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.

Geometric cues for directing the differentiation of mesenchymal stem cells.

Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature-and with each occupying the same area-display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.

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.

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9.

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.