Carolyn E. Fiskerstrand

An intronic polymorphic domain often associated with susceptibility to affective disorders has allele dependent differential enhancer activity in embryonic stem cells

Variable number tandem repeats (VNTR) within non‐coding regions of a number of genes have been correlated with susceptibility to various disease states. In particular, a VNTR polymorphism of a 16 or 17 bp element within intron 2 of the human serotonin transporter gene has been correlated with a predisposition to affective disorders. We have demonstrated that this region will support differential levels of reporter gene expression in differentiating embryonic stem cells, this being dependent on the presence of 10 or 12 copies of the repeat. The VNTR domain can therefore act as a transcriptional regulator, a property which potentially contributes to disease susceptibility.

The dopamine transporter gene (SLC6A3) variable number of tandem repeats domain enhances transcription in dopamine neurons.

The dopamine (DAT) and serotonin (SERT) transporter genes both contain variable number of tandem repeats (VNTR) in non‐coding gene regions which have been correlated with a predisposition to a variety of CNS disorders. There is considerable homology between individual DAT and SERT repeat DNA sequences, which is reflected in their ability to compete with each other for specific protein binding as demonstrated by electrophoretic mobility shift assay. The SERT VNTR has recently been shown to act as a transcriptional enhancer. Because of the similarities between SERT and DAT VNTRs, the DAT VNTR may also enhance transcription. This study demonstrates by lipid transfection into an immortalized dopaminergic cell line and biolistic transfection into dopamine neurons in neonatal rat midbrain slices that the human nine‐repeat DAT VNTR can enhance transcription. This enhancing activity suggests that the DAT VNTR may play a role in regulation of DAT gene expression.

The serotonin transporter intronic VNTR enhancer correlated with a predisposition to affective disorders has distinct regulatory elements within the domain based on the primary DNA sequence of the repeat unit

We have demonstrated that a variable number tandem repeat domain (VNTR) within intron 2 of the serotonin transporter gene is a transcriptional regulatory domain which is potentially correlated with a predisposition to affective disorders and other behavioural conditions. This correlation based on copy number of the VNTR alone (nine, 10 or 12 copies of 16/17 base‐pair element) has been controversial and not reproduced in all studies. We demonstrate that individual repeat elements within the VNTR domain differ in their enhancer activity in an embryonic stem cell model. This has implications for both the mechanism by which these VNTRs are correlated with the progression of the disease and suggests that clinical analysis should now be extended to correlate sequence variation within the VNTR with the disorder. The latter may resolve some of the conflicting data published to date.

Behavioural changes in the rat following infection with varicella-zoster virus

Following the establishment of a chronic varicella-zoster virus infection in the rat, behavioural allodynia and hyperalgesia were observed in the injected, but not the contralateral hind limb up to 33 days post-infection. This model may prove useful in investigating mechanisms involved in the establishment of post-herpetic neuralgia.

The molecular biology of preprotachykinin-A gene expression.

The expression of neuropeptides is largely tissue-specific and under strictly regulated and complex control. In view of the diversity of neuronal phenotypes, with concomitant plasticity of gene expression within any phenotype, it is obvious that there is coordinated activation and repression of genes. One of the central observations from these studies is that neuropeptide gene expression is dependent upon the combinatorial interaction of multiple transcription factors with the regulatory elements which determine mRNA synthesis. These factors mediate both tissue specific and stimulus inducible gene expression. We will illustrate some of the mechanisms that regulate neuropeptide gene expression utilizing our own studies on the rat preprotachykinin-A gene (rPPT) and, where appropriate, expand on the generality of these findings to other neuropeptide genes.

Molecular models to analyse preprotachykinin-A expression and function

Towards an understanding of the mechanisms controlling Preprotachykinin A (PPT) expression we have generated a variety of molecular models to determine the mechanisms regulating both the tissue-specific and stimulus-inducible expression of the PPT gene. The approaches used include transgenic and virus vector models complementing biochemical analysis of promoter interactions with transcription factors. We have identified and characterised a yeast artificial chromosome (YAC) containing the human PPT gene and generated transgenic mouse lines containing multiple copies of this chromosome on a normal mouse genetic background. This resulted in a pattern of expression in the nervous system remarkably similar to that reported for PPT mRNA in rodents. In addition, this transgenic model has been constructed in such a manner to allow for over expression of tachykinins based on the number of extra alleles in the transgenic mouse. These animals allow us to further examine the function of the tachykinins and acts as a useful complement to existing PPT ablated mice. In vitro we have introduced the proximal PPT promoter in reporter gene constructs into adult neurones in both DRG and the CNS by an adenoassociated virus (AAV) vector or by biolistic transfection respectively. Using the AAV vector we have demonstrated that the proximal promoter can mediate the effects of NGF in adult rat DRG. These models allow us to delineate transcriptional domains involved in the physiological and pathological expression of the PPT gene.

An intronic domain within the rat preprotachykinin-A gene containing a CCCT repetitive motif acts as an enhancer in differentiating embryonic stem cells

Previous attempts by several groups to clone fragments containing intron 2 of the rat preprotachykinin-A gene have generated deletions of various sizes. We have determined that these deletions occur within a specific region of the intron spanning a CCCT tandem repeat domain. We show that this intronic domain is able to support reporter gene expression in mouse embryonic stem (ES) cells that have been induced to differentiate but not in undifferentiated ES cells. No significant expression was observed in the HeLa clonal cell line. This demonstrates that this intron 2 domain is a highly restrictive enhancer and may be associated with differentiation.

Identification of a novel multifunctional structural domain in the herpes simplex virus type 1 genome: implications for virus latency.

A domain, previously termed RE1, exists within the herpes simplex virus type 1 genome potentially influencing expression of immediate early genes and the latency associated transcripts. This domain consists of 10 tandem copies of a CT-rich sequence. We demonstrate that this domain binds multiple host-cell factors that may allow RE1 to act either as a transcriptional regulator and/or to affect nucleosomal and DNA structure in the latent genome.

A role for the octamer-binding protein in preprotachykinin-a gene expression

A rat Preprotachykinin-A promoter fragment has been previously identified which supports reporter gene activity in primary cultures of adult dorsal root ganglion neurons. That study demonstrated that two promoter domains which exhibit enhancer activity in these neurons are bound by the same classes of transcription factors. Further, the two domains exhibit similarities with respect to the relationship of bound transcription factors within each domain. This suggests that these domains may function in an identical manner or may act synergistically to regulate gene expression. These domains contain recognition motifs for at least three classes of transcription factors: octamer-binding proteins, Sp1-related proteins and an as yet unidentified but distinct factor. The definition of an octamer-binding protein site within these domains is of interest, as this class of factor has recently been suggested as mediating the effect of nerve growth factor in sensory neurons. Nerve growth factor is a well-characterized regulator of preprotachykinin-A gene expression. Definition of these sites within the promoter allows for the design of rational experiments to address the mechanism of transcriptional regulation of the rat preprotachykinin-A gene.

A regulatory domain spanning the repeat sequence RE1 from herpes simplex virus type 1 has cell specific differential functions in trigeminal neurons and fibroblasts

In this report we demonstrate that the herpes simplex virus type 1 reiteration element 1 (RE1) (nt: 117158–117353) in concert with its flanking sequences is both a cell specific and stimulus inducible regulatory domain. This region of the virus genome and specifically the RE1 supports differential reporter gene expression in both baby hamster kidney cells and disassociated rat trigeminal ganglia and is present within a region that is implicated in regulating latency of the virus in neuronal cells. Further we demonstrate that this locus is a transcriptional regulatory domain and a target for the transcription factor CCCTC binding protein.