Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development

Gadd45 proteins have been implicated in the cellular response to physiological or environmental stress and the accompanying cell cycle arrest, DNA repair, cell survival and senescence or apoptosis. Although their molecular function is well studied, the expression and role of Gadd45 genes during embryonic development in mice is largely unknown. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development. In situ hybridizations on sectioned and whole mouse embryos show most prominent Gadd45a expression in the tip of the closing neural tube, the cranial and dorsal root ganglia and the somites. Mouse Gadd45b is expressed strongly in the chorion, but only weakly in the embryo proper, including somites and limb buds. Murine Gadd45g expression strongly resembles Xenopus and medaka fish expression in primary neuron precursors and post-mitotic neurons, indicating a conserved role for Gadd45g in vertebrate neurogenesis. Additionally, Gadd45 genes show conserved expression during somitogenesis. In summary, Gadd45 genes are expressed in evolutionary conserved, but also divergent domains, which predominantly encompass areas of cell differentiation, consistent with their established function in growth arrest and DNA demethylation.     

Mammalian DNA demethylation

DNA cytosine methylation is a reversible epigenetic mark regulating gene expression. Aberrant methylation profiles are concomitant with developmental defects and cancer. Numerous studies in the past decade have identified enzymes and pathways responsible for active DNA demethylation both on a genome-wide as well as gene-specific scale. Recent findings have strengthened the idea that 5-methylcytosine oxidation catalyzed by members of the ten-eleven translocation (Tet1–3) oxygenases in conjunction with replication-coupled dilution of the conversion products causes the majority of genome-wide erasure of methylation marks during early development. In contrast, short and long patch DNA excision repair seems to be implicated mainly in gene-specific demethylation. Growth arrest and DNA damage-inducible protein 45 a (Gadd45a) regulates gene-specific demethylation within regulatory sequences of limited lengths raising the question of how such site specificity is achieved. A new study identified the protein inhibitor of growth 1 (Ing1) as a reader of the active chromatin mark histone H3 lysine 4 trimethylation (H3K4me3). Ing1 binds and directs Gadd45a to target sites, thus linking the histone code with DNA demethylation.     

Role of Gadd45a in Wip1-dependent regulation of intestinal tumorigenesis

Conversion of intestinal stem cells into tumor-initiating cells is an early step in Apc^Min-induced polyposis. Wild-type p53-induced phosphatase 1 (Wip1)-dependent activation of a DNA damage response and p53 has a permanent role in suppression of stem cell conversion, and deletion of Wip1 lowers the tumor burden in Apc^Min mice. Here we show that cyclin-dependent kinase inhibitor 2a, checkpoint kinase 2, and growth arrest and DNA damage gene 45a (Gadd45a) exert critical functions in the tumor-resistant phenotype of Wip1-deficient mice. We further identified Gadd45a as a haploinsufficient gene in the regulation of Wip1-dependent tumor resistance in mice. Gadd45a appears to function through its ability to activate the Jnk-dependent signaling pathway that in turn is a necessary mediator of the proapoptotic functions of p53 that respond to activation of the β-catenin signaling pathway. We propose that silencing of Gadd45a is sufficient to override p53 activation in the presence of active β-catenin under conditions of an enhanced DNA damage response.     

Daily Variation in Global and Local DNA Methylation in Mouse Livers

DNA methylation is one of the best-characterized epigenetic modifications and has an important biological relevance. Here we showed that global DNA methylation level in mouse livers displayed a daily variation where the peak phases occurred during the end of the day and the lowest level at the beginning of the day in the light-dark or dark-dark cycles. Typical repeat sequence long interspersed nucleotide element-1 (LINE-1) had a similar methylation rhythm to global DNA. DNA methyltransferase 3A (DNMT3A) and ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) brought a relative forward daily variation to global DNA methylation, and the temporary change in ratio of SAM to SAH had no influence on the DNA methylation level. The rhythm of global DNA methylation was lost and DNA methylation level was increased in Per1^-/-Per2^-/- double knockout mice, which were in accordance with changes of Dnmt3a mRNA levels and its rhythm. Our results suggest that the daily variation in global DNA methylation was associated with the change of Dnmt3a expression rather than ratio of SAM to SAH.     

Gemcitabine functions epigenetically by inhibiting repair mediated DNA demethylation

Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. In mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation.     

Gadd45a promotes DNA demethylation through TDG

Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)—initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway.     

Gadd45a is an RNA binding protein and is localized in nuclear speckles

Background

The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids.

Principal Findings

Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function.

Significance

The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle.     

DNA excision repair proteins and Gadd45 as molecular players for active DNA demethylation

DNA cytosine methylation represents an intrinsic modification signal of the genome that plays important roles in heritable gene silencing, heterochromatin formation and certain transgenerational epigenetic inheritance. In contrast to the process of DNA methylation that is catalyzed by specific classes of methyltransferases, molecular players underlying active DNA demethylation have long been elusive. Emerging biochemical and functional evidence suggests that active DNA demethylation in vertebrates can be mediated through DNA excision repair enzymes, similar to the well-known repair-based DNA demethylation mechanism in Arabidopsis. As key regulators, non-enzymatic Gadd45 proteins function to recruit enzymatic machineries and promote coupling of deamination, base and nucleotide-excision repair in the process of DNA demethylation. In this article, we review recent findings and discuss functional and evolutionary implications of such mechanisms underlying active DNA demethylation.     

Gadd45a and Gadd45g regulate neural development and exit from pluripotency in Xenopus

Gadd45 genes encode a small family of multifunctional stress response proteins, mediating cell proliferation, apoptosis, DNA repair and DNA demethylation. Their role during embryonic development is incompletely understood. Here we identified Xenopus Gadd45b, compared Gadd45a, Gadd45b and Gadd45g expression during Xenopus embryogenesis, and characterized their gain and loss of function phenotypes. Gadd45a and Gadd45g act redundantly and double Morpholino knock down leads to pleiotropic phenotypes, including shortened axes, head defects and misgastrulation. In contrast, Gadd45b, which is expressed at very low levels, shows little effect upon knock down or overexpression. Gadd45ag double Morphants show reduced neural cell proliferation and downregulation of pan-neural and neural crest markers. In contrast, Gadd45ag Morphants display increased expression of multipotency marker genes including Xenopus oct4 homologs as well as gastrula markers, while mesodermal markers are downregulated. The results indicate that Gadd45ag are required for early embryonic cells to exit pluripotency and enter differentiation.     

Gadd45a deletion aggravates hematopoietic stem cell dysfunction in ATM-deficient mice

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM^-/-) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM^-/- HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM^-/- mice. Instead, ATM and Gadd45a double knockout (ATM^-/- Gadd45a^-/-) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM^-/- HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM^-/- Gadd45a^-/- HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM^-/- Gadd45a^-/- mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM^-/- mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM^-/- HSCs.     

Gadd45-induced cell cycle G2/M arrest for improved transient gene expression in Chinese hamster ovary cells

Gadd45 is a p53-regulated protein and is involved in cell cycle arrest in the G2/M phase. In an effort to improve transient gene expression (TGE) in Chinese hamster ovary (CHO) cells, the effect of Gadd45-induced cell cycle arrest on TGE in CHO cells was investigated using the two different expression vectors encoding Fcfusion protein and recombinant antibody. To regulate the expression of Gadd45 in CHO cells, the CHO-TREx-gadd45 cell line was established using the T-REx system controlled by doxycycline. During the cultures for TGE, Gadd45 overexpression severely inhibited cell growth, but significantly enhanced TGE. Compared with the culture without Gadd45 overexpression, the TGE of Fc fusion protein and humanized antibody were increased by 111 and 93%, respectively. The enhanced TGE, despite the cell growth arrest induced by Gadd45 overexpression, was due to the significantly increased specific productivity, resulting from enhanced transfection efficiency, increased cell size, and active DNA demethylation. Taken together, the data obtained here demonstrate that Gadd45-induced cell cycle arrest in G2/M phase can significantly enhance TGE in CHO cells.     

Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan

Progressive attrition of telomeres triggers DNA damage response (DDR) and limits the regenerative capacity of adult stem cells during mammalian aging. Intriguingly, telomere integrity is not only determined by telomere length but also by the epigenetic status of telomeric/sub‐telomeric regions. However, the functional interplay between DDR induced by telomere shortening and epigenetic modifications in aging remains unclear. Here, we show that deletion of Gadd45a improves the maintenance and function of intestinal stem cells (ISCs) and prolongs lifespan of telomerase‐deficient mice (G3Terc^?/?). Mechanistically, Gadd45a facilitates the generation of a permissive chromatin state for DDR signaling by inducing base excision repair‐dependent demethylation of CpG islands specifically at sub‐telomeric regions of short telomeres. Deletion of Gadd45a promotes chromatin compaction in sub‐telomeric regions and attenuates DDR initiation at short telomeres of G3Terc^?/? ISCs. Treatment with a small molecule inhibitor of base excision repair reduces DDR and improves the maintenance and function of G3Terc^?/? ISCs. Taken together, our study proposes a therapeutic approach to enhance stem cell function and prolong lifespan by targeting epigenetic modifiers.     

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α−/− cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α−/− cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α−/− cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian, and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G2/M "arrest," showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis, and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL.p53^-/- mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^-/- mice show chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfb/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfb/Smad pathway.     

Emerging roles of TET proteins and 5-Hydroxymethylcytosines in active DNA demethylation and beyond

Cytosine methylation is the major epigenetic modification of metazoan DNA. Although there is strong evidence that active DNA demethylation occurs in animal cells, the molecular details of this process are unknown. The recent discovery of the TET protein family (TET1–3) 5-methylcytosine hydroxylases has provided a new entry point to reveal the identity of the long-sought DNA demethylase. Here, we review the recent progress in understanding the function of TET proteins and 5-hydroxymethylcytosine (5hmC) through various biochemical and genomic approaches, the current evidence for a role of 5hmC as an early intermediate in active DNA demethylation and the potential functions of TET proteins and 5hmC beyond active DNA demethylation. We also discuss how future studies can extend our knowledge of this novel epigenetic modification.Key words: TET1, 5-hydroxymethylcytosine, active DNA demethylation, epigenetic, DNA methylation, hippocampus, electroconvulsive stimulation, Gadd45b, BER     

TLR2-ICAM1-Gadd45α Axis Mediates the Epigenetic Effect of Selenium on DNA Methylation and Gene Expression in Keshan Disease

Keshan disease (KD) is a fatal dilated cardiomyopathy with unknown etiology, and selenium deficiency is considered the main cause of KD. Several observations implicate a role for altered DNA methylation in selenium deficiency-related diseases. The aim of the present study was to investigate the epigenetic effects of selenium (Se) on DNA methylation and gene expression in Keshan disease. Using methylated DNA immunoprecipitation chip (MeDIP-Chip) and quantitative RT-PCR, we identified two inflammatory-related genes (TLR2 and ICAM1) that were differentially methylated and expressed between normal individuals and KD patients. Results from DNA methylation profile between KD patients and normal individuals showed that selenium deficiency decreased methylation of CpG islands in promoter regions of TLR2 and ICAM1 and upregulated messenger RNA (mRNA) and protein levels of TLR2 and ICAM1. In rat animal model of Keshan disease, selenite treatment could increase TLR2 and ICAM1 promoter methylation, suppress these genes expression, and reduce infiltration of myocardial inflammatory cells. In cell culture model of Keshan disease, we found 5-Aza-dC (DNMT1 inhibitor) treatment in the presence of selenium-reduced mRNA and protein levels of DNMT1 regardless of TLR2 and ICAM1 promoter methylation status and expression levels of these genes. Selenite treatment suppressed the expression of the Gadd45α, TLR2, and ICAM1 in a concentration-dependent manner, while selenium deficiency increased the expression of the Gadd45α, TLR2, and ICAM1 and decreased TLR2 and ICAM1 promoter methylation level in a time-dependent manner. Our results revealed that TLR2-ICAM1-Gadd45α axis might play an important role in gene-specific active DNA demethylation during inflammatory response in myocardium.     

Plasticity of DNA methylation in mouse T cell activation and differentiation

Background

Circulating CD4⁺ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4⁺ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results

Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion

We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.     

Neural tube development requires the cooperation of p53- and Gadd45a-associated pathways

BACKGROUND: Numerous genetically engineered mouse models for neural tube defects (NTDs) exist, and some of the implicated proteins are functionally related. For example, the growth arrest and DNA damage-inducible protein Gadd45a and tumor suppressor p53 are functionally similar, and both are involved in neural tube development (Gadd45a- and Trp53-null embryos show low levels of exencephaly). To assess their roles in neural tube development, we generated double-null mice from Gadd45a- and Trp53-null mice, as well as from cyclin-dependent kinase inhibitor (Cdkn1a) (p21)-null and xeroderma pigmentosum group C (XPC)-null mice that do not show spontaneous exencephaly. METHODS: Gadd45a-, Trp53-, Cdkn1a-, and XPC-null mice were crossed to generate several double-null mouse models. Embryos (embryonic day [ED] 16-18) from the single- and double-null crosses were scored for NTDs. RESULTS: Deletion of both Gadd45a and Trp53 in mice increased exencephaly frequencies compared to the deletion of either single gene (34.0% in Gadd45a/Trp53-null compared to 8.4% and 9.1% in the Gadd45a- and Trp53-null embryos, respectively). Furthermore, although deletion of another p53-regulated gene, Cdkn1a, is not associated with exencephaly, in conjunction with Gadd45a deletion, the exencephaly frequencies are increased (30.5% in the Gadd45a/Cdkn1a-null embryos) and are similar to those in the Gadd45a/Trp53-null embryos. Although XPC deletion increased exencephaly frequencies in Trp53-null embryos, XPC deletion did not increase the exencephaly frequencies in Gadd45a-null embryos. CONCLUSIONS: The increased genetic liability to exencephaly in the Gadd45a/Trp53- and Gadd45a/Cdkn1a-null embryos may be related to the disruption of multiple cellular pathways associated with Gadd45a and p53.