Functional organization of an Mbp enhancer exposes striking transcriptional regulatory diversity within myelinating glia.

In mammals, large caliber axons are ensheathed by myelin, a glial specialization supporting axon integrity and conferring accelerated and energy-efficient action potential conduction. Myelin basic protein (MBP) is required for normal myelin elaboration with maximal mbp transcription in oligodendrocytes requiring the upstream M3 enhancer. To further characterize the mechanism regulating mbp transcription, we defined M3 structure/function relationships by evaluating its evolutionary conservation, DNA footprints and the developmental programing conferred in mice by M3 derivatives. Multiple M3 regulatory element combinations were found to drive expression in oligodendrocytes and Schwann cells with a minimal 129 bp sequence conferring expression in oligodendrocytes throughout myelin elaboration, maintenance and repair. Unexpectedly, M3 derivatives conferred markedly different spatial and temporal expression programs thus illuminating striking transcriptional heterogeneity within post-mitotic oligodendrocytes. Finally, one M3 derivative engaged only during primary myelination, not during adult remyelination, demonstrating that transcriptional regulation in the two states is not equivalent.

ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress.

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10µM), or large inclusions (≥10µM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS.

Gene targeting of mouse Tardbp negatively affects Masp2 expression.

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function. To investigate this hypothesis, we attempted to generate a mouse conditional knockout of the Tardbp gene using the classical Cre-loxP technology. Even though heterozygote mice for the targeted allele were successfully generated, we were unable to obtain homozygotes. Here we show that although the targeting vector was specifically designed to not overlap with Tardbp adjacent genes, the homologous recombination event affected the expression of a downstream gene, Masp2. This may explain the inability to obtain homozygote mice with targeted Tardbp.

Hypermethylation of the CpG island near the G4C2 repeat in ALS with a C9orf72 expansion.

The G4C2 repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G4C2 repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5' of the G4C2 repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD).

RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS.

TDP-43 is a predominantly nuclear DNA/RNA binding protein involved in transcriptional regulation and RNA processing. TDP-43 is also a component of the cytoplasmic inclusion bodies characteristic of amyotrophic lateral sclerosis (ALS) and of frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). We have investigated the premise that abnormalities of TDP-43 in disease would be reflected by changes in processing of its target RNAs. To this end, we have firstly identified RNA targets of TDP-43 using UV-Cross-Linking and Immunoprecipitation (UV-CLIP) of SHSY5Y cells, a human neuroblastoma cell line. We used conventional cloning strategies to identify, after quality control steps, 127 targets. Results show that TDP-43 binds mainly to introns at UG/TG repeat motifs (49%) and polypyrimidine rich sequences (17.65%). To determine if the identified RNA targets of TDP-43 were abnormally processed in ALS versus control lumbar spinal cord RNA, we performed RT-PCR using primers designed according to the location of TDP-43 binding within the gene, and prior evidence of alternative splicing of exons adjacent to this site. Of eight genes meeting these criteria, five were differentially spliced in ALS versus control. This supports the premise that abnormalities of TDP-43 in ALS are reflected in changes of RNA processing.

Regulatory modules function in a non-autonomous manner to control transcription of the mbp gene.

Multiple regulatory modules contribute to the complex expression programs realized by many loci. Although long thought of as isolated components, recent studies demonstrate that such regulatory sequences can physically associate with promoters and with each other and may localize to specific sub-nuclear transcription factories. These associations provide a substrate for putative interactions and have led to the suggested existence of a transcriptional interactome. Here, using a controlled strategy of transgenesis, we analyzed the functional consequences of regulatory sequence interaction within the myelin basic protein (mbp) locus. Interactions were revealed through comparisons of the qualitative and quantitative expression programs conferred by an allelic series of 11 different enhancer/inter-enhancer combinations ligated to a common promoter/reporter gene. In a developmentally contextual manner, the regulatory output of all modules changed markedly in the presence of other sequences. Predicted by transgene expression programs, deletion of one such module from the endogenous locus reduced oligodendrocyte expression levels but unexpectedly, also attenuated expression of the overlapping golli transcriptional unit. These observations support a regulatory architecture that extends beyond a combinatorial model to include frequent interactions capable of significantly modulating the functions conferred through regulatory modules in isolation.

Separate proteolipid protein/DM20 enhancers serve different lineages and stages of development.

The gene encoding DM20 emerged in cartilaginous fish, descending from a bilaterian ancestor of the M6 proteolipid gene family. Its proteolipid protein (PLP) isoform appeared in amphibians, contains an additional 35 amino acids, and, in the mammalian CNS, is the dominant myelin protein in which it confers an essential neuroprotective function. During development, the DM20 isoform is prominent in a number of tissues, and plp/DM20 transcripts are detected in multiple progenitor populations, including those that continue to express plp/DM20 as they differentiate into myelinating oligodendrocytes. The locus also encodes isoforms with extended leader sequences that accumulate in the cell bodies of several types of neurons. Here, to locate and characterize regulatory sequences controlling the complex plp/DM20 transcription program, putative regulatory sequences, suggested by interspecies conservation, were ligated individually to a minimally promoted eGFPlacZ reporter gene. These constructs were inserted in single copy at a common site adjacent to the hypoxanthine-guanine phosphoribosyltransferase locus in embryonic stem cells and their in vivo expression programs were compared in transgenic mice. Most expressed developmental and cell-specific subprograms accommodated within the known expression phenotype of the endogenous plp/DM20 locus, thus defining multiple components of the combinatorial mechanism controlling its normal temporal and cell-specific program. Along with previously characterized nervous system enhancers, those described here should help expose the content and configuration of elements that are operational in multiple glial and neuronal lineages. The transgenic lines derived here also provide effective markers for multiple stages of glial and neuronal lineage progression.

Functional organization of a Schwann cell enhancer.

Myelin basic protein (MBP) gene expression is conferred in oligodendrocytes and Schwann cells by different upstream enhancers. In Schwann cells, expression is controlled by a 422 bp enhancer lying -9 kb from the gene. We show here that it contains 22 mammalian conserved motifs > or =6 bp. To investigate their functional significance, different combinations of wild-type or mutated motifs were introduced into reporter constructs that were inserted in single copy at a common hypoxanthine phosphoribosyltransferase docking site in embryonic stem cells. Lines of transgenic mice were derived, and the subsequent qualitative and quantitative expression phenotypes were compared at different stages of maturation. In the enhancer core, seven contiguous motifs cooperate to confer Schwann cell specificity while different combinations of flanking motifs engage, at different stages of Schwann cell maturation, to modulate expression level. Mutation of a Krox-20 binding site reduces the level of reporter expression, whereas mutation of a potential Sox element silences reporter expression. This potential Sox motif was also found conserved in other Schwann cell enhancers, suggesting that it contributes widely to regulatory function. These results demonstrate a close relationship between phylogenetic footprints and regulatory function and suggest a general model of enhancer organization. Finally, this investigation demonstrates that in vivo functional analysis, supported by controlled transgenesis, can be a robust complement to molecular and bioinformatics approaches to regulatory mechanisms.

Nonhemolytic, nonmotile gram-positive rods indicative of Bacillus anthracis.

We report a 40-year-old female patient who was admitted to the hospital because of a left ovarian mass torsion. A nonhemolytic, nonmotile Bacillus, suspicious of Bacillus anthracis, was isolated from a blood culture. We discuss the evaluation that led to the final identification of the bacterium as B. megaterium.