Genomic Structure and Functional Characterisation of the Promoters of Human and Mouse nogo/rtn4

Abstract

The reticulon-family member Nogo-A is a potent neurite growth inhibitory protein in vitro and may play a role in the restriction of axonal regeneration after injury and of structural plasticity in the CNS of higher vertebrates. Of the three major isoforms of Nogo, Nogo-A is mostly expressed in the brain, Nogo-B is found in a ubiquitous pattern, and Nogo-C is most highly expressed in muscle. Seven additional splice-variants derived both from differential splicing and differential promoter usage have been identified. Analysis of the TATA-less Nogo-A/B promoter (P1) shows that conserved GC-boxes and a CCAAT-box within the first 500 bp upstream of the transcription start are responsible for its regulation. No major differences in the methylation status of the P1 CpG-island in tissues expressing or not expressing Nogo-A/B could be detected, suggesting that silencer elements are involved in the regulation. The specific expression pattern of Nogo-A/B is due to differential splicing. The basal Nogo-C promoter (P2) is regulated by a proximal and a distal element. The 5′UTR of Nogo-C harbours a negative control element. These data may help to identify factors that can modulate Nogo transcription, thus offering an alternative approach for Nogo neutralisation.

Introduction

Proteins that inhibit neurite growth appear to contribute in a major fashion to the lack of axonal regeneration after lesion in the adult central nervous system (CNS) of higher vertebrates. Neurite growth inhibitory factors were first demonstrated and are enriched in CNS white matter and myelin.¹ In culture, neurite growth cones collapse when they contact the surface of oligodendrocytes.2., 3. Major players in the inhibition process appear to include a high M_r protein called NI-250, the myelin-associated glycoprotein MAG and several proteoglycans of myelin.

Amino acid sequence determination of purified bNI-220⁴ followed by cDNA cloning identified a protein referred to as Nogo-A.5., 6., 7. Nogo is a novel member of the reticulon (RTN) family of proteins named after the main subcellular localisation of RTN-1 in the endoplasmic reticulum.8., 9. These proteins share a conserved 188 amino acid residues (aa) long C-terminal reticulon-homology domain (Pfam PF02453) with two large hydrophobic regions of unknown function. The N-terminal sequences share some physico-chemical properties but differ amongst the reticulon family members. Nogo-A colocalises not only with ER but also with Golgi markers (T.O. et al., unpublished results) and is present on oligodendrocyte plasma membranes (ibid.). Another reticulon member (RTN2-C) was shown to be associated with muscular Z-disks.¹⁰

Reticulon proteins occur in many tissues (especially RTN3 and Nogo-B/RTN4-B1), suggesting a basic function in cell physiology (T.O. et al., unpublished results). For Nogo-A, RTN1-A, RTN1-C and RTN2-A, highest expression levels are detected in nervous tissue.¹¹ Nogo-A is mainly found in oligodendrocytes, but particularly during development also in DRG and sympathetic ganglia, motor neurons, hippocampal pyramidal cells and Purkinje cells.¹¹ RTN2-C and Nogo-C/RTN4-C are particularly enriched in skeletal muscle.¹²

Knowledge about the regulation of reticulons at the mRNA and protein level is scarce. Mouse rtn1 transcription is upregulated by ethanol.¹³ In the rat, RTN1-A (rS-Rex-b) is induced by interleukin 1 (IL-1) in cultured sympathetic neurons.¹⁴ Acute thyroid hormone (TH) treatment suppresses RTN1-A in foetal and adult brain.¹⁵ Since ethanol interferes with TH signalling (percutaneous ethanol injections are clinically used to treat hyperthyroidism¹⁶), the ethanol and TH pathways could converge.

rtn1 is located on human chromosome 14q21→14q22;¹⁷ rtn2 on chromosome 19q13.3,¹⁸ and rtn3 on chromosome 11q13.¹⁹ A human pseudogene of rtn3 exists on chromosome 4¹⁹ (T.O. et al., unpublished results). nogo/rtn4 (KIAA0886) was mapped to chromosome 2p16.1→2p16.3²⁰ and to position 2p14→2p13 by radiation hybrid mapping.²¹

Here we determine and compare the genomic structure and organisation of the human and mouse nogo gene and its transcripts, including a functional analysis of regulatory elements in their promoters, and a comparison of their conserved non-coding sequences.

In order to understand the complex spatial and temporal expression pattern of the nogo transcripts and the respective changes followed after an injury, it is of outstanding interest to know the underlying molecular mechanisms regulating Nogo expression on a transcriptional and a translational level. Furthermore, the loss of regeneration capacity of axons in the adult CNS has been “acquired” relatively recently during higher vertebrate evolution. Close analysis of the gene structure of nogo might give insights into the evolutionary process responsible for the birth of one of these candidate components.