No abstract

To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (F 0) genes. PROG stimulated the P0 promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a FROG antagonist, did not abolish the effect of PIROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of FROG receptor, PROG did not stimulate the P0 and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by FROG of promoter activity of two peripheral myelin protein genes is Schwann-celI specific. Key Words: Progesterone-Schwann cell-P0 and peripheral myelin protein-22 gene promoters.

The gene encoding protein zero (P 0 ), the most abundant protein of peripheral nervous system myelin, is expressed uniquely in Schwann cells. Previous studies have demonstrated that much of the cell type specificity of this expression is due to transcriptional control elements in the 1.1-kilobase pair 5-regulatory region of the gene. We have now analyzed this region and have identified a set of functional elements in the 500 base pairs proximal to the transcription start site. DNA sequence conservation within the 5 regions of the human, mouse, and rat P 0 genes correlates closely with the results of promoter deletion analysis of the 1.1-kilobase pair region assayed in Schwann cell cultures and reveals a potent proximal region from position ؊350 to ؉45. Sites of protein/DNA interaction within the proximal 500 base pairs of the promoter were identified by footprinting assays. Functional transcriptional elements were identified within the protected regions in the proximal promoter by mutation and transient transfection analysis in P 0 -expressing cell lines. The core (or basal) P 0 promoter is identified as two regulatory elements, a G/Crich element that binds nuclear factor Sp1 and a CAAT box that binds NF-Y. These core elements are essential for the transcription observed from the transfected promoter in cultured Schwann cells.The myelin sheath is a specialized membranous organelle of the vertebrate nervous system. Elaborated by oligodendrocytes in the central nervous system and by Schwann cells in the peripheral nervous system (PNS), 1 this organelle consists of a large sheet of plasma membrane that is repeatedly wrapped and very tightly compacted around axons (1). Myelin is essential for the rapid conduction of action potentials in vertebrates, and human diseases that compromise the integrity of the sheath (e.g. peripheral neuropathies in the PNS and multiple sclerosis in the central nervous system) are generally debilitating (2). The elaboration of myelin requires a substantial biosynthetic up-regulation of plasma membrane lipids and proteins, several of which are unique to the myelin sheath (3). In the PNS, the most abundant of these is protein zero (P 0 ), a 31-kilodalton, immunoglobulin-related, transmembrane glycoprotein that accounts for greater than 50% of the protein in mammalian PNS myelin (4). The mRNA encoding this protein is estimated to account for nearly 8% of the poly(A) RNA in actively myelinating Schwann cells (5).A variety of studies have demonstrated that P 0 is essential for peripheral nerve development and function. Mice in which the P 0 gene has been deleted exhibit a severe peripheral neuropathy that includes hypomyelination, loss of motor control, tremors, and grossly abnormal myelin sheaths (6, 7). Furthermore, mutations in the P 0 gene account for a variety of inherited human peripheral neuropathies, including Charcot-MarieTooth disease type 1B, Dejerine-Sottas syndrome, and congenital hypomyelination (8 -12).High level P 0 expression is a distinctive feature of terminal differentiatio...

The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DRa gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DRa promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. DNA sequence comparisons have revealed that all human and murine class II genes possess two short, highly conserved sequences located in the region 5' of the transcription start site (15). The length of the spacer region between the two consensus sequences is also highly conserved. These consensus sequences, referred to as the X and Y (or A and B) boxes, are putative cis-acting regulatory elements involved in the control of class II gene expression.Our laboratory was the first to report that the class II boxes are required for promoter function in a human class II gene, HLA-DRcx, and that a DNA probe containing these consensus sequences binds to a nuclear protein that exhibits wide tissue distribution (16). We have now extended this investigation by determining that the specific sites of protein-DNA contact are contained in the Y box. Mutation of these contact sites reduces promoter activity, providing evidence that protein-Y box interactions are critical for DRot promoter function. Such a correlation between protein-DNA interaction sites and promoter activity has not been previously demonstrated for human class II MHC genes.

The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-KB/Rel binding sites located at -109 to -118 (Ii KB-1) and -163 to -172 (Ii KB-2) from the transcription start site. We report here that the differential function of each of these NF-KB/Rel sites in several distinct cell types depends on cell-specific binding of NF-KB/Rel transcription factors. Ii KB-1 is a positive regulatory element in B-cell lines and in the li-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glial cell lines. In vivo protein-DNA contacts are detectable at Ii KB-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-KB/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii KB-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again