Papers by Julia Zeitlinger
Paused RNA polymerase II inhibits new transcriptional initiation
Nature genetics, Jan 15, 2017
RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginnin... more RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. However, it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.
Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis
Genome Biology, 2016
Drosophila dorso-ventral (DV) patterning is one of the best-understood regulatory networks to dat... more Drosophila dorso-ventral (DV) patterning is one of the best-understood regulatory networks to date, and illustrates the fundamental role of enhancers in controlling patterning, cell fate specification, and morphogenesis during development. Histone acetylation such as H3K27ac is an excellent marker for active enhancers, but it is challenging to obtain precise locations for enhancers as the highest levels of this modification flank the enhancer regions. How to best identify tissue-specific enhancers in a developmental system de novo with a minimal set of data is still unclear. Using DV patterning as a test system, we develop a simple and effective method to identify tissue-specific enhancers de novo. We sample a broad set of candidate enhancer regions using data on CREB-binding protein co-factor binding or ATAC-seq chromatin accessibility, and then identify those regions with significant differences in histone acetylation between tissues. This method identifies hundreds of novel DV enhancers and outperforms ChIP-seq data of relevant transcription factors when benchmarked with mRNA expression data and transgenic reporter assays. These DV enhancers allow the de novo discovery of the relevant transcription factor motifs involved in DV patterning and contain additional motifs that are evolutionarily conserved and for which the corresponding transcription factors are expressed in a DV-biased fashion. Finally, we identify novel target genes of the regulatory network, implicating morphogenesis genes as early targets of DV patterning. Taken together, our approach has expanded our knowledge of the DV patterning network even further and is a general method to identify enhancers in any developmental system, including mammalian development.
Drosophila poised enhancers are generated during tissue patterning with the help of repression
Histone modifications are frequently used as markers for enhancer states, but how to interpret en... more Histone modifications are frequently used as markers for enhancer states, but how to interpret enhancer states in the context of embryonic development is not clear. The poised enhancer signature, involving H3K4me1 and low levels of H3K27ac, has been reported to mark inactive enhancers that are poised for future activation. However, future activation is not always observed, and alternative reasons for the widespread occurrence of this enhancer signature have not been investigated. By analyzing enhancers during dorsal-ventral (DV) axis formation in the Drosophila embryo, we find that the poised enhancer signature is specifically generated during patterning in the tissue where the enhancers are not induced, including at enhancers that are known to be repressed by a transcriptional repressor. These results suggest that, rather than serving exclusively as an intermediate step before future activation, the poised enhancer state may be a mark for spatial regulation during tissue patterning. We discuss the possibility that the poised enhancer state is more generally the result of repression by transcriptional repressors.
Cori meets Dobzhansky: Evolution and Gene Expression in St. Louis
BioEssays, 2015
St. Louis and its famous Gateway Arch were the setting of the Special Symposium: Evolution and Co... more St. Louis and its famous Gateway Arch were the setting of the Special Symposium: Evolution and Core Processes in Gene Regulation, sponsored by the American Society for Biochemistry and Molecular Biology. Biochemists and evolutionary biologists highlighted growing connections between studies of biochemical mechanism and natural selection on gene expression.
Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation
Genome Research, 2015
The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime ... more The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility but the mechanisms by which this occurs are poorly understood. Here we analyze the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). Our results show that early enhancers are characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. We envision that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner.
Development
Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Dr... more Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Drosophila JNK pathway, D-Fos and the phosphatase Puckered (Puc). To identify principles of epithelial closure processes, we studied another cell sheet movement that we term thorax closure, the joining of the parts of the wing imaginal discs which give rise to the adult thorax during metamorphosis. In thorax closure a special row of margin cells express puc and accumulate prominent actin fibres during midline attachment. Genetic data indicate a requirement of D-Fos and the JNK pathway for thorax closure, and a negative regulatory role of Puc. Furthermore, puc expression co-localises with elevated levels of D-Fos, is reduced in a JNK or D-Fos loss-of-function background and is ectopically induced after JNK activation. This suggests that Puc acts downstream of the JNK pathway and D-Fos to mediate a negative feed-back loop. Therefore, the molecular circuitry required for thorax closure is ver...
A computational pipeline for comparative ChIP-seq analyses
Nature Protocols, 2011
Chromatin immunoprecipitation (ChIP) followed by deep sequencing can now easily be performed acro... more Chromatin immunoprecipitation (ChIP) followed by deep sequencing can now easily be performed across different conditions, time points and even species. However, analyzing such data is not trivial and standard methods are as yet unavailable. Here we present a protocol to systematically compare ChIP-sequencing (ChIP-seq) data across conditions. We first describe technical guidelines for data preprocessing, read mapping, read-density visualization and peak calling. We then describe methods and provide code with specific examples to compare different data sets across species and across conditions, including a threshold-free approach to measure global similarity, a strategy to assess the binary conservation of binding events and measurements for quantitative changes of binding. We discuss how differences in binding can be related to gene functions, gene expression and sequence changes. Once established, this protocol should take about 2 d to complete and be generally applicable to many data sets.
Genes & Development, 1997
Drosophila Jun is shown to be involved in different signal transduction pathways and developmenta... more Drosophila Jun is shown to be involved in different signal transduction pathways and developmental decisions. Dorsal closure, a morphogenetic process occurring during Drosophila embryogenesis, is regulated by Hemipterous (Hep) and Basket (Bsk), homologs of JNKK and JNK, respectively. Embryos lacking Jun activity exhibit a dorsal closure phenotype, very similar to that of bsk and hep mutants, indicating that Jun is a target of Hep/Bsk signaling. In eye and wing development Jun participates in a separate signaling pathway that is comprised of Ras, Raf, and the ERK-type kinase Rolled. In contrast to the strict requirement for Jun in dorsal closure, its role in the eye is redundant but can be uncovered by mutations in other signaling components. The redundant function of Jun in eye development may contribute to the precision of photoreceptor differentiation and ommatidial assembly.
Developmental Dynamics, 1996
Retinoic acid and its isoforms are considered to be endogenous compounds which regulate embryonic... more Retinoic acid and its isoforms are considered to be endogenous compounds which regulate embryonic development. In the work reported here we have determined which retinoids are present in zebrafish embryos and how their levels change throughout development and into adulthood. All-trans-RA is present and its level does not change significantly during embryogenesis. We failed to detect other retinoic acid isomers such as 9-cis-RA and 4-0xo-RA, but we did observe a rapid rise in the level of didehydroretinol after gastrulation. The most striking result is that the zebrafish embryo, like Xenopus and tunicates, contains a vast excess of t-retinal whereas the embryos of higher vertebrates have an excess of t-retinol. However, as the zebrafish grows, the levels of t-retinol rise so that by adulthood t-retinol and t-retinal concentrations are more equivalent, indicating a changing pattern of retinoid metabolism with growth. To examine the significance of the use of t-retinal as a precursor of t-RA we treated embryos with disulphiram, an inhibitor of retinaldehyde dehydrogenase. This resulted in embryos with an undulating notochord and correspondingly abnormal somites and ventral floor plate. In contrast to this effect, 4-methylpyrazole, which inhibits alcohol dehydrogenases, had no effect on development. This effect of disulphiram suggests that t-RA may be involved in the establishment of the anteroposterior axis of the embryo. o 1996 wiley-Liss, Inc.
Developmental Biology, 2010
Development, 2014
The rapid expansion of genomics methods has enabled developmental biologists to address fundament... more The rapid expansion of genomics methods has enabled developmental biologists to address fundamental questions of developmental gene regulation on a genome-wide scale. These efforts have demonstrated that transcription of developmental control genes by RNA polymerase II (Pol II) is commonly regulated at the transition to productive elongation, resulting in the promoter-proximal accumulation of transcriptionally engaged but paused Pol II prior to gene induction. Here we review the mechanisms and possible functions of Pol II pausing and their implications for development.
Cell, 2013
Paused RNA polymerase (Pol II) is a pervasive feature of Drosophila embryos and mammalian stem ce... more Paused RNA polymerase (Pol II) is a pervasive feature of Drosophila embryos and mammalian stem cells, but its role in development is uncertain. Here, we demonstrate that a spectrum of paused Pol II determines the ''time to synchrony''-the time required to achieve coordinated gene expression across the cells of a tissue. To determine whether synchronous patterns of gene activation are significant in development, we manipulated the timing of snail expression, which controls the coordinated invagination of $1,000 mesoderm cells during gastrulation. Replacement of the strongly paused snail promoter with moderately paused or nonpaused promoters causes stochastic activation of snail expression and increased variability of mesoderm invagination. Computational modeling of the dorsal-ventral patterning network recapitulates these variable and bistable gastrulation profiles and emphasizes the importance of timing of gene activation in development. We conclude that paused Pol II and transcriptional synchrony are essential for coordinating cell behavior during morphogenesis.
Massive zygotic transcription begins in many organisms during the midblastula transition when the... more Massive zygotic transcription begins in many organisms during the midblastula transition when the cell cycle of the dividing egg slows down. A few genes are transcribed before this stage but how this differential activation is accomplished is still an open question. We have performed ChIP-seq experiments on tightly staged Drosophila embryos and show that massive recruitment of RNA polymerase II (Pol II) with widespread pausing occurs de novo during the midblastula transition. However, ∼100 genes are strongly occupied by Pol II before this timepoint and most of them do not show Pol II pausing, consistent with a requirement for rapid transcription during the fast nuclear cycles. This global change in Pol II pausing correlates with distinct core promoter elements and associates a TATA-enriched promoter with the rapid early transcription. This suggests that promoters are differentially used during the zygotic genome activation, presumably because they have distinct dynamic properties.
ChIP-nexus enables improved detection of in vivo transcription factor binding footprints
Nature biotechnology, Jan 9, 2015
Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transc... more Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins-human TBP and Drosophila NFkB, Twist and Max-shows that it outperforms existing ChIP protocols in resolution and specificity, pinpoints relevant binding sites within enhancers containing multiple binding motifs, and allows for the analysis of in vivo binding specificities. Notably, we show that Max frequently interacts with DNA sequences next to its motif, and that this binding pattern correlates with local DNA-sequence features such as DNA sh...
the end of M and early G1. It is not yet clear whether this model, developed using a small set of... more the end of M and early G1. It is not yet clear whether this model, developed using a small set of genes, will Nine Cambridge Center Cambridge, Massachusetts 02142 extrapolate to regulation of all cell cycle genes. Microarray analysis has revealed that the expression levels of approximately 800 genes vary in a periodic fashion during the yeast cell cycle (Cho et al., 1998; Spellman et al., 1998), but little is known about the regulation of most of these genes. The set of genes controlled 200 Technology Square Cambridge, Massachusetts 02139 by MBF and SBF has recently been identified by using a genome-wide binding method, confirming that these factors are largely bound to genes expressed in late G1 and revealing how sets of functionally related genes are Summary regulated during this time (Iyer et al., 2001). Identification of the genes regulated by all nine transcription factors Genome-wide location analysis was used to determine how the yeast cell cycle gene expression program is
Nature Genetics, 2007
Regulation of gene expression is integral to the development and survival of all organisms. Trans... more Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter 1 , followed by initiation of RNA synthesis and the transition to productive elongation 2-4 . In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region 5-13 . To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues.
Transcriptional regulation of the cell cycle
Genome Biology, 2001
Analysis of the regulation of the yeast cell cycle by nine cell-cycle transcriptional activators
Genes & development, Jan 15, 2007
Genetic studies have identified numerous sequence-specific transcription factors that control dev... more Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. We determined the genome-wide occupancy of the dorsoventral (DV) determinants Dorsal, Twist, and Snail in the Drosophila embryo using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip). The in vivo binding of these proteins correlate tightly with the limits of known enhancers. Our analysis predicts substantially more target genes than previous estimates, and includes Dpp signaling components and anteroposterior (AP) segmentation determinants. Thus, the ChIP-chip data uncover a much larger than expected regulatory network, which integrates diverse patterning processes during development.
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Papers by Julia Zeitlinger