Maike Stam - Academia.edu (original) (raw)

Papers by Maike Stam

Methods in Molecular Biology

Enhancers are important regulators of gene expression during numerous crucial processes including... more Enhancers are important regulators of gene expression during numerous crucial processes including tissue differentiation across development. In plants, their recent molecular characterization revealed their capacity to activate the expression of several target genes through the binding of transcription factors. Nevertheless, identifying these target genes at a genome-wide level remains a challenge, in particular in species with large genomes, where enhancers and target genes can be hundreds of kilobases away. Therefore, the contribution of enhancers to regulatory network is still poorly understood in plants. In this study, we investigate the enhancer-driven regulatory network of two maize tissues at different stages: leaves at seedling stage and husks (bracts) at flowering. Using a systems biology approach, we integrate genomic, epigenomic and transcriptomic data to model the regulatory relationship between transcription factors and their potential target genes. We identify regulato...

Current Protocols in Plant Biology

Journal of Bioinformatics and Computational Biology

The circular chromosome conformation capture technique followed by sequencing (4C-seq) has been u... more The circular chromosome conformation capture technique followed by sequencing (4C-seq) has been used in a number of studies to investigate chromosomal interactions between DNA fragments. Computational pipelines have been developed and published that offer various possibilities of 4C-seq data processing and statistical analysis. Here, we present an overview of four of such pipelines (fourSig, FourCSeq, 4C-ker and w4Cseq) taking into account the most important stages of computations. We provide comparisons of the methods and discuss their advantages and possible weaknesses. We illustrate the results with the use of data obtained for two different species, in a study devoted to vernalization control in Arabidopsis thaliana by the FLOWERING LOCUS C (FLC) gene and to long-range chromatin interactions in mouse embryonic stem cells.

Plant Methods

Background: The chromosome conformation capture (3C) technique is a method to study chromatin int... more Background: The chromosome conformation capture (3C) technique is a method to study chromatin interactions at specific genomic loci. Initially established for yeast the 3C technique has been adapted to plants in recent years in order to study chromatin interactions and their role in transcriptional gene regulation. As the plant scientific community continues to implement this technology, a discussion on critical controls, validations steps and interpretation of 3C data is essential to fully benefit from 3C in plants. Results: Here we assess the reliability and robustness of the 3C technique for the detection of chromatin interactions in Arabidopsis. As a case study, we applied this methodology to the genomic locus of a floral integrator gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and demonstrate the need of several controls and standard validation steps to allow a meaningful interpretation of 3C data. The intricacies of this promising but challenging technique are discussed in depth. Conclusions: The 3C technique offers an interesting opportunity to study chromatin interactions at a resolution infeasible by microscopy. However, for interpretation of 3C interaction data and identification of true interactions, 3C technology demands a stringent experimental setup and extreme caution.

Journal of visualized experiments : JoVE, Jan 28, 2018

When generating transgenic plants, generally the objective is to have stable expression of a tran... more When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2-0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bac...

Genome Biology

Background: While most cells in multicellular organisms carry the same genetic information, in ea... more Background: While most cells in multicellular organisms carry the same genetic information, in each cell type only a subset of genes is being transcribed. Such differentiation in gene expression depends, for a large part, on the activation and repression of regulatory sequences, including transcriptional enhancers. Transcriptional enhancers can be located tens of kilobases from their target genes, but display characteristic chromatin and DNA features, allowing their identification by genome-wide profiling. Here we show that integration of chromatin characteristics can be applied to predict distal enhancer candidates in Zea mays, thereby providing a basis for a better understanding of gene regulation in this important crop plant. Result: To predict transcriptional enhancers in the crop plant maize (Zea mays L. ssp. mays), we integrated available genome-wide DNA methylation data with newly generated maps for chromatin accessibility and histone 3 lysine 9 acetylation (H3K9ac) enrichment in young seedling and husk tissue. Approximately 1500 intergenic regions, displaying low DNA methylation, high chromatin accessibility and H3K9ac enrichment, were classified as enhancer candidates. Based on their chromatin profiles, candidate sequences can be classified into four subcategories. Tissue-specificity of enhancer candidates is defined based on the tissues in which they are identified and putative target genes are assigned based on tissue-specific expression patterns of flanking genes. Conclusions: Our method identifies three previously identified distal enhancers in maize, validating the new set of enhancer candidates and enlarging the toolbox for the functional characterization of gene regulation in the highly repetitive maize genome.

Annals of Botany

In genetically modified plants, the introduced transgenes are sometimes not expressed. They can b... more In genetically modified plants, the introduced transgenes are sometimes not expressed. They can be silenced. Transgenes can also cause the silencing of endogenous plant genes if they are sufficiently homologous, a phenomenon known as co-suppression. Silencing occurs transcriptionally and post-transcriptionally but silencing of endogenous genes seems predominantly post-transcriptional. If viral transgenes are introduced and silenced, the posttranscriptional process also prevents homologous RNA viruses from accumulating ; this is a means of generating virus-resistant plants. A major goal of current research is to dissect the mechanism(s) of these sequence-homologydependent gene silencing phenomena. Various factors seem to play a role, including DNA methylation, transgene copy number and the repetitiveness of the transgene insert, transgene expression level, possible production of aberrant RNAs, and ectopic DNA-DNA interactions. The causal relationship between these factors and the link between transcriptional and post-transcriptional silencing is not always clear. In this review we discuss various observations associated with gene silencing and attempt to relate them.

Plasmid, 2017

When generating transgenic plants, one of the objectives is to achieve stable expression of the t... more When generating transgenic plants, one of the objectives is to achieve stable expression of the transgene. Transgene silencing can be avoided by single copy integration of the transgene. Binary systems that predominantly result in single copy integrations, such as BIBAC vectors, are also single-copy in E. coli, the organism in which the T-DNA to be delivered to the plant is assembled. Although a low-copy number is important for stable maintenance of large DNA fragments in E. coli, it hampers cloning into the vector due to a low DNA yield. Here we describe BIBAC vectors to which Gateway site-specific recombination sites are added. These sites provide a fast and easy introduction of sequences of interest into any vector. Our Gateway-compatible BIBAC vectors are available with two selectable markers for plants - resistance to Basta (BIBAC-BAR-GW) and DsRed fluorescence in the seed coat (BIBAC-RFP-GW). Using the BIBAC-BAR-GW vector we have generated different fluorescence-based reporter constructs that, when delivered to plant cells, can be used to study and optimize precise, template-dependent site-specific genome editing by CRISPR-Cas9, TALENs or ZFP-nuclease complexes, and oligonucleotide-directed mutagenesis. We have generated 59 reporter lines in A. thaliana with our reporter constructs, and for the lines carrying single T-DNA integrations (32 out of 59) we have determined the integrity of the integrations, their genomic locations and the expression level of the reporters. Similarly to its original counterpart, BIBAC-BAR-GW generates single T-DNA integrations in Arabidopsis with 50% efficiency, and 90% of those are intact. The reporter constructs in the independent transgenic lines exhibit only an up to 3-fold difference in expression level. These features combined with an easy manipulation of the vector due to the added Gateway sites make the BIBAC-GW vectors an attractive tool for generating transgenic plants.

lnhibition of flower pigmentation in transgenic petunia plants was previously accomplished by exp... more lnhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35s promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the vira1 CaMV 35s promoter was in- sufficiently expressed in cell types of this organ in which

Plant J, 2000

The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. ... more The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between as-RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is posttranscriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T-DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the ®rst class contains a single copy (S) T-DNA of which the as-Chs gene is transcribed, although several-fold lower than the endogenous Chs genes. As there are not suf®cient as-RNAs to degrade every mRNA, we speculate that silencing is induced by double-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as-Chs transgenes were proximal to the centre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.

Paramutation is the heritable transfer of epigenetic information from one allele of a gene to ano... more Paramutation is the heritable transfer of epigenetic information from one allele of a gene to another allele of the same gene. In general, the consequence of this trans-communication is a change in gene expression. Paramutation has been observed in plants, fungi and mammals, but is most extensively studied in maize thanks to the long-standing history of maize genetics. For decades,

ChIP-seq is a powerful and innovative technique to study protein-DNA interaction in living organi... more ChIP-seq is a powerful and innovative technique to study protein-DNA interaction in living organisms. ChIP is more commonly used in Human and animal tissues comparing to plants. Diagenode has deve- loped a reliable ChIP-seq protocol for plants: “The Plant ChIP-seq kit”. This kit was optimized for the model plant Arabidopsis thaliana . As plant tissues can vary greatly between families and species, new ChIP protocols need to be optimized for several model plant tissues. This work constitutes the first step of the optimization process of a commercial protocol valid for monocots by Diagenode, represented by the EpiTRAITS fellow Wassim Lakhal. It was realized in the University of Amsterdam (UvA), Swammer- dam Institute for Life Sciences, Plant Development & Epigenetics group that have expertise in ChIP on maize, within EpiTRAITS project.

Epigenetic gene regulation confers stability of gene expression patterns through cell divisions w... more Epigenetic gene regulation confers stability of gene expression patterns through cell divisions while allowing changes in expression in response to environmental or developmental cues. Changes in epigenetic gene regulation are a major cause for trait variation in crops. EpiTRAITS European project focuses on one of the key plant traits, flowering, which is controlled by various epigenetic mechanisms. The scientific program aims to bridge the gap between fundamental and applied research by translating results from epigenetic research in model organisms to improved technologies for crop breeding and molecular diagnostic tools. Diagenode, as a leading company specialised in Epigenetics and Human molecular diagnostics, is the only company providing a complete solution for epigenetics research, including state-of-the-art products and technologies for DNA sonication, best-in-class antibodies, and high-quality kits for chromatin immunoprecipitation followed by High throughput sequencing (Ch...