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.

Involvement of N-cadherin in the protective effect of glial cell line-derived neurotrophic factor on dopaminergic neuron damage.

The aim of this study was to further confirm that glial cell line-derived neurotrophic factor (GDNF) exerts a neuro-protective effect on dopaminergic neurons (DAs) and to investigate the protective mechanism. Cadherins are calcium-dependent adhesion proteins, and N-cadherins are found in neurons. Our study attempted to ascertain whether GDNF activates the PI3K/Akt signaling pathway through the mediation of N-cadherin to confer a protective effect on DAs. Flow cytometry and Hoechst 33258 staining results indicated that the apoptosis rate of damaged neurocytes increased following interference of N-cadherin expression. Immunoblotting results demonstrated that the amount of phosphorylated Akt (p-Akt) in the cytoplasm decreased, while the total Akt quantity remained unchanged following interference of N-cadherin expression. The immunohistochemical staining results demonstrated that the levels of total N-cadherin, phosphorylated N-cadherin (Tyr860) and p-Akt decreased; however, the amount of total Akt remained unchanged. In addition, we also demonstrated that Tyr860 and p-Akt levels were reduced in a GDNF dose-dependent manner with the phosphorylation level peaking at GDNF dose of 50 ng/ml (in vitro) and 50 ng/4 µl (in vivo), and also in a time-dependent manner with the phosphorylation level peaking at 15 min (in vitro) and 30 min (in vivo). Statistical analysis also showed that changes in the phosphorylation levels of Tyr860 and p-Akt demonstrated a positive correlation. Collectively, GDNF activates the PI3K/Akt pathway via N-cadherin to protect DAs.

Systematic comparison of constitutive promoters and the doxycycline-inducible promoter.

Constitutive promoters are used routinely to drive ectopic gene expression. Here, we carried out a systematic comparison of eight commonly used constitutive promoters (SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems, and COPIA and ACT5C for Drosophila systems). We also included in the comparison the TRE promoter, which can be activated by the rtTA transcriptional activator in a doxycycline-inducible manner. To make our findings representative, we conducted the comparison in a variety of cell types derived from several species. We found that these promoters vary considerably from one another in their strength. Most promoters have fairly consistent strengths across different cell types, but the CMV promoter can vary considerably from cell type to cell type. At maximal induction, the TRE promoter is comparable to a strong constitutive promoter. These results should facilitate more rational choices of promoters in ectopic gene expression studies.