The evolution of lipophilin genes from invertebrates to tetrapods: DM-20 cannot replace proteolipid protein in CNS myelin.

The proteolipid protein (PLP) gene encodes two myelin-specific protein isoforms, DM-20 and PLP, which are members of the highly conserved lipophilin family of transmembrane proteins. While the functions of this family are poorly understood, the fact that null mutations of the PLP gene cause leukodystrophy in man is testament to the importance of DM-20 and PLP in normal CNS function. PLP differs from DM-20 by the presence of a 35 amino acid domain exposed to the cytoplasm, which is not encoded by other lipophilin genes and appears to have arisen in amphibians approximately 300 million years before present. However, the lipophilin gene family can be traced back at least 550 million years and is represented in Drosophila and silkworms. Thus, from an evolutionary perspective PLP can reasonably be anticipated to perform functions in CNS myelin that cannot be accomplished by other lipophilins. Herein we use a novel knock-in strategy to generate mice expressing wild-type levels of a Plp gene that has been modified to encode only DM-20. Although DM-20 is incorporated into functional compact myelin sheaths in young animals, our data show that the 35 amino acid PLP-specific peptide is required to engender the normal myelin period and to confer long-term stability on this multilamellar membrane.

CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice.

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.

Disrupted proteolipid protein trafficking results in oligodendrocyte apoptosis in an animal model of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease resulting from mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Distinguishing features of PMD include pleiotropy and a range of disease severities among patients. Previously, we demonstrated that, when expressed in transfected fibroblasts, many naturally occurring mutant PLP alleles encode proteins that accumulate in the endoplasmic reticulum and are not transported to the cell surface. In the present communication, we show that oligodendrocytes in an animal model of PMD, the msd mouse, accumulate Plp gene products in the perinuclear region and are unable to transport them to the cell surface. Another important aspect of disease in msd mice is oligodendrocyte cell death, which is increased by two- to threefold. We demonstrate in msd mice that this death occurs by apoptosis and show that at the time oligodendrocytes die, they have differentiated, extended processes that frequently contact axons and are expressing myelin structural proteins. Finally, we define a hypothesis that accounts for pathogenesis in most PMD patients and animal models of this disease and, moreover, can be used to develop potential therapeutic strategies for ameliorating the disease phenotype.

Erythroid Krüppel-like factor exhibits an early and sequentially localized pattern of expression during mammalian erythroid ontogeny.

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific transcription factor that mediates activation via binding to a 9 base pair sequence that encompasses the CACCC element, one of a trio of evolutionarily conserved sequence motifs that are functionally important for transcription of red cell-specific genes. Molecular analyses have delineated the specificity of its interaction and activation through the CAC site at the adult beta-globin promoter. However, its expression and distribution during murine ontogeny have not been established. To address these issues, we have focused on biological aspects of EKLF expression by examining the onset and localization of its mRNA during murine development by using reverse transcription/polymerase chain reaction (RT/PCR) analysis of differentiating embryonic stem cells and in situ analyses of normal developing embryos. In addition, we have monitored the presence of EKLF protein by blot analysis of whole-cell extracts derived from circulating cells and embryonic tissue. Our studies show that EKLF mRNA is first expressed at the neural plate stage (day 7.5) within primitive erythroid cells at the very beginning of blood island formation in the yolk sac. EKLF is then expressed by day 9 in the hepatic primordia and remains high in the liver, which becomes the sole source of EKLF mRNA in the 14.5 day fetus. Concomitantly with EKLF mRNA, EKLF protein is also expressed in primitive erythroid cells and in the fetal liver. Finally, EKLF expression in the adult spleen is strictly localized to the red pulp. These studies demonstrate that EKLF is a specific, early marker of erythroid differentiation consistent with its requirement for later globin (and possibly other red cell gene-specific) expression. In addition, EKLF exhibits alternate, sequentially active sites of expression within regions known to harbor hematopoietic precursors during murine ontogeny. Thus, EKLF expression exhibits biological properties that, in addition to previous molecular and more recent genetic studies, augment the evidence in favor of its important role in erythroid cell-specific expression.

The erythroid Krüppel-like factor transactivation domain is a critical component for cell-specific inducibility of a beta-globin promoter.

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.

Analyses of beta-thalassemia mutant DNA interactions with erythroid Krüppel-like factor (EKLF), an erythroid cell-specific transcription factor.

We describe functional tests and molecular modeling of erythroid Krüppel-like factor (EKLF) interactions with its DNA binding site. EKLF, a zinc finger-containing, erythroid-specific transcription factor, binds and transactivates from the CACCC element, an evolutionarily conserved DNA sequence present within a large number of erythroid-specific promoters and enhancers. This DNA binding element is the site of naturally occurring point mutations that give rise to beta-thalassemia. We have directly tested whether CAC site point mutations (including two of the beta-thalassemia mutants) affect EKLF transactivation and DNA binding function. In vivo analyses demonstrate that EKLF is unable to transactivate a reporter plasmid that contains these mutations. In vitro analyses reveal a 40-100-fold decrease in binding affinity for these sites that accounts for the in vivo observations. The homology between the three EKLF and Zif268 zinc fingers and their conserved sequence-specific contacts to their target site allowed us to formulate a molecular model of the EKLF/CAC site complex, based primarily on energy minimization/refinement of the Zif268/DNA co-crystal structure. These models suggest that both specific and nonspecific hydrogen bonding play a critical role in the ability of EKLF to prefer binding to its cognate site. Analysis of sequence-specific contacts by EKLF to its target site within the beta-globin promoter verified the residues predicted to be important by the functional and modeling data. Together these results demonstrate that EKLF displays a strong discriminatory ability among potential DNA target sites consistent with the beta-thalassemia data. They also suggest that lack of EKLF binding to these sites may play a determining role in its phenotype, and they strengthen the evidence in favor of EKLF's proposed role in erythroid-specific transcriptional activation through the CACCC elements.