
Transcription regulation is important for nearly all cellular processes. To understand how transcription is regulated by different regulatory complexes, DNA microarray expression analysis is used to determine the genome-wide changes in mRNA levels upon deletion of individual factors that belong to different transcription regulatory complexes. We an...

Network

Cited By

Projects

Project (1)
Project
Fluorescence imaging of transcription in living cells allows us to visualize the spatiotemporal dynamics of gene regulation in the nucleus. The goal of this research is to develop and implement tools to monitor all relevant length and time scales required for researchers to characterize complex and transient biological processes related to human health and disease.