Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 ß: identification of miR-153 target genes with functions related to IA-2ß in pancreas and brain.

AIMS/HYPOTHESIS: We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2ß. We also identified miR-153 target genes that correlated with IA-2ß localisation and function. METHODS: A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2ß, quantitative PCR analysis of miR-153 and Ia-2ß (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2ß single knockout and Ia-2/Ia-2ß double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. RESULTS: Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2ß, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2ß in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2ß knockout and Ia-2/Ia-2ß double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2ß expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. CONCLUSIONS/INTERPRETATION: This study suggests the involvement of miR-153, IA-2ß and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.

A distal Schwann cell-specific enhancer mediates axonal regulation of the Oct-6 transcription factor during peripheral nerve development and regeneration.

The POU domain transcription factor Oct-6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct-6 is under the control of axonal signals. Identification of the cis-acting elements necessary for Oct-6 gene regulation is an important step in deciphering the complex signalling between Schwann cells and axons governing myelination. Here we show that a fragment distal to the Oct-6 gene, containing two DNase I-hypersensitive sites, acts as the Oct-6 Schwann cell-specific enhancer (SCE). The SCE is sufficient to drive spatially and temporally correct expression, during both normal peripheral nerve development and regeneration. We further demonstrate that a tagged version of Oct-6, driven by the SCE, rescues the peripheral nerve phenotype of Oct-6-deficient mice. Thus, our isolation and characterization of the Oct-6 SCE provides the first description of a cis-acting genetic element that responds to converging signalling pathways to drive myelination in the peripheral nervous system.

Transcriptional regulation of the POU gene Oct-6 in Schwann cells.

Genetic evidence suggests that the POU transcription factor Oct-6 plays a pivotal role as an intracellular regulator of Schwann cell differentiation. In the absence of Oct-6 function Schwann cells are generated in appropriate numbers and these cells differentiate normally up to the promyelin stage at which they transiently arrest. During peripheral nerve development Oct-6 expression is initiated in Schwann cell precursors and is strongly upregulated in promyelin cells. Oct-6 expression is subsequently extinguished in terminally differentiating Schwann cells. Thus, identification and characterisation of the DNA elements involved in this stage specific regulation may lead us to the signaling cascade and the axon-derived signals that drive Schwann cell differentiation and initiate myelination. Here we present experiments that aim at identifying such regulatory sequences.

Comparison of sequence and function of the Oct-6 genes in zebrafish, chicken and mouse.

To examine the role of the Oct-6 gene in Schwann cell differentiation we have cloned and characterized the chicken and zebrafish homologues of the mouse Oct-6 gene. While highly homologous in the Pit1-Oct1/2-Unc86 (POU) domain, sequence similarities are limited outside this domain. Both genes are intronless and both proteins lack the amino acid repeats that are a characteristic feature of the mammalian Oct-6 proteins. However as in mammals, the aminoterminal parts of the chicken and zebrafish Oct-6 proteins are essential for transactivation of octamer containing promoters. By immunohistochemistry we have found that the chicken Oct-6 protein is expressed in late embryonic ensheathing Schwann cells of the sciatic nerve and is rapidly downregulated when myelination proceeds. This expression profile in glial cells is identical to that in the mouse and rat. Furthermore the zebrafish Oct-6 homolog is expressed in the posterior lateral nerve at a time when it contains actively myelinating Schwann cells. Thus despite extensive primary sequence divergence among the vertebrate Oct-6 proteins, the expression of the chicken and zebrafish Oct-6 proteins is consistent with the notion that Oct-6 functions as a 'competence factor' in promyelin cells to execute the myelination program.

The POU factor Oct-6 and Schwann cell differentiation.

The POU transcription factor Oct-6, also known as SCIP or Tst-1, has been implicated as a major transcriptional regulator in Schwann cell differentiation. Microscopic and immunochemical analysis of sciatic nerves of Oct-6(-/-) mice at different stages of postnatal development reveals a delay in Schwann cell differentiation, with a transient arrest at the promyelination stage. Thus, Oct-6 appears to be required for the transition of promyelin cells to myelinating cells. Once these cells progress past this point, Oct-6 is no longer required, and myelination occurs normally.