generating-transgenic-plants-with-single-copy-insertions-using-bibac

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

University of Amsterdam

There is rapidly growing evidence that folding of the chromatin fibre inside the interphase nucleus has an important role in the regulation of gene expression. In particular, the formation of loops mediated by the interaction between specific regulatory elements, for instance enhancers and promoters, is crucial in gene control. Biochemical studies that were based on the chromosome conformation capture (3C) technology have confirmed that eukaryotic genomes are highly looped. Insight into the underlying principles comes from polymer models that explore the properties of the chromatin fibre inside the nucleus. Recent models indicate that chromatin looping can explain various properties of interphase chromatin, including chromatin compaction and compartmentalisation of chromosomes. Entropic effects have a key role in these models. In this Commentary, we give an overview of the recent conjunction of ideas regarding chromatin looping in the fields of biology and polymer physics. Starting from simple linear polymer models, we explain how specific folding properties emerge upon introducing loops and how this explains a variety of experimental observations. We also discuss different polymer models that describe chromatin folding and compare them to experimental data. Experimentally testing the predictions of such polymer models and their subsequent improvement on the basis of measurements provides a solid framework to begin to understand how our genome is folded and how folding relates to function.

Chromatin folding--from biology to polymer models and back.

Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type-specific transcription. We know remarkably little about the principles that control chromosome folding. Here we explore the view that intrachromosomal interactions, forming a complex pattern of loops, are a key element in chromosome folding. CTCF and cohesin are two abundant looping proteins of interphase chromosomes of higher eukaryotes. To investigate the role of looping in large-scale (supra Mb) folding of human chromosomes, we knocked down the gene that codes for CTCF and the one coding for Rad21, an essential subunit of cohesin. We measured the effect on chromosome folding using systematic 3D fluorescent in situ hybridization (FISH). Results show that chromatin becomes more compact after reducing the concentration of these two looping proteins. The molecular basis for this counter-intuitive behaviour is explored by polymer modelling usingy the Dynamic Loop model (Bohn M, Heermann DW (2010) Diffusion-driven looping provides a consistent framework for chromatin organization. PLoS ONE 5: e12218.). We show that compaction can be explained by selectively decreasing the number of short-range loops, leaving long-range looping unchanged. In support of this model prediction it has recently been shown by others that CTCF and cohesin indeed are responsible primarily for short-range looping. Our results suggest that the local and the overall changes in of chromosome structure are controlled by a delicate balance between short-range and long-range loops, allowing easy switching between, for instance, open and more compact chromatin states.

Depletion of the chromatin looping proteins CTCF and cohesin causes chromatin compaction: insight into chromatin folding by polymer modelling.

Bacterial genomes are functionally organized and compactly folded into a structure referred to as bacterial chromatin or the nucleoid. An important role in genome folding is attributed to Nucleoid-Associated Proteins, also referred to as bacterial chromatin proteins. Although a lot of molecular insight in the mechanisms of operation of these proteins has been generated in the test tube, knowledge on genome organization in the cellular context is still lagging behind severely. Here, we discuss important advances in the understanding of three-dimensional genome organization due to the application of Chromosome Conformation Capture and super-resolution microscopy techniques. We focus on bacterial chromatin proteins whose proposed role in genome organization is supported by these approaches. Moreover, we discuss recent insights into the interrelationship between genome organization and genome activity/stability in bacteria.

Bacterial chromatin: converging views at different scales.

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.

BIBAC-GW-based vectors for generating reporter lines for site-specific genome editing in planta.

The epigenome is a heritable layer of information not encoded in the DNA sequence of the genome, but in chemical modifications of DNA or histones. These chemical modifications, together with transcription factors, operate as spatiotemporal regulators of genome activity. Dissecting epigenome function requires controlled site-specific alteration of epigenetic information. Such control can be obtained using designed DNA-binding platforms associated with effector domains to function as targeted transcription factors or epigenetic modifiers. Here, we review the use of dCas9 as a novel and versatile tool for fundamental studies on epigenetic landscapes, chromatin structure and transcription regulation, and the potential of this approach in basic research in these fields.

Article	Year
Chromatin folding--from biology to polymer models and back. Journal of cell science\| PubMed ID: 21378305	2011
Depletion of the chromatin looping proteins CTCF and cohesin causes chromatin compaction: insight into chromatin folding by polymer modelling. PLoS computational biology\| PubMed ID: 25299688	2014
Bacterial chromatin: converging views at different scales. Current opinion in cell biology\| PubMed ID: 26942688	2016
BIBAC-GW-based vectors for generating reporter lines for site-specific genome editing in planta. Plasmid\| PubMed ID: 28034789	2017
dCas9: A Versatile Tool for Epigenome Editing. Current issues in molecular biology\| PubMed ID: 28879853	2018

Mariliis Tark-Dame

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