Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate Central Nervous System.

BACKGROUND: Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS. RESULTS: Consistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC. CONCLUSIONS: Although seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals.

Comparative gene expression profiling between optic nerve and spinal cord injury in Xenopus laevis reveals a core set of genes inherent in successful regeneration of vertebrate central nervous system axons.

BACKGROUND: The South African claw-toed frog, Xenopus laevis, is uniquely suited for studying differences between regenerative and non-regenerative responses to CNS injury within the same organism, because some CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs. Tissues from these CNS regions (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) were used in a three-way RNA-seq study of axotomized CNS axons to identify potential core gene expression programs for successful CNS axon regeneration. RESULTS: Despite tissue-specific changes in expression dominating the injury responses of each tissue, injury-induced changes in gene expression were nonetheless shared between the two axon-regenerative CNS regions that were not shared with the non-regenerative region. These included similar temporal patterns of gene expression and over 300 injury-responsive genes. Many of these genes and their associated cellular functions had previously been associated with injury responses of multiple tissues, both neural and non-neural, from different species, thereby demonstrating deep phylogenetically conserved commonalities between successful CNS axon regeneration and tissue regeneration in general. Further analyses implicated the KEGG adipocytokine signaling pathway, which links leptin with metabolic and gene regulatory pathways, and a novel gene regulatory network with genes regulating chromatin accessibility at its core, as important hubs in the larger network of injury response genes involved in successful CNS axon regeneration. CONCLUSIONS: This study identifies deep, phylogenetically conserved commonalities between CNS axon regeneration and other examples of successful tissue regeneration and provides new targets for studying the molecular underpinnings of successful CNS axon regeneration, as well as a guide for distinguishing pro-regenerative injury-induced changes in gene expression from detrimental ones in mammals.

A novel role for the nuclear localization signal in regulating hnRNP K protein stability in vivo.

hnRNP K is a highly conserved nucleocytoplasmic shuttling protein, which associates with RNAs through synergistic binding via its three KH domains. hnRNP K is required for proper nuclear export and translational control of its mRNA targets, and these processes are controlled by hnRNP K's movement between subcellular compartments. Whereas the nuclear export and localization of hnRNP K that is associated with mRNP complexes has been well studied, the trafficking of hnRNP K that is unbound to mRNA has yet to be elucidated. To that end, we expressed an EGFP-tagged RNA binding-defective form of hnRNP K in intact Xenopus embryos, and found it was rapidly degraded in vivo. Deleting hnRNP K's nuclear localization signal (NLS), which contains two prospective ubiquitination sites, rescued the protein from degradation. These data demonstrate a novel activity for the NLS of hnRNP K in regulating the protein's stability in vivo when it is unbound to nucleic acids.

Phosphorylation of heterogeneous nuclear ribonucleoprotein K at an extracellular signal-regulated kinase phosphorylation site promotes neurofilament-medium protein expression and axon outgrowth in Xenopus.

Post-transcriptional control of cytoskeletal genes fine-tunes the supply of structural materials to growing axons in response to extracellular cues. In Xenopus, heterogeneous nuclear ribonucleoprotein K (hnRNPK) plays a crucial role in the nuclear export and translation of multiple cytoskeletal-related mRNAs required for axon outgrowth, and as a substrate of multiple kinases, is thus a likely molecular target of cell signaling pathways regulating such outgrowth. To study the role of hnRNPK's phosphorylation by extracellular signal-regulated kinase (ERK) in Xenopus axon outgrowth, we identified the only ERK1 phosphorylation site on Xenopus hnRNPK (S257; homologous with S284 of human hnRNPK) using an in vitro phosphorylation assay and tested its function in vivo by expressing phosphomimetic (S257D) and phosphodeficient (S257A) forms of hnRNPK in Xenopus embryos. Although neither form altered hnRNPK nuclear export, only the phosphomimetic form significantly rescued both neurofilament protein expression and axon outgrowth from hnRNPK knockdown. This finding represents a previously unidentified function of phosphorylation at this phylogenetically conserved site and implicates hnRNPK as an intracellular molecular target of ERK-mediated signaling in axon outgrowth.

New York State TrueAllele ® casework validation study.

DNA evidence can pose interpretation challenges, particularly with low-level or mixed samples. It would be desirable to make full use of the quantitative data, consider every genotype possibility, and objectively produce accurate and reproducible DNA match results. Probabilistic genotype computing is designed to achieve these goals. This validation study assessed TrueAllele(®) probabilistic computer interpretation on 368 evidence items in 41 test cases and compared the results with human review of the same data. Whenever there was a human result, the computer's genotype was concordant. Further, the computer produced a match statistic on 81 mixture items (for 87 inferred matching genotypes) in the test cases, while human review reported a statistic on 25 of these items (30.9%). Using match statistics to quantify information, probabilistic genotyping was shown to be sensitive, specific, and reproducible. These results demonstrate that objective probabilistic genotyping of biological evidence can reliably preserve DNA identification information.

Validating TrueAllele® DNA mixture interpretation.

DNA mixtures with two or more contributors are a prevalent form of biological evidence. Mixture interpretation is complicated by the possibility of different genotype combinations that can explain the short tandem repeat (STR) data. Current human review simplifies this interpretation by applying thresholds to qualitatively treat STR data peaks as all-or-none events and assigning allele pairs equal likelihood. Computer review, however, can work instead with all the quantitative data to preserve more identification information. The present study examined the extent to which quantitative computer interpretation could elicit more identification information than human review from the same adjudicated two-person mixture data. The base 10 logarithm of a DNA match statistic is a standard information measure that permits such a comparison. On eight mixtures having two unknown contributors, we found that quantitative computer interpretation gave an average information increase of 6.24 log units (min = 2.32, max = 10.49) over qualitative human review. On eight other mixtures with a known victim reference and one unknown contributor, quantitative interpretation averaged a 4.67 log factor increase (min = 1.00, max = 11.31) over qualitative review. This study provides a general treatment of DNA interpretation methods (including mixtures) that encompasses both quantitative and qualitative review. Validation methods are introduced that can assess the efficacy and reproducibility of any DNA interpretation method. An in-depth case example highlights 10 reasons (at 10 different loci) why quantitative probability modeling preserves more identification information than qualitative threshold methods. The results validate TrueAllele(®) DNA mixture interpretation and establish a significant information improvement over human review.