Purkinje Cell Protein Research Articles

Beta receptor isoforms are not essential for thyroid hormone-dependent acceleration of PCP-2 and myelin basic protein gene expression in the developing brains of neonatal mice

In rat pups, thyroid hormone dependent brain development coincides with the appearance of the thyroid hormone receptor (TR) β1 isoform. This finding led to the suggestion that TR β1 plays an essential role in brain development. The recent availability of a mouse TR β knockout strain allowed us to test this possibility by determining whether TR β is essential for the normal developmental pattern of expression of two thyroid hormone regulated brain genes, myelin basic protein (MBP), and Purkinje cell protein 2 (Pcp-2). Northern analysis of total mRNA from the brains of wild-type mice established that, as in the rat pup, the initial rate of rise of the MBP and Pcp-2 mRNA is slowed in the hypothyroid state. Supporting the effectiveness of TR β gene deletion was the finding that the thiiodothyronine (T3) nuclear binding capacity in the livers and brains of knockout animals was consistent with the fractional contribution of TR β1 to total binding capacity in the wild-type tissues. Further, no TR β1 could be detected by isoform-specific immunoprecipitation of nuclear receptor extracts. However, deletion of the functional TR β in the TR β knockout mice did not affect the normal ontogeny of expression of the Pcp-2 and MBP genes in the postnatal pup. We conclude that TR β is not essential for the normal developmental expression of these T3 dependent brain genes.

Purkinje cell protein-2 cis-elements mediate repression of T3-dependent transcriptional activation

Previous studies in our laboratory show that triiodothyronine upregulates expression of the cerebellar Purkinje cell-specific gene Pcp-2 during the first 2 weeks of rat neonatal life. A specific thyroid hormone response element, the A1 TRE, mediates this regulation. The finding that the contiguous 68 bases (−267/−199) of the Pcp-2 promoter 3′ to the A1 TRE repressed T3 response in transactivation studies suggested that this sequence could play a role in preventing premature T3-dependent activation of Pcp-2 in the fetus. We now show that deletion of this region resulted in enhanced T3-dependent activation of the native Pcp-2 promoter. The sequence is not a generalized silencer since it does not alter basal activity of mouse mammary tumor virus (MMTV) or thymidine kinase (TK) promoters. Deletion and linker scanning studies indicate that the 5′ 30 bases of the −267/−199 region mediate most of the response silencing activity. The −267/−199 region also attenuates T3-induced transactivation mediated by other TREs. Gel shift analysis reveals that nuclear proteins from fetal but not adult brains complex with the −267/−199 region, supporting the hypothesis that this region binds proteins that suppress Pcp-2 expression early in brain development.

Transcriptional regulation of a Purkinje cell-specific gene through a functional interaction between ROR alpha and RAR.

The orphan nuclear receptor ROR alpha is highly expressed in the Purkinje cells of the cerebellum during the postnatal development of brain. A recent observation has been made that the ROR alpha gene is disrupted in staggerer mice-which show a cell-autonomous defect in the development of the Purkinje cells. In order to understand the functions of ROR alpha in cerebellar development, I attempted to identify its target genes. Transient expression study demonstrated that transcription of the Purkinje cell protein-2 (Pcp-2) gene is activated by ROR alpha, which binds as a monomer to a single half-site motif (RORE) within the promoter region. Its transcription was also activated by retinoic acid receptor (RAR) which binds as a heterodimer with RXR to a retinoic acid responsive element (RARE) in the downstream region. Interestingly, the ROR alpha-mediated transcription is further activated synergistically by RAR. That the Pcp-2 gene is a target of ROR alpha, and is suggested that its transcription is also regulated by RAR.

A Purkinje cell protein-2 intronic thyroid hormone response element binds developmentally regulated thyroid hormone receptor-nuclear protein complexes.

Two thyroid hormone response elements (TREs), designated A1 TRE (-295/-268) and B1 TRE (+207/+227), have been identified within the Purkinje cell-expressed Pcp-2 gene. Previous studies have characterized the A1 TRE (Zou et al., 1994). This article analyzes the structural and functional characteristics of the intronic B1 TRE. The B1 sequence contains four overlapping TRE half-sites. The 3' DR4 motif, consisting of the second and forth half-sites, is responsible for the T3 induction observed with the B1 sequence. Gel-shift analysis reveals developmentally regulated complexes that are abundant in the fetus and at birth and then fall precipitously in the neonate bind to B1. The observed time-course of these complexes varies inversely with the rise in Pcp-2 expression, thus raising the possibility that the complexes may represent inhibitory factors. Supershift analysis indicates that endogenous TR alpha 1 is present in the fetal nuclear protein complexes that bind to B1. Competition analysis also indicates the second B1 TRE half-site is important in binding the TR alpha 1-TRAP complexes. These studies suggest that the B1 sequence may bind potential TR alpha 1-TRAP repressor complexes in the fetus, whereas in the neonate, these TRE sites may be involved in the activation of Pcp-2 by binding other TR-TRAP-activating complexes.

Identification of Natural Monomeric Response Elements of the Nuclear Receptor RZR/ROR. THEY ALSO BIND COUP-TF HOMODIMERS

The receptor RZR/ROR is an important member of the nuclear receptor superfamily and has recently been shown to be the nuclear receptor for the pineal gland hormone melatonin. RZR/ROR binds as a monomer to DNA, and the human 5-lipoxygenase gene has been identified as the first RZR/ROR/melatonin-responding gene. Another prominent nuclear receptor is COUP-TF, which binds as a dimer to DNA. In this study, the sequences of known promoter regions of genes that may be involved in the physiological action of melatonin have been screened for putative monomeric RZR/ROR response elements. The binding of RZR/ROR and COUP-TF was compared and quantified on a set of 12 putative response elements. Interestingly, COUP-TF homodimers were found to bind with high affinity to some of the monomeric RZR/ROR response elements. Four RZR/ROR response elements, found in the genes of the mouse bifunctional enzyme, the rat bone sialoprotein, mouse Purkinje cell protein 2, and human p21(WAF1/CIP1), were shown to be inducible by melatonin under conditions of low constitutive activity. Surprisingly, the constitutive activity of COUP-TF was also stimulated by an unknown serum compound. The novel Purkinje cell protein 2 and p21(WAF1/CIP1) RZR/ROR/melatonin-responding genes may be the key for understanding the role of RZR/RORalpha in the mouse mutation staggerer and the antiproliferative action of melatonin, respectively.

Purkinje cell protein-2 regulatory regions and transgene expression in cerebellar compartments.

The Purkinje cell protein 2 (Pcp-2) is expressed in cerebellar Purkinje cells and retinal bipolar neurons. To illuminate how Pcp-2 expression is restricted to only two neuronal types and to derive tools to express heterologous genes in these neuronal subpopulations, genomic sequences of the mouse Pcp-2 gene have been cloned and flanking sequences have been evaluated as a source of neuron-specific regulatory elements. An upstream region with homology to other genes expressed in neurons was identified and a hybrid gene containing this sequence was constructed by ligating 0.4 kb of upstream and 0.3 kb of downstream Pcp-2-flanking DNA to lacZ. Transgenic mice bearing this construct exhibited beta-galactosidase in a wide array of neuron types, suggesting that this sequence may play an important role in specifying neuronal expression. Addition of a further 3.1 kb of Pcp-2 upstream sequences restricted expression of beta-galactosidase to a small number of neuron types and most notably to Purkinje cells within parasagitally oriented cerebellar compartments. The presence of elements lying within the 3.1-kb upstream region and acting to specifically restrict Pcp-2 expression is therefore suggested. Moreover, as beta-galactosidase was not expressed in the bipolar cells of these transgenic mice, retinal expression of the endogenous Pcp-2 gene must involve elements in addition to those conferring expression within Purkinje cells.