Publications

The John Innes Centre Publications Repository contains details of all publications resulting from our researchers.

The repository also includes Open Access publications, which can be identified by the icons found on search results.

 Green open access publications are marked by the PDF icon. Click on the publication title, or the PDF icon, and read a pre-print PDF version of the publication.  Gold open access publications have the gold open padlock icon. You can read the full version of these papers on the publishing journal’s website without a subscription. 

The creation of this publications repository was funded by BBSRC.

Search Results

Show > [ 25, 50, 75, 100, All ]

Abstract

Single-cell quantification of transcription kinetics and variability promotes a mechanistic understanding of gene regulation. Here, using single-molecule RNA fluorescence in situ hybridization and mathematical modeling, we dissect cellular RNA dynamics for Arabidopsis FLOWERING LOCUS C (FLC). FLC expression quantitatively determines flowering time and is regulated by antisense (COOLAIR) transcription. In cells without observable COOLAIR expression, we quantify FLC transcription initiation, elongation, intron processing, and lariat degradation, as well as mRNA release from the locus and degradation. In these heterogeneously sized cells, FLC mRNA number increases linearly with cell size, resulting in a large cell-to-cell variability in transcript level. This variation is accounted for by cell-size-dependent, Poissonian FLC mRNA production, but not by large transcriptional bursts. In COOLAIR-expressing cells, however, antisense transcription increases with cell size and contributes to FLC transcription decreasing with cell size. Our analysis therefore reveals an unexpected role for antisense transcription in modulating the scaling of transcription with cell size.

Duncan S., Olsson T. G., Hartley M., Dean C., Rosa S. (2017)

Single Molecule RNA FISH in Arabidopsis Root Cells

Bio-Protocol (7) e2240

Publisher's version: 10.21769/BioProtoc.2240

ID: 56157

read more

Abstract

Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule RNA fluorescence in situ hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript (Raj et al., 2008). This approach is simple to implement and has the advantage that it can be readily applied to any genetic background.

Abstract

Genes targeted by Polycomb repressive complex 2 (PRC2) are regulated in cis by chromatin modifications and also in trans by diffusible regulators such as transcription factors. Here, we introduce a mathematical model in which transcription directly antagonizes Polycomb silencing, thereby linking these cis- and trans-regulatory inputs to gene expression. The model is parameterized by recent experimental data showing that PRC2-mediated repressive chromatin modifications accumulate extremely slowly. The model generates self-perpetuating, bistable active and repressed chromatin states that persist through DNA replication, thereby ensuring high-fidelity transmission of the current chromatin state. However, sufficiently strong, persistent activation or repression of transcription promotes switching between active and repressed chromatin states. We observe that when chromatin modification dynamics are slow, transient pulses of transcriptional activation or repression are effectively filtered, such that epigenetic memory is retained. Noise filtering thus depends on slow chromatin dynamics and may represent an important function of PRC2-based regulation.

Abstract

Genes targeted by Polycomb repressive complex 2 (PRC2) are regulated in cis by chromatin modifications and also in trans by diffusible regulators such as transcription factors. Here, we introduce a mathematical model in which transcription directly antagonizes Polycomb silencing, thereby linking these cis- and trans-regulatory inputs to gene expression. The model is parameterized by recent experimental data showing that PRC2-mediated repressive chromatin modifications accumulate extremely slowly. The model generates self-perpetuating, bistable active and repressed chromatin states that persist through DNA replication, thereby ensuring high-fidelity transmission of the current chromatin state. However, sufficiently strong, persistent activation or repression of transcription promotes switching between active and repressed chromatin states. We observe that when chromatin modification dynamics are slow, transient pulses of transcriptional activation or repression are effectively filtered, such that epigenetic memory is retained. Noise filtering thus depends on slow chromatin dynamics and may represent an important function of PRC2-based regulation.

Abstract

Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription.

Hawkes E. J., Hennelley S. P., Novikova I. V., Irwin J. A., Dean C., Sanbonmatsu K. Y. (2016)

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

Cell Reports (16) 3087-3096

Publisher's version: 10.1016/j.celrep.2016.08.045

ID: 55121

read more

Abstract

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role forCOOLAIR transcripts rather than, or in addition to, antisense transcription.

Duncan S., Olsson T., Hartley M., Dean C., Rosa S. (2016)

A method for detecting single mRNA molecules in Arabidopsis thaliana

Plant Methods (12) 13

Publisher's version: 10.1186/s13007-016-0114-x

ID: 54993

read more

Abstract

Background: Despite advances in other model organisms, there are currently no techniques to explore cell-to-cell variation and sub-cellular localization of RNA molecules at the single-cell level in plants.Results: Here we describe a method for imaging individual mRNA molecules in Arabidopsis thalianaroot cells using multiple singly labeled oligonucleotide probes. We demonstrate detection of both mRNA and nascent transcripts of the housekeeping gene Protein Phosphatase 2A. Our image analysis pipeline also enables quantification of mRNAs that reveals the frequency distribution of transcripts per cell underlying the population mean.Conclusion: This method allows single molecule RNA in situ to be exploited as a powerful tool for studying gene regulation in plants.

Yang H., Howard M., Dean C. (2016)

Physical coupling of activation and derepression activities to maintain an active transcriptional state at FLC.

Proceedings of the National Academy of Sciences of the United States of America (113) 9369-74

Publisher's version: 10.1073/pnas.1605733113

ID: 55008

read more

Abstract

Establishment and maintenance of gene expression states is central to development and differentiation. Transcriptional and epigenetic mechanisms interconnect in poorly understood ways to determine these states. We explore these mechanisms through dissection of the regulation of Arabidopsis thaliana FLOWERING LOCUS C (FLC). FLC can be present in a transcriptionally active state marked by H3K36me3 or a silent state marked by H3K27me3. Here, we investigate the trans factors modifying these opposing histone states and find a physical coupling in vivo between the H3K36 methyltransferase, SDG8, and the H3K27me3 demethylase, ELF6. Previous modeling has predicted this coupling would exist as it facilitates bistability of opposing histone states. We also find association of SDG8 with the transcription machinery, namely RNA polymerase II and the PAF1 complex. Delivery of the active histone modifications is therefore likely to be through transcription at the locus. SDG8 and ELF6 were found to influence the localization of each other on FLC chromatin, showing the functional importance of the interaction. In addition, both influenced accumulation of the associated H3K27me3 and H3K36me3 histone modifications at FLC We propose the physical coupling of activation and derepression activities coordinates transcriptional activity and prevents ectopic silencing.

Irwin J. A., Soumpourou E., Lister C., Ligthart J. D., Kennedy S., Dean C. (2016)

Nucleotide polymorphism affecting FLC expression underpins heading date variation in horticultural brassicas.

Plant Journal (N/A) N/A

Publisher's version: 10.1111/tpj.13221

ID: 53464

read more

Abstract

Variation in flowering time and response to overwintering has been exploited to breed brassica vegetables that can be harvested year-round. Our knowledge of flowering time control now enables investigation of the molecular basis of this important variation. Here, we show that a major determinant of heading date variation in Brassica oleracea is from variation in vernalization response through allelic variation at FLOWERING LOCUS C.C2 (BoFLC4). We characterize two alleles of BoFLC.C2 that are both functional and confer a requirement for vernalization, but they show distinct expression dynamics in response to cold. Complementation experiments in A. thaliana revealed the allelic variation is due to cis polymorphism at BoFLC.C2, which quantitatively influences the degree of cold-induced epigenetic silencing. This results in one allelic variant conferring consistently later heading under both glasshouse and field conditions through reduced environmental sensitivity. Our results suggest that breeding of brassica varieties for commercially valuable variation in heading date has been achieved through selection of cis polymorphism at FLC; similar to that underpinning natural variation in A. thaliana. This understanding will allow selection of alleles with distinct sensitivities to cold and robust heading dates under variable climatic conditions and facilitate breeding of varieties more resistant to climate change. This article is protected by copyright. All rights reserved.

Wu Z., Ietswaart R., Liu F., Yang H., Howard M., Dean C. (2016)

Quantitative regulation of FLC via coordinated transcriptional initiation and elongation.

Proceedings of the National Academy of Sciences of the United States of America (113) 218-23

Publisher's version: 10.1073/pnas.1518369112

ID: 52474

read more

Abstract

The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ~25-fold with elongation rate varying ~8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.

Duncan S., Holm S., Questa J., Irwin J., Grant A., Dean C. (2015)

Seasonal shift in timing of vernalization as an adaptation to extreme winter.

ELife (4) 4532801

Publisher's version: 10.7554/eLife.06620

ID: 51899

read more

Abstract

The requirement for vernalization, a need for prolonged cold to trigger flowering, aligns reproductive development with favorable spring conditions. In Arabidopsis thaliana vernalization depends on the cold-induced epigenetic silencing of the floral repressor locus FLC. Extensive natural variation in vernalization response is associated with A. thaliana accessions collected from different geographical regions. Here, we analyse natural variation for vernalization temperature requirement in accessions, including those from the northern limit of the A. thaliana range. Vernalization required temperatures above 0°C and was still relatively effective at 14°C in all the accessions. The different accessions had characteristic vernalization temperature profiles. One Northern Swedish accession showed maximum vernalization at 8°C, both at the level of flowering time and FLC chromatin silencing. Historical temperature records predicted all accessions would vernalize in autumn in N. Sweden, a prediction we validated in field transplantation experiments. The vernalization response of the different accessions was monitored over three intervals in the field and found to match that when the average field temperature was given as a constant condition. The vernalization temperature range of 0-14°C meant all accessions fully vernalized before snowfall in N. Sweden. These findings have important implications for understanding the molecular basis of adaptation and for predicting the consequences of climate change on flowering time.

Abstract

Analysis of how seasonal cues influence the timing of the floral transition has revealed many important principles for how epigenetic regulation can integrate a variety of environmental cues with developmental signals. The study of the pathways that necessitate overwintering in plants and their ability to respond to prolonged cold (the vernalization requirement and response pathways) has elaborated different chromatin regulatory pathways and the involvement of noncoding RNAs. The major target of these vernalization pathways in Arabidopsis (Arabidopsis thaliana) is Flowering Locus C (FLC). A relatively simple picture of FLC regulation is emerging of a few core complexes and mechanisms that antagonize each other's actions. This balance provides a fine degree of control that has nevertheless permitted evolution of a wide range of natural variation in vernalization in Arabidopsis. Similar simple routes of adaptation may underlie life history variation between species.

Berry S., Hartley M., Olsson T. S., Dean C., Howard M. (2015)

Local chromatin environment of a Polycomb target gene instructs its own epigenetic inheritance.

eLife (4) e07205

Publisher's version: 10.7554/eLife.07205

ID: 50254

read more

Abstract

Inheritance of gene expression states is fundamental for cells to 'remember' past events, such as environmental or developmental cues. The conserved Polycomb Repressive Complex 2 (PRC2) maintains epigenetic repression of many genes in animals and plants and modifies chromatin at its targets. Histones modified by PRC2 can be inherited through cell division. However, it remains unclear whether this inheritance can direct long-term memory of individual gene expression states (cis memory) or instead if local chromatin states are dictated by the concentrations of diffusible factors (trans memory). By monitoring the expression of two copies of the Arabidopsis Polycomb target gene FLOWERING LOCUS C (FLC) in the same plants, we show that one copy can be repressed while the other is active. Furthermore, this 'mixed' expression state is inherited through many cell divisions as plants develop. These data demonstrate that epigenetic memory of FLC expression is stored not in trans but in cis.

Berry S., Dean C. (2015)

Environmental perception and epigenetic memory: mechanistic insight through FLC.

Plant Journal (10.1111/tpj.12869) epub

Publisher's version: 10.1111/tpj.12869

ID: 51292

read more

Abstract

Chromatin plays a central role in orchestrating gene regulation at the transcriptional level. However, our understanding of how chromatin states are altered in response to environmental and developmental cues, and then maintained epigenetically over many cell divisions, remains poor. The floral repressor gene FLOWERING LOCUS C (FLC) in Arabidopsis thaliana is a useful system to address these questions. FLC is transcriptionally repressed during exposure to cold temperatures, allowing studies of how environmental conditions alter expression states at the chromatin level. FLC repression is also epigenetically maintained during subsequent development in warm conditions, so that exposure to cold may be remembered. This memory depends on molecular complexes that are highly conserved among eukaryotes, making FLC not only interesting as a paradigm for understanding biological decision-making in plants, but also an important system for elucidating chromatin-based gene regulation more generally. In this review, we summarize our understanding of how cold temperature induces a switch in the FLC chromatin state, and how this state is epigenetically remembered. We also discuss how the epigenetic state of FLC is reprogrammed in the seed to ensure a requirement for cold exposure in the next generation.

Angel A., Song J., Yang H., Questa J. I., Dean C., Howard M. (2015)

Vernalizing cold is registered digitally at FLC.

Proceedings of the National Academy of Sciences of the United States of America (112) 41464151

Publisher's version: 10.1073/pnas.1503100112

ID: 49365

read more

Abstract

A fundamental property of many organisms is an ability to sense, evaluate, and respond to environmental signals. In some situations, generation of an appropriate response requires long-term information storage. A classic example is vernalization, where plants quantitatively sense long-term cold and epigenetically store this cold-exposure information to regulate flowering time. In Arabidopsis thaliana, stable epigenetic memory of cold is digital: following long-term cold exposure, cells respond autonomously in an all-or-nothing fashion, with the fraction of cells that stably silence the floral repressor FLOWERING LOCUS C (FLC) increasing with the cold exposure duration. However, during cold exposure itself it is unknown whether vernalizing cold is registered at FLC in individual cells in an all-or-nothing (digital) manner or is continuously varying (analog). Using mathematical modeling, we found that analog registration of cold temperature is problematic due to impaired analog-to-digital conversion into stable memory. This disadvantage is particularly acute when responding to short cold periods, but is absent when cold temperatures are registered digitally at FLC. We tested this prediction experimentally, exposing plants to short periods of cold interrupted with even shorter warm breaks. For FLC expression, we found that the system responds similarly to both interrupted and uninterrupted cold, arguing for a digital mechanism integrating long-term temperature exposure.

Li P., Tao Z., Dean C. (2015)

Phenotypic evolution through variation in splicing of the noncoding RNA COOLAIR

Genes and Development (29) 1-6

Publisher's version: /10.1101/gad.258814.115

ID: 49350

read more

Abstract

The extent to which natural polymorphisms in noncoding sequences have functional consequences is still unknown. A large proportion of the natural variation in flowering in Arabidopsis thaliana accessions is due to noncoding cis polymorphisms that define distinct haplotypes of FLOWERING LOCUS C (FLC). Here, we show that a single natural intronic polymorphism in one haplotype affects FLC expression and thus flowering by specifically changing splicing of the FLC antisense transcript COOLAIR. Altered antisense splicing increases FLC expression via a cotranscriptional mechanism involving capping of the FLC nascent transcript. Single noncoding polymorphisms can therefore be a major contributor to phenotypic evolution through modulation of noncoding transcripts.

Csorba T., Questa J. I., Sun Q., Dean C. (2014)

Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization.

Proceedings of the National Academy of Sciences of the United States of America (111) 16160-5

Publisher's version: 10.1073/pnas.1419030111

ID: 48918

read more

Abstract

Long noncoding RNAs (lncRNAs) have been proposed to play important roles in gene regulation. However, their importance in epigenetic silencing and how specificity is determined remain controversial. We have investigated the cold-induced epigenetic switching mechanism involved in the silencing of Arabidopsis thaliana Flowering Locus C (FLC), which occurs during vernalization. Antisense transcripts, collectively named COOLAIR, are induced by prolonged cold before the major accumulation of histone 3 lysine 27 trimethylation (H3K27me3), characteristic of Polycomb silencing. We have found that COOLAIR is physically associated with the FLC locus and accelerates transcriptional shutdown of FLC during cold exposure. Removal of COOLAIR disrupted the synchronized replacement of H3K36 methylation with H3K27me3 at the intragenic FLC nucleation site during the cold. Consistently, genetic analysis showed COOLAIR and Polycomb complexes work independently in the cold-dependent silencing of FLC. Our data reveal a role for lncRNA in the coordinated switching of chromatin states that occurs during epigenetic regulation.

Crevillén P., Yang H., Cui X., Greeff C., Trick M., Qiu Q., Cao X., Dean C. (2014)

Epigenetic reprogramming that prevents transgenerational inheritance of the vernalized state.

Nature (515) 587-90

Publisher's version: 10.1038/nature13722

ID: 48941

read more

Abstract

The reprogramming of epigenetic states in gametes and embryos is essential for correct development in plants and mammals. In plants, the germ line arises from somatic tissues of the flower, necessitating the erasure of chromatin modifications that have accumulated at specific loci during development or in response to external stimuli. If this process occurs inefficiently, it can lead to epigenetic states being inherited from one generation to the next. However, in most cases, accumulated epigenetic modifications are efficiently erased before the next generation. An important example of epigenetic reprogramming in plants is the resetting of the expression of the floral repressor locus FLC in Arabidopsis thaliana. FLC is epigenetically silenced by prolonged cold in a process called vernalization. However, the locus is reactivated before the completion of seed development, ensuring the requirement for vernalization in every generation. In contrast to our detailed understanding of the polycomb-mediated epigenetic silencing induced by vernalization, little is known about the mechanism involved in the reactivation of FLC. Here we show that a hypomorphic mutation in the jumonji-domain-containing protein ELF6 impaired the reactivation of FLC in reproductive tissues, leading to the inheritance of a partially vernalized state. ELF6 has H3K27me3 demethylase activity, and the mutation reduced this enzymatic activity in planta. Consistent with this, in the next generation of mutant plants, H3K27me3 levels at the FLC locus stayed higher, and FLC expression remained lower, than in the wild type. Our data reveal an ancient role for H3K27 demethylation in the reprogramming of epigenetic states in plant and mammalian embryos.

Zhu D., Rosa S., Dean C. (2014)

Nuclear Organization Changes and the Epigenetic Silencing of FLC during Vernalization

Journal of Molecular Biology (427 ) 659-669

Publisher's version: 10.1016/j.jmb.2014.08.025

ID: 48566

read more

Abstract

Changes in nuclear organization are considered an important complement to trans-acting factors, histone modifications and non-coding RNAs in robust and stable epigenetic silencing. However, how these multiple layers interconnect mechanistically to reinforce each other's activity is still unclear. A system providing long timescales facilitating analysis of these interconnections is vernalization. This involves the Polycomb-mediated epigenetic silencing of flowering locus C (FLC) that occurs as Arabidopsis plants are exposed to prolonged cold. Analysis of changes in nuclear organization during vernalization has revealed that disruption of a gene loop and physical clustering of FLC loci are part of the vernalization mechanism. These events occur at different times and thus contribute to distinct aspects of the silencing mechanism. The physical clustering of FLC loci is tightly correlated with the accumulation of specific Polycomb complexes/H3K27me3 at a localized intragenic site during the cold. Since the quantitative nature of vernalization is a reflection of a bistable cell autonomous switch in an increasing number of cells, this correlation suggests a tight connection between the switching mechanism and changes in nuclear organization. This integrated picture is likely to be informative for many epigenetic mechanisms.

Li P., Filiault D., Box M., Kerdaffrec E., Oosterhout C., Wilczek A., Schmitt A., McMullan M., Bergelson J., Nordborg M., Dean C. (2014)

Multiple FLC haplotypes defined by independent cis-regulatory variation underpin life history diversity in Arabidopsis thaliana

Genes and Development (28) 1635-1640

Publisher's version: 10.1101/gad.245993.114

ID: 48565

read more

Abstract

Relating molecular variation to phenotypic diversity is a major goal in evolutionary biology. In Arabidopsis thaliana, FLOWERING LOCUS C (FLC) is a major determinant of the variation in vernalization- the acceleration of flowering by prolonged cold. Here, we analyse the worldwide FLC haplotype structure and identify five predominant FLC haplotypes defined by non-coding sequence variation. Genetic and transgenic experiments show they are functionally distinct with non-coding polymorphism influencing the rate of FLC epigenetic silencing. This non-coding variation at a single locus accounts for a large proportion of natural variation in vernalization of A. thaliana accessions. These multiple, functionally distinct FLC haplotypes appear to contribute to the broad geographical range of A. thaliana accessions.

Abstract

Post-translational modifications of histone tails are an important factor regulating chromatin structure and gene expression. Epigenetic memory systems have been predicted to involve mutually exclusive histone modifications that through positive feedback mechanisms generate bistable states. How the states are interconverted is not understood and whether the histone modifications are sufficient for epigenetic memory is still greatly debated. We have exploited the process of vernalization, the slow quantitative epigenetic silencing of Arabidopsis FLC induced by cold, to detail with fine temporal and spatial resolution the dynamics of histone modifications during an epigenetic switch. The profiles of H3K36me3, H3K4me3 and H3K4me2 at FLC throughout the vernalization process were compared to H3K27me3, which accumulates at a local nucleation region during the cold and spreads across the locus on return to the warm. We find for many phases of the vernalization process H3K36me3 and H3K27me3 show opposing profiles in the FLC nucleation region and gene body, that H3K36me3 and H3K27me3 rarely co-exist on the same histone tail, and that this antagonism is functionally important. A lack of H3K36me3 results in a fully silenced state at FLC even in the absence of cold. We therefore propose that H3K36me3 is the opposing modification to H3K27me3 in the Polycomb-mediated silencing of FLC. However, the lack of an absolute mirror profile predicted from modelling suggests their antagonistic roles contribute a necessary but not sufficient component of the mechanism enabling switching between, and inheritance of, epigenetic states.

Song J., Rutjens B., Dean C. (2014)

Detecting histone modifications in plants.

Methods Molecular Biology (1112) 165-75

Publisher's version: 10.1007/978-1-62703-773-0_11

ID: 47819

read more

Abstract

Histone modifications play an essential role in chromatin-associated processes including gene regulation and epigenetic inheritance. It is therefore very important to quantitatively analyze histone modifications at both the single gene and whole genome level. Here, we describe a robust chromatin immunoprecipitation (ChIP) method for Arabidopsis, which could be adapted for other plant species. This method is compatible with multiple downstream applications including qPCR, tilling arrays, and high-throughput sequencing.

Wang Z. W., Wu Z., Raitskin O., Sun Q., Dean C. (2014)

Antisense-mediated FLC transcriptional repression requires the P-TEFb transcription elongation factor.

Proceedings of the National Academy of Sciences of the United States of America (111) 7468-73

Publisher's version: 10.1073/pnas.1406635111

ID: 47816

read more

Abstract

The functional significance of noncoding transcripts is currently a major question in biology. We have been studying the function of a set of antisense transcripts called COOLAIR that encompass the whole transcription unit of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Alternative polyadenylation of COOLAIR transcripts correlates with different FLC sense expression states. Suppressor mutagenesis aimed at understanding the importance of this sense-antisense transcriptional circuitry has identified a role for Arabidopsis cyclin-dependent kinase C (CDKC;2) in FLC repression. CDKC;2 functions in an Arabidopsis positive transcription elongation factor b (P-TEFb) complex and influences global RNA polymerase II (Pol II) Ser(2) phosphorylation levels. CDKC;2 activity directly promotes COOLAIR transcription but does not affect an FLC transgene missing the COOLAIR promoter. In the endogenous gene context, however, the reduction of COOLAIR transcription by cdkc;2 disrupts a COOLAIR-mediated repression mechanism that increases FLC expression. This disruption then feeds back to indirectly increase COOLAIR expression. This tight interconnection between sense and antisense transcription, together with differential promoter sensitivity to P-TEFb, is central to quantitative regulation of this important floral repressor gene.

Marquardt S., Raitskin O., Wu Z., Liu F., Sun Q., Dean C. (2014)

Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription.

Molecular Cell (54) 156-65

Publisher's version: 10.1016/j.molcel.2014.03.026

ID: 47373

read more

Abstract

Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

JIC Cookie Policy. We use cookies on this site to enhance your user experience. By clicking any link on this page you are giving your consent for us to set cookies. You can find out more about the cookies by clicking here.

Accept cookies