Embryonic stem cells induce pluripotency in somatic cell fusion through biphasic reprogramming.

It is a long-held paradigm that cell fusion reprograms gene expression but the extent of reprogramming and whether it is affected by the cell types employed remain unknown. We recently showed that the silencing of somatic genes is attributable to either trans-acting cellular environment or cis-acting chromatin context. Here, we examine how trans- versus cis-silenced genes in a somatic cell type behave in fusions to another somatic cell type or to embryonic stem cells (ESCs). We demonstrate that while reprogramming of trans-silenced somatic genes occurs in both cases, reprogramming of cis-silenced somatic genes occurs only in somatic-ESC fusions. Importantly, ESCs reprogram the somatic genome in two distinct phases: trans-reprogramming occurs rapidly, independent of DNA replication, whereas cis-reprogramming occurs with slow kinetics requiring DNA replication. We also show that pluripotency genes Oct4 and Nanog are cis-silenced in somatic cells. We conclude that cis-reprogramming capacity is a fundamental feature distinguishing ESCs from somatic cells.

Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells.

Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed "cis-silenced" (or "occluded") genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease.

Architectural dynamics of the meiotic spindle revealed by single-fluorophore imaging.

Bipolarity of the meiotic spindle, required for proper chromosome segregation, is maintained throughout cell division despite rapid microtubule turnover. How this is achieved has remained mysterious, as determining the organization of individual spindle microtubules has been difficult. Here, we develop single-fluorophore speckle imaging to examine microtubule organization in the vertebrate meiotic spindle. We find that the mean length of microtubules is approximately 40% of spindle length. Long and short filaments distribute randomly throughout the spindle and those in close proximity can move in the same direction with highly heterogeneous velocities. The ratio between microtubule and spindle lengths remains unchanged as spindles elongate upon dynein-dynactin inhibition. However, maintaining this ratio depends on proper kinesin-5 function. Our data suggest that force transmission within the spindle must be understood in terms of the crosslinking dynamics of a tiled array of individual filaments, most of which do not span the distance from the pole to the metaphase plate.

Examining how the spatial organization of chromatin signals influences metaphase spindle assembly.

During cell division, the proper assembly of a microtubule-based bipolar spindle depends on signals from chromatin. However, it is unknown how the spatial organization of chromatin signals affects spindle morphology. Here, we use paramagnetic chromatin beads, and magnetic fields for their alignment in cell-free extracts, to examine the spatial components of signals that regulate spindle assembly. We find that for linear chromatin-bead arrays that vary by eightfold in length, metaphase spindle size and shape are constant. Our findings indicate that, although chromatin provides cues for microtubule formation, metaphase spindle organization, which is controlled by microtubule-based motors, is robust to changes in the shape of chromatin signals.

Probing the mechanical architecture of the vertebrate meiotic spindle.

Accurate chromosome segregation during meiosis depends on the assembly of a microtubule-based spindle of proper shape and size. Current models for spindle-size control focus on reaction diffusion-based chemical regulation and balance in activities of motor proteins. Although several molecular perturbations have been used to test these models, controlled mechanical perturbations have not been possible. Here we report a piezoresistive dual cantilever-based system to test models for spindle-size control and examine the mechanical features, such as deformability and stiffness, of the vertebrate meiotic spindle. We found that meiotic spindles prepared in Xenopus laevis egg extracts were viscoelastic and recovered their original shape in response to small compression. Larger compression resulted in plastic deformation, but the spindle adapted to this change, establishing a stable mechanical architecture at different sizes. The technique we describe here may also be useful for examining the micromechanics of other cellular organelles.

Chromatin analysis of occluded genes.

We recently described two opposing states of transcriptional competency. One is termed 'competent' whereby a gene is capable of responding to trans-acting transcription factors of the cell, such that it is active if appropriate transcriptional activators are present, though it can also be silent if activators are absent or repressors are present. The other is termed 'occluded' whereby a gene is silenced by cis-acting, chromatin-based mechanisms in a manner that blocks it from responding to trans-acting factors, such that it is silent even when activators are present in the cellular milieu. We proposed that gene occlusion is a mechanism by which differentiated cells stably maintain their phenotypic identities. Here, we describe chromatin analysis of occluded genes. We found that DNA methylation plays a causal role in maintaining occlusion for a subset of occluded genes. We further examined a variety of other chromatin marks typically associated with transcriptional silencing, including histone variants, covalent histone modifications and chromatin-associated proteins. Surprisingly, we found that although many of these marks are robustly linked to silent genes (which include both occluded genes and genes that are competent but silent), none is linked specifically to occluded genes. Although the observation does not rule out a possible causal role of these chromatin marks in occlusion, it does suggest that these marks might be secondary effect rather than primary cause of the silent state in many genes.

Systematic identification of cis-silenced genes by trans complementation.

A gene's transcriptional output is the combined product of two inputs: diffusible factors in the cellular milieu acting in trans, and chromatin state acting in cis. Here, we describe a strategy for dissecting the relative contribution of cis versus trans mechanisms to gene regulation. Referred to as trans complementation, it entails fusing two disparate cell types and searching for genes differentially expressed between the two genomes of fused cells. Any differential expression can be causally attributed to cis mechanisms because the two genomes of fused cells share a single homogenized milieu in trans. This assay uncovered a state of transcriptional competency that we termed 'occluded' whereby affected genes are silenced by cis-acting mechanisms in a manner that blocks them from responding to the trans-acting milieu of the cell. Importantly, occluded genes in a given cell type tend to include master triggers of alternative cell fates. Furthermore, the occluded state is maintained during cell division and is extraordinarily stable under a wide range of physiological conditions. These results support the model that the occlusion of lineage-inappropriate genes is a key mechanism of cell fate restriction. The identification of occluded genes by our assay provides a hitherto unavailable functional readout of chromatin state that is distinct from and complementary to gene expression status.

Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles.

During cell division metaphase spindles maintain constant length, whereas spindle microtubules continuously flux polewards, requiring addition of tubulin subunits at microtubule plus-ends, polewards translocation of the microtubule lattice, and removal of tubulin subunits from microtubule minus-ends near spindle poles. How these processes are coordinated is unknown. Here, we show that dynein/dynactin, a multi-subunit microtubule minus-end-directed motor complex, and NuMA, a microtubule cross-linker, regulate spindle length. Fluorescent speckle microscopy reveals that dynactin or NuMA inhibition suppresses microtubule disassembly at spindle poles without affecting polewards microtubule sliding. The observed uncoupling of these two components of flux indicates that microtubule depolymerization is not required for the microtubule transport associated with polewards flux. Inhibition of Kif2a, a KinI kinesin known to depolymerize microtubules in vitro, results in increased spindle microtubule length. We find that dynein/dynactin contribute to the targeting of Kif2a to spindle poles, suggesting a model in which dynein/dynactin regulate spindle length and coordinate flux by maintaining microtubule depolymerizing activities at spindle poles.

Par6alpha signaling controls glial-guided neuronal migration.

Neuronal migrations along glial fibers provide a primary pathway for the formation of cortical laminae. To examine the mechanisms underlying glial-guided migration, we analyzed the dynamics of cytoskeletal and signaling components in living neurons. Migration involves the coordinated two-stroke movement of a perinuclear tubulin 'cage' and the centrosome, with the centrosome moving forward before nuclear translocation. Overexpression of mPar6alpha disrupts the perinuclear tubulin cage, retargets PKCzeta and gamma-tubulin away from the centrosome, and inhibits centrosomal motion and neuronal migration. Thus, we propose that during neuronal migration the centrosome acts to coordinate cytoskeletal dynamics in response to mPar6alpha-mediated signaling.

Evidence for a critical role of gene occlusion in cell fate restriction.

The progressive restriction of cell fate during lineage differentiation is a poorly understood phenomenon despite its ubiquity in multicellular organisms. We recently used a cell fusion assay to define a mode of epigenetic silencing that we termed "occlusion", wherein affected genes are silenced by cis-acting chromatin mechanisms irrespective of whether trans-acting transcriptional activators are present. We hypothesized that occlusion of lineage-inappropriate genes could contribute to cell fate restriction. Here, we test this hypothesis by introducing bacterial artificial chromosomes (BACs), which are devoid of chromatin modifications necessary for occlusion, into mouse fibroblasts. We found that BAC transgenes corresponding to occluded endogenous genes are expressed in most cases, whereas BAC transgenes corresponding to silent but non-occluded endogenous genes are not expressed. This indicates that the cellular milieu in trans supports the expression of most occluded genes in fibroblasts, and that the silent state of these genes is solely the consequence of occlusion in cis. For the BAC corresponding to the occluded myogenic master regulator Myf5, expression of the Myf5 transgene on the BAC triggered fibroblasts to acquire a muscle-like phenotype. These results provide compelling evidence for a critical role of gene occlusion in cell fate restriction.

A zebrafish coxsackievirus and adenovirus receptor homologue interacts with coxsackie B virus and adenovirus.

In this study, a zebrafish homologue of the coxsackievirus and adenovirus receptor (CAR) protein was identified. Although the extracellular domain of zebrafish CAR (zCAR) is less than 50% identical to that of human CAR (hCAR), zCAR mediated infection of transfected cells by both adenovirus type 5 and coxsackievirus B3. CAR residues interacting deep within the coxsackievirus canyon are highly conserved in zCAR and hCAR, which is consistent with the idea that receptor contacts within the canyon are responsible for coxsackievirus attachment. In contrast, CAR residues contacting the south edge of the canyon are not conserved, suggesting that receptor interaction with the viral "puff region" is not essential for attachment.