Epigenetic Studies Point to DNA Replication/Repair Genes as a Basis for the Heritable Nature of Long Term Complications in Diabetes.

Metabolic memory (MM) is defined as the persistence of diabetic (DM) complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of the DNA replication/DNA metabolism process group (with upregulation of the apex1, mcm2, mcm4, orc3, lig1, and dnmt1 genes) were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6-13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of the DNA replication/repair process group best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes.

Use of zebrafish as a model to investigate the role of epigenetics in propagating the secondary complications observed in diabetes mellitus.

Diabetes mellitus (DM) is classified as a disease of metabolic dysregulation predicted to affect over 400 million individuals world-wide by 2030. The debilitating aspects of this disease are the long term complications involving microvascular and macrovascular pathologies. These long term complications are related to the clinical phenomenon of metabolic memory (MM) that is defined as the persistence of diabetic complications even after glycemic control has been pharmacologically achieved. The persistent nature of MM has invoked involvement of epigenetic processes. Current research with the DM/MM zebrafish model as described in this review as well as human and mammalian studies has established that changes in DNA methylation patterns appear to contribute to tissue dysfunctions associated with DM. This review will describe studies on an adult zebrafish model of type I diabetes mellitus that allows analysis of both the hyperglycemic (HG or DM) phase and MM phase of the disease. The review will discuss the model in regards to: 1) its hyperglycemic phase, 2) its MM phase, 3) biochemical õpathways underlying changes in DNA methylation patterns observed in the model, 4) loci specific changes in DNA methylation patterns, and 5) strengths of the adult zebrafish model as compared to other MM animal models.

Inhibition of poly-ADP ribose polymerase enzyme activity prevents hyperglycemia-induced impairment of angiogenesis during wound healing.

We previously reported a zebrafish model of type I diabetes mellitus (DM) that can be used to study the hyperglycemic (HG) and metabolic memory (MM) states within the same fish. Clinically, MM is defined as the persistence of diabetic complications even after glycemic control is pharmacologically achieved. In our zebrafish model, MM occurs following ß-cell regeneration, which returns fish to euglycemia. During HG, fish acquire tissue deficits reflective of the complications seen in patients with DM and these deficits persist after fish return to euglycemia (MM). The unifying mechanism for the induction of diabetic complications involves a cascade of events that is initiated by the HG stimulation of poly-ADP ribose polymerase enzyme (Parp) activity. Additionally, recent evidence shows that the HG induction of Parp activity stimulates changes in epigenetic mechanisms that correlate with the MM state and the persistence of complications. Here we report that wound-induced angiogenesis is impaired in DM and remains impaired when fish return to a euglycemic state. Additionally, inhibition of Parp activity prevented the HG-induced wound angiogenesis deficiency observed. This approach can identify molecular targets that will provide potential new avenues for therapeutic discovery as angiogenesis imbalances are associated with all HG-damaged tissues.

An assay for lateral line regeneration in adult zebrafish.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Parp inhibition prevents ten-eleven translocase enzyme activation and hyperglycemia-induced DNA demethylation.

Studies from human cells, rats, and zebrafish have documented that hyperglycemia (HG) induces the demethylation of specific cytosines throughout the genome. We previously documented that a subset of these changes become permanent and may provide, in part, a mechanism for the persistence of complications referred to as the metabolic memory phenomenon. In this report, we present studies aimed at elucidating the molecular machinery that is responsible for the HG-induced DNA demethylation observed. To this end, RNA expression and enzymatic activity assays indicate that the ten-eleven translocation (Tet) family of enzymes are activated by HG. Furthermore, through the detection of intermediates generated via conversion of 5-methyl-cytosine back to the unmethylated form, the data were consistent with the use of the Tet-dependent iterative oxidation pathway. In addition, evidence is provided that the activity of the poly(ADP-ribose) polymerase (Parp) enzyme is required for activation of Tet activity because the use of a Parp inhibitor prevented demethylation of specific loci and the accumulation of Tet-induced intermediates. Remarkably, this inhibition was accompanied by a complete restoration of the tissue regeneration deficit that is also induced by HG. The ultimate goal of this work is to provide potential new avenues for therapeutic discovery.

A zebrafish model of diabetes mellitus and metabolic memory.

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined. We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Impaired tissue regeneration corresponds with altered expression of developmental genes that persists in the metabolic memory state of diabetic zebrafish.

As previously reported by our laboratory, streptozocin-induced diabetes mellitus (DM) in adult zebrafish results in an impairment of tissue regeneration as monitored by caudal fin regeneration. Following streptozocin withdrawal, a recovery phase occurs to reestablish euglycemia, via pancreatic beta-cell regeneration. However, DM-associated impaired fin regeneration continues indefinitely in the metabolic memory (MM) state, allowing for subsequent molecular analysis of the underlying mechanisms of MM. This study focuses on elucidating the molecular basis that explains the DM-associated impaired fin regeneration and why it persists into the MM state with the aim of better understanding MM. Using a combination of microarray analysis and bioinformatics approaches, our study found that of the 14,900 transcripts analyzed, aberrant expression of 71 genes relating to tissue developmental and regeneration processes were identified in DM fish and the altered expression of these 71 genes persisted in MM fish. Key regulatory genes of major development and signal transduction pathways were identified among this group of 71. The aberrant expression of key regulatory genes in the DM state that persist into the MM state provides a plausible explanation on how hyperglycemia induced impaired fin regeneration in the adult zebrafish DM/MM model.

Metabolic memory and chronic diabetes complications: potential role for epigenetic mechanisms.

Recent estimates indicate that diabetes mellitus currently affects more than 10 % of the world's population. Evidence from both the laboratory and large scale clinical trials has revealed that prolonged hyperglycemia induces chronic complications which persist and progress unimpeded even when glycemic control is pharmaceutically achieved via the phenomenon of metabolic memory. The epigenome is comprised of all chromatin modifications including post translational histone modification, expression control via miRNAs and the methylation of cytosine within DNA. Modifications of these epigenetic marks not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters. As such, these processes have gained much attention as potential molecular mechanisms underlying metabolic memory and chronic diabetic complications. Here we present a review of the very recent literature published pertaining to this subject.

Heritable transmission of diabetic metabolic memory in zebrafish correlates with DNA hypomethylation and aberrant gene expression.

Metabolic memory (MM) is the phenomenon whereby diabetes complications persist and progress after glycemic recovery is achieved. Here, we present data showing that MM is heritable and that the transmission correlates with hyperglycemia-induced DNA hypomethylation and aberrant gene expression. Streptozocin was used to induce hyperglycemia in adult zebrafish, and then, following streptozocin withdrawal, a recovery phase was allowed to reestablish a euglycemic state. Blood glucose and serum insulin returned to physiological levels during the first 2 weeks of the recovery phase as a result of pancreatic ß-cell regeneration. In contrast, caudal fin regeneration and skin wound healing remained impaired to the same extent as in diabetic fish, and this impairment was transmissible to daughter cell tissue. Daughter tissue that was never exposed to hyperglycemia, but was derived from tissue that was, did not accumulate AGEs or exhibit increased levels of oxidative stress. However, CpG island methylation and genome-wide microarray expression analyses revealed the persistence of hyperglycemia-induced global DNA hypomethylation that correlated with aberrant gene expression for a subset of loci in this daughter tissue. Collectively, the data presented here implicate the epigenetic mechanism of DNA methylation as a potential contributor to the MM phenomenon.

Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus.

The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background.

De novo formation of a subnuclear body.

The mammalian cell nucleus contains structurally stable functional compartments. We show here that one of them, the Cajal body (CB), can be formed de novo. Immobilization on chromatin of both CB structural components, such as coilin, and functional components of the CB, such as the SMN complex, spliceosomal small nuclear ribonucleoproteins (RNPs), small nucleolar RNPs, and small Cajal body-specific RNPs, is sufficient for the formation of a morphologically normal and apparently functional CB. Biogenesis of the CB does not follow a hierarchical assembly pathway and exhibits hallmarks of a self-organizing structure.

Conservation of a masked nuclear export activity of La proteins and its effects on tRNA maturation.

La is an RNA-processing-associated phosphoprotein so highly conserved that the human La protein (hLa) can replace the tRNA-processing function of the fission yeast La protein (Sla1p) in vivo. La proteins contain multiple trafficking elements that support interactions with RNAs in different subcellular locations. Prior data indicate that deletion of a nuclear retention element (NRE) causes nuclear export of La and dysfunctional processing of associated pre-tRNAs that are spliced but 5' and 3' unprocessed, with an accompanying decrease in tRNA-mediated suppression, in fission yeast. To further pursue these observations, we first identified conserved residues in the NREs of hLa and Sla1p that when substituted mimic the NRE deletion phenotype. NRE-defective La proteins then deleted of other motifs indicated that RNA recognition motif 1 (RRM1) is required for nuclear export. Mutations of conserved RRM1 residues restored nuclear accumulation of NRE-defective La proteins. Some RRM1 mutations restored nuclear accumulation, prevented disordered pre-tRNA processing, and restored suppression, indicating that the tRNA-related activity of RRM1 and its nuclear export activity could be functionally separated. When mapped onto an hLa structure, the export-sensitive residues comprised surfaces distinct from the RNA-binding surface of RRM1. The data indicate that the NRE has been conserved to mask or functionally override an equally conserved nuclear export activity of RRM1. The data suggest that conserved elements mediate nuclear retention, nuclear export, and RNA-binding activities of the multifunctional La protein and that their interrelationship contributes to the ability of La to engage its different classes of RNA ligands in different cellular locations.

Mouse and human La proteins differ in kinase substrate activity and activation mechanism for tRNA processing.

The La protein interacts with a variety of small RNAs as well as certain growth-associated mRNAs such as Mdm2 mRNA. Human La (hLa) phosphoprotein is so highly conserved that it can replace the tRNA processing function of the fission yeast La protein in vivo. We used this system, which is based on tRNA-mediated suppression (TMS) of ade6-704 in S. pombe, to compare the activities of mouse and human La proteins. Prior studies indicate that hLa is activated by phosphorylation of serine-366 by protein kinase CK2, neutralizing a negative effect of a short basic motif (SBM). First, we report the sequence mapping of the UGA stop codon that requires suppressor tRNA for TMS, to an unexpected site in S. pombe ade6-704. Next, we show that, unlike hLa, native mLa is unexpectedly inactive for TMS, although its intrinsic activity is revealed by deletion of its SBM. We then show that mLa is not phosphorylated by CK2, accounting for the mechanistic difference between mLa and hLa. We found a PKA/PKG target sequence in mLa (S199) that is not present in hLa, and show that PKA/PKG efficiently phosphorylates mLa S199 in vitro. A noteworthy conclusion that comes from this work is that this fission yeast system can be used to gain insight into differences in control mechanisms used by La proteins of different mammalian species. Finally, RNA binding assays indicate that while mutation of mLa S199 has little effect on pre-tRNA binding, it substantially decreases binding to a probe derived from Mdm2 mRNA. In closing, we note that species-specific signaling through La may be relevant to the La-dependent Mdm2 pathways of p53 metabolism and cancer progression in mice and humans.

Separate RNA-binding surfaces on the multifunctional La protein mediate distinguishable activities in tRNA maturation.

By sequence-specific binding to 3' UUU-OH, the La protein shields precursor (pre)-RNAs from 3' end digestion and is required to protect defective pre-transfer RNAs from decay. Although La is comprised of a La motif and an RNA-recognition motif (RRM), a recent structure indicates that the RRM beta-sheet surface is not involved in UUU-OH recognition, raising questions as to its function. Progressively defective suppressor tRNAs in Schizosaccharomyces pombe reveal differential sensitivities to La and Rrp6p, a 3' exonuclease component of pre-tRNA decay. 3' end protection is compromised by mutations to the La motif but not the RRM surface. The most defective pre-tRNAs require a second activity of La, in addition to 3' protection, that requires an intact RRM surface. The two activities of La in tRNA maturation map to its two conserved RNA-binding surfaces and suggest a modular model that has implications for its other ligands.

The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells.

The La protein is a target of autoantibodies in patients suffering from Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La+/-intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM.

Human La is found at RNA polymerase III-transcribed genes in vivo.

The human La autoantigen can bind to nascent RNA transcripts and has also been postulated to act as an RNA polymerase III (pol III) transcription initiation and termination factor. Here, we show by chromatin immunoprecipitation (ChIP) that La is associated with pol III-transcribed genes in vivo. In contrast, the Ro autoantigen, which can also bind pol III transcripts, is not found at these genes. The putative pol III transcription factors NF1 and TFIIA are also not detected at class III genes. Binding of La remains when transcription is repressed at mitosis and does not correlate with the presence of polymerase at the gene. However, gene occupancy depends on the phosphorylation status of La, with the less prevalent, unphosphorylated form being found selectively on pol III templates.

Mutations in the RNA polymerase III subunit Rpc11p that decrease RNA 3' cleavage activity increase 3'-terminal oligo(U) length and La-dependent tRNA processing.

Termination by RNA polymerase III (Pol III) produces RNAs whose 3' oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3' exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3' cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNASerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3' cleavage activity and produce pre-tRNASerUGAM transcripts with elongated 3'-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNASerUGAM contains too few 3' Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3'-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.

Nonphosphorylated human La antigen interacts with nucleolin at nucleolar sites involved in rRNA biogenesis.

La is a RNA-binding protein implicated in multiple pathways related to the production of tRNAs, ribosomal proteins, and other components of the translational machinery (D. J. Kenan and J. D. Keene, Nat. Struct. Mol. Biol. 11:303-305, 2004). While most La is phosphorylated and resides in the nucleoplasm, a fraction is in the nucleolus, the site of ribosome production, although the determinants of this localization are incompletely known. In addition to its conserved N-terminal domain, human La harbors a C-terminal domain that contains an atypical RNA recognition motif and a short basic motif (SBM) adjacent to phosphoserine-366. We report that nonphosphorylated La (npLa) is concentrated in nucleolar sites that correspond to the dense fibrillar component that harbors nascent pol I transcripts as well as fibrillarin and nucleolin, which function in early phases of rRNA maturation. Affinity purification and native immunoprecipitation of La and fluorescence resonance energy transfer in the nucleolus reveal close association with nucleolin. Moreover, La lacking the SBM does not localize to nucleoli. Lastly, La exhibits SBM-dependent, phosphorylation-sensitive interaction with nucleolin in a yeast two-hybrid assay. The data suggest that interaction with nucleolin is, at least in part, responsible for nucleolar accumulation of La and that npLa may be involved in ribosome biogenesis.

CK2 is responsible for phosphorylation of human La protein serine-366 and can modulate rpL37 5'-terminal oligopyrimidine mRNA metabolism.

La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3' UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5' regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S(366)) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5' untranslated regions comprised of terminal oligopyrimidine (5'TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S(366) represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S(366) phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5'TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A(366) than with La S(366), concomitant with La A(366)-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S(366) in vivo, that this limits 5'TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery.

Differential phosphorylation and subcellular localization of La RNPs associated with precursor tRNAs and translation-related mRNAs.

The La protein facilitates the production of tRNAs in the nucleus and the translation of specific mRNAs in the cytoplasm. We report that human La that is phosphorylated on serine 366 (pLa) is nucleoplasmic and associated with precursor tRNAs and other nascent RNA polymerase III transcripts while nonphosphorylated (np)La is cytoplasmic and associated with a subset of mRNAs that contain 5'-terminal oligopyrimidine (5'TOP) motifs known to control protein synthesis. Thus, La ribonucleoproteins (RNP) exist in distinct states that differ in subcellular localization, serine 366 phosphorylation, and associated RNAs. These results are consistent with a model in which the relative concentrations of the La S366 isoforms in different subcellular compartments in conjunction with the relative concentrations of specific RNA ligands in these compartments determine the differential association of npLa and pLa with their respective classes of associated RNAs.