Suppression of MeCP2beta expression inhibits neurite extension in PC12 cells.

Regulation of gene expression is critical to the proper development of neuronal cells. The methyl-CpG binding protein 2 (MeCP2) operates as a transcriptional repressor by facilitating histone deacetylation and DNA methylation-dependent transcriptional silencing. This study examined the importance of MeCP2 in the regulation of neurite formation in PC12 cells. Expression of MeCP2 increased in a time-dependent manner after induction of neuronal differentiation. Expression was assessed at both the transcriptional and translation levels, and reached a maximum at 24 h post-induction. In addition, a marked inhibition of neurite extension and proper localization of a marker for synapse formation, synapsin I, were observed when MeCP2 expression was decreased by the addition of an antisense morpholino oligomer directed to the translational initiation site for MeCP2beta. The removal of the antisense oligomer allowed neurite extension to progress. However, the addition of antisense oligomer to previously differentiated PC12 cells did not affect established neurite processes. Taken collectively, our results indicate a role for MeCP2beta early in the events of neurite formation and that the relative levels of MeCP2alpha and MeCP2beta may be different in early differentiating neurons than is found in the adult brain. In addition, unique functions may exist for the two isoforms of MeCP2. Our results indicate that the inhibition of neurite elaboration caused by a reduction in MeCP2 may be reversible.

BMP-1-mediated proteolytic processing of alternatively spliced isoforms of collagen type XI.

Collagen type XI is a minor constituent of heterotypic collagen fibrils of developing cartilage and plays a regulatory role in fibril diameter. Collagen type XI is a heterotrimer composed of the alpha1, alpha2 and alpha3 chains. The mRNA encoding exons 6a, 6b and 8 of the alpha1 chain are expressed alternatively to generate six possible isoforms. The 6b-containing isoform has the most restricted distribution of all isoforms. It is first localized in the developing long bone, where mineralized tissue initially forms, and is later restricted to regions of cartilage that will be subsequently converted into bone. Bone morphogenetic protein 1 (BMP-1) and related proteins cleave procollagens I-III, V and VII, yielding triple-helical molecules that associate into collagen fibrils. The present study demonstrates that the alpha1 chain of collagen type XI can serve as a substrate for BMP-1. In addition, the efficiency with which BMP-1 processes different isoforms of the alpha1 chain varies. The amino acid sequence adjacent to the processing site influences the rate and extent of processing, as do sequences further away. Smaller fragments identified from cartilage extracts indicated that processing by BMP-1, in combination with other processing enzymes, generates small fragments of p6b-containing isoforms.