We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term followup Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.T he landmark Diabetes Control and Complications Trial (DCCT; 1983-1993 clearly showed that intensive (INT) glycemic control profoundly reduces the development and progression of microvascular complications in type 1 diabetes (T1D). The DCCT participants were subsequently followed in the Epidemiology of Diabetes Interventions and Complications (EDIC) Study (1994 to present), during which all subjects were advised to practice INT treatment. Surprisingly, those previously assigned to conventional (CONV) therapy continued to develop complications, such as nephropathy, retinopathy, and macrovascular diseases, at significantly higher rates than the previous INT therapy group, despite nearly similar HbA1c levels during the EDIC Study (1-3). This persistence of benefit from early application of INT therapy, called "metabolic memory," is an enigma in the field of T1D: recent studies have suggested the involvement of epigenetic mechanisms (4-9).Epigenetics is the study of mostly heritable changes in gene expression and phenotype that occur without alterations in the underlying DNA sequence. Epigenetic states are affected by environmental factors, such as aberrant nutrition and metabolic states (4, 6-8, 10). DNA methylation (DNA-me; the classic epigenetic mark) and posttranslational modifications (PTMs) of histo...

Ig class switch recombination (CSR) and somatic hypermutation serve to diversify antibody responses and are orchestrated by the activity of activation-induced cytidine deaminase and many proteins involved in DNA repair and genome surveillance. Msh5, a gene encoded in the central MHC class III region, and its obligate heterodimerization partner Msh4 have a critical role in regulating meiotic homologous recombination and have not been implicated in CSR. Here, we show that MRL/lpr mice carrying a congenic H-2 b/b MHC interval exhibit several abnormalities regarding CSR, including a profound deficiency of IgG3 in most mice and long microhomologies at Ig switch (S) joints. We found that Msh5 is expressed at low levels on the H-2 b haplotype and, importantly, a similar long S joint microhomology phenotype was observed in both Msh5 and Msh4-null mice. We also present evidence that genetic variation in MSH5 is associated with IgA deficiency and common variable immune deficiency (CVID) in humans. One of the human MSH5 alleles identified contains two nonsynonymous polymorphisms, and the variant protein encoded by this allele shows impaired binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA deficiency patients carrying disease-associated MSH5 alleles show increased donor/acceptor microhomology, involving pentameric DNA repeat sequences and lower mutation rates than controls. Our findings suggest that Msh4/5 heterodimers contribute to CSR and support a model whereby Msh4/5 promotes the resolution of DNA breaks with low or no terminal microhomology by a classical nonhomologous end-joining mechanism while possibly suppressing an alternative microhomology-mediated pathway.immunoglobulin subclass deficiency ͉ mismatch repair ͉ Msh4 A fter appropriate stimulation, B cells undergo class switch recombination (CSR), whereby the functionally rearranged V(D)J DNA segment is recombined with a downstream Ig constant region segment. The biochemistry of CSR is complex and involves the B cell-specific gene activation-induced cytidine deaminase, which initiates both CSR and somatic hypermutation (1). CSR also requires many ubiquitously expressed genes important for detecting DNA mismatches and breaks and regulating DNA repair (2). CSR occurs at specific DNA segments called switch (S) regions, which lie upstream of each constant region and contain hotspots for activation-induced cytidine deaminase-mediated cytosine deamination. The ligation of the S region with the downstream S regions is carried out by protein factors that comprise the nonhomologous end joining machinery for DNA repair (1, 2).Mismatch repair proteins play a critical role in safeguarding genetic stability. The key proteins for initiation of eukaryotic mismatch repair are homologues of bacterial MutS and MutL. In mammals, there are five MutS (Msh2, Msh3, Msh4, Msh5, and Msh6) and four MutL (Mlh1, Mlh3, Pms1, and Pms2) homologues. Each Mut homologue acts at the DNA repair or recombination site by forming heterodimers; Msh2-Msh6 (MutS␣), Msh2-Msh3 (MutS␤), M...

SignificanceMice that have homozygous deletion of the p53 tumor suppressor protein universally die of malignancy, generally before 6 months of age. We show that hemizygous deficiency of RALBP1 (RLIP76 or Rlip) confers a degree of protection from spontaneous malignancy that has never previously been observed. This discovery introduces a paradigm for p53 function, in which Rlip plays a central role as an effector that appears necessary for the cancer susceptibility of p53 null mice. Because p53 loss has a powerful effect on genomic instability that contributes to the initiation and promotion of cancers and to drug and radiation resistance in humans, our findings provide a method for prevention and therapy of p53-deficient cancer.

COHCAP (City of Hope CpG Island Analysis Pipeline) is an algorithm to analyze single-nucleotide resolution DNA methylation data produced by either an Illumina methylation array or targeted bisulfite sequencing. The goal of the COHCAP algorithm is to identify CpG islands that show a consistent pattern of methylation among CpG sites. COHCAP is currently the only DNA methylation package that provides integration with gene expression data to identify a subset of CpG islands that are most likely to regulate downstream gene expression, and it can generate lists of differentially methylated CpG islands with ∼50% concordance with gene expression from both cell line data and heterogeneous patient data. For example, this article describes known breast cancer biomarkers (such as estrogen receptor) with a negative correlation between DNA methylation and gene expression. COHCAP also provides visualization for quality control metrics, regions of differential methylation and correlation between methylation and gene expression. This software is freely available at https://sourceforge.net/projects/cohcap/.

The frequencies of nonselected mutations among adaptive Lac؉ revertants of Escherichia coli strains with and without the error-prone DNA polymerase IV (Pol IV) were compared. This frequency was more than sevenfold lower in the Pol IV-defective strain than in the wild-type strain. Thus, the mutations that occur during hypermutation are due to Pol IV.

The DNA mismatch repair (MMR) pathway is one of the most critical genome surveillance systems for governing faithful transmission of genetic information during DNA replication. The functional necessity of this pathway in humans is partially reflected by the tight link between MMR gene mutations and the development of hereditary nonpolyposis colorectal cancer. Increasing evidence has suggested a broad involvement of MMR proteins in various aspects of DNA metabolism beyond the scope of DNA mismatch correction, such as in the processes of DNA damage response and homologous recombination. Though evidence is presently lacking for potential functional involvement of hMSH4 and hMSH5 in MMR, these two proteins are thought to play roles in meiotic and mitotic DNA double strand break (DSB) repair and DNA damage responses in human cells.

Although increasing evidence has suggested that the hMSH5 protein plays an important role in meiotic and mitotic DNA recombinational repair, its precise functions in recombination and DNA damage response are presently elusive. Here we show that the interaction between hMSH5 and cAbl confers ionizing radiation (IR)-induced apoptotic response by promoting c-Abl activation and p73 accumulation, and these effects are greatly enhanced in cells expressing hMSH5 P29S (i.e. the hMSH5 variant possessing a proline to serine change within the N-terminal (Px) 5 dipep-tide repeat). Our current study provides the first evidence that the (Px) 5 dipeptide repeat plays an important role in modulating the interaction between hMSH5 and c-Abl and alteration of this dipeptide repeat in hMSH5 P29S leads to increased IR sensitivity owing to enhanced caspase-3-mediated apoptosis. In addition, RNAi-mediated hMSH5 silencing leads to the reduction of apoptosis in IR-treated cells. In short, this study implicates a role for hMSH5 in DNA damage response involving c-Abl and p73, and suggests that mutations impairing this process could significantly affect normal cellular responses to anti-cancer treatments.

The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.