Super-resolution re-scan second harmonic generation microscopy.

Second harmonic generation microscopy (SHG) is generally acknowledged as a powerful tool for the label-free three-dimensional visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Despite the great need, progress in super-resolved SHG imaging lags behind the developments reported over the past years in fluorescence-based optical nanoscopy. In this work, we demonstrate super-resolved re-scan SHG, qualitatively and quantitatively showing on collagenous tissues the available resolution advantage over the diffraction limit. We introduce as well super-resolved re-scan two-photon excited fluorescence microscopy, an imaging modality not explored to date.

dCas9: A Versatile Tool for Epigenome Editing.

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.

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

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.

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

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.

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

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.

A physical approach to segregation and folding of the Caulobacter crescentus genome.

Bacterial genomes are functionally organized. This organization is dynamic and globally changing throughout the cell cycle. Upon initiation of replication of the chromosome, the two origins segregate and move towards their new location taking along the newly replicated genome. Caulobacter crescentus employs a dedicated active partitioning (Par) system to move one copy of the parS centromere to the distal pole, while the other stays at the stalked pole. In this issue of Molecular Microbiology, Hong and McAdams describe studies on the speed of segregation of parS and regions up to 150 kb away. They show clear differences in segregation rates between parS and 50 kb flanking regions versus regions further away. To assess segregation rates the authors track fluorescent markers during movement using time-lapse microscopy. The relation between genomic and physical distance of pairs of markers reflects how the genome is folded. This relation permits testing experimental data against models from polymer physics. Such models are helpful in understanding principles of genome folding. Although long used in studies on eukaryotes, this approach has rarely been applied to bacteria. Finally, the authors give the first direct evidence for a role of the bacterial chromatin protein HU in folding the genome in vivo.

Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target.

Homologous recombination (HR) has a major impact in bacterial evolution. Most of the knowledge about the mechanisms and control of HR in bacteria has been obtained in fast growing bacteria. However, in their natural environment bacteria frequently meet adverse conditions which restrict the growth of cells. We have constructed a test system to investigate HR between a plasmid and a chromosome in carbon-starved populations of the soil bacterium Pseudomonas putida restoring the expression of phenol monooxygenase gene pheA. Our results show that prolonged starvation of P. putida in the presence of phenol stimulates HR. The emergence of recombinants on selective plates containing phenol as an only carbon source for the growth of recombinants is facilitated by reactive oxygen species and suppressed by DNA mismatch repair enzymes. Importantly, the chromosomal location of the HR target influences the frequency and dynamics of HR events. In silico analysis of binding sites of nucleoid-associated proteins (NAPs) revealed that chromosomal DNA regions which flank the test system in bacteria exhibiting a lower HR frequency are enriched in binding sites for a subset of NAPs compared to those which express a higher frequency of HR. We hypothesize that the binding of these proteins imposes differences in local structural organization of the genome that could affect the accessibility of the chromosomal DNA to HR processes and thereby the frequency of HR.

Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida.

The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif(r) mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

3D genome organization: a role for phase separation and loop extrusion?

In eukaryotes, genomic information is encoded in chromosomes, which occupy distinct territories within the nucleus. Inside these territories, chromosomes are folded in a hierarchical set of topological structures, called compartments, topologically associated domains and loops. Phase separation and loop extrusion are the mechanisms indicated to mediate the 3D organization of the genome, and gene activity and epigenetic marks determine the activity level of the formed chromatin domains. The main difference between plants and animals may be the absence of canonical insulator elements in plants. Comparison across plant species indicates that the identification of chromatin domains is affected by genome size, gene density, and the linear distribution of genes and transposable elements.

Visualization and Quantification of Cell-to-cell Movement of Proteins in Nicotiana benthamiana.

Cell-to-cell movement of proteins through plasmodesmata is a widely-established mechanism for intercellular signaling in plants. Current techniques to study intercellular protein translocation rely on single-cell transformation using particle bombardment or transgenic lines expressing photo-inducible fluorophores. The method presented here allows visualization and objective quantification of (effector) protein movement between N. benthamiana leaf cells. Agroinfiltration is performed using a single binary vector encoding a GFP-tagged protein of interest that is either mobile or non-mobile (MP; non-MP), together with an ER-anchored mCherry. Upon creation of mosaic-like transformation patterns, cell-to-cell movement of the MP can be followed by monitoring translocation of the GFP signal from mCherry labeled transformed cells into neighboring non-transformed cells. This process can be visualized using confocal microscopy and quantified following protoplast isolation and flow cytometric cell analysis. This method overcomes the limitations of existing methods as it allows rapid and objective quantification of protein translocation without the need of creating transgenic plants.

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 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 bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Bacterial chromatin: converging views at different scales.

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.

The visualization of large organized chromatin domains enriched in the H3K9me2 mark within a single chromosome in a single cell.

Despite considerable efforts, our understanding of the organization of higher order chromatin conformations in single cells and how these relate to chromatin marks remains poor. We have earlier invented the Chromatin In Situ Proximity (ChrISP) technique to determine proximities between chromatin fibers within a single chromosome. Here we used ChrISP to identify chromosome 11-specific hubs that are enriched in the H3K9me2 mark and that project toward the nuclear membrane in finger-like structures. Conversely, chromosome 11-specfic chromatin hubs, visualized by the presence of either H3K9me1 or H3K9me3 marks, are chromosome-wide and largely absent at the nuclear periphery. As the nuclear periphery-specific chromatin hubs were lost in the induced reduction of H3K9me2 levels, they likely represent Large Organization Chromatin in Lysine Methylation (LOCK) domains, previously identified by ChIP-seq analysis. Strikingly, the downregulation of the H3K9me2/3 marks also led to the chromosome-wide compaction of chromosome 11, suggesting a pleiotropic function of these features not recognized before. The ChrISP-mediated visualization of dynamic chromatin states in single cells thus provides an analysis of chromatin structures with a resolution far exceeding that of any other light microscopic technique.