DNMT1 Y495C mutation interferes with maintenance methylation of imprinting control regions.

Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E) is a rare autosomal dominant neurological disorder due to missense mutations in DNA methyltransferase 1 (DNMT1). To investigate the nature of the dominant effect, we compared methylomes of transgenic R1wtDnmt1 and R1Dnmt1Y495C mouse embryonic stem cells (mESCs) overexpressing WT and the mutant mouse proteins respectively, with the R1 (wild-type) cells. In case of R1Dnmt1Y495C, 15 out of the 20 imprinting control regions were hypomethylated with transcript level dysregulation of multiple imprinted genes in ESCs and neurons. Non-imprinted regions, minor satellites, major satellites, LINE1 and IAP repeats were unaffected. These data mirror the specific imprinting defects associated with transient removal of DNMT1 in mESCs, deletion of the maternal-effect DNMT1o variant in preimplantation mouse embryos, and in part, reprogramming to naïve human iPSCs. This is the first DNMT1 mutation demonstrated to specifically affect Imprinting Control Regions (ICRs), and reinforces the differences in maintenance methylation of ICRs over non-imprinted regions. Consistent with nervous system abnormalities in the HSAN1E disorder and involvement of imprinted genes in normal development and neurogenesis, R1Dnmt1Y495C cells showed dysregulated pluripotency and neuron marker genes, and yielded more slender, shorter, and extensively branched neurons. We speculate that R1Dnmt1Y495C cells produce predominantly dimers containing mutant proteins, leading to a gradual and specific loss of ICR methylation during early human development.

Generation of a transgenic mouse embryonic stem cell line overexpressing DNMT1.

DNMT1 overexpression is reported in disorders like schizophrenia, bipolar, epilepsy and multiple cancer types. Here, we used non-homologous recombination to generate R1Dnmt1WT-1, a mouse embryonic stem cell (ESC) line carrying a Dnmt1 cDNA transgene to achieve about two-fold overexpression. This ESC line showed increased transcript levels of Sox2 pluripotency marker. R1Dnmt1WT-1 embryoid bodies showed increased levels of Lefty1 (endoderm), Tbxt and Acta2 (mesoderm), and Pax6 (ectoderm) transcripts. This new line showed normal karyotype and microsatellite profiles making it useful in studying carcinogenesis and abnormal neurogenesis due to DNMT1 overexpression.

Dysregulation of schizophrenia-associated genes and genome-wide hypomethylation in neurons overexpressing DNMT1.

Aim: To study the effects of DNMT1 overexpression on transcript levels of genes dysregulated in schizophrenia and on genome-wide methylation patterns. Materials & methods: Transcriptome and DNA methylome comparisons were made between R1 (wild-type) and Dnmt1tet/tet mouse embryonic stem cells and neurons overexpressing DNMT1. Genes dysregulated in both Dnmt1tet/tet cells and schizophrenia patients were studied further. Results & conclusions: About 50% of dysregulated genes in patients also showed altered transcript levels in Tet/Tet neurons in a DNA methylation-independent manner. These neurons unexpectedly showed genome-wide hypomethylation, increased transcript levels of Tet1 and Apobec 1-3 genes and increased activity and copy number of LINE-1 elements. The observed similarities between Tet/Tet neurons and schizophrenia brain samples reinforce DNMT1 overexpression as a risk factor.

Lay abstract DNMT1 controls cytosine methylation, which is often associated with reduced gene expression. Increased levels of DNMT1 is a risk factor for schizophrenia but information on the affected genes is limited. In this study, ∼50% of genes with altered levels of messenger RNAs in schizophrenia patients were also altered in neurons with increased DNMT1. Surprisingly, the neurons with higher DNMT1 levels showed genome-wide decrease in methylation. These findings uncover a new type of gene dysregulation that is independent of DNMT1's catalytic activity.

Generation of a transgenic mouse embryonic stem cell line expressing Dnmt1Y495C mutation associated with HSAN1E disorder.

DNMT1 Y495C is the most common mutation associated with hereditary sensory and autonomic neuropathy type 1E, and dementia. Here we employed non-homologous recombination and generated a mouse embryonic stem cell line carrying a transgene expressing DNMT1 Y495C mutation in the wild-type background. The resultant cell line, Dnmt1Y495C-1 showed increased transcript levels of the Oct4 and Sox2 pluripotency markers and Gata6 and Pax6 germ layer markers. This cell line showed normal karyotype, expression of the mutant Dnmt1 and wild-type transcripts in approximately equal ratios and is a useful model for studying the abnormal neurogenesis due to the DNMT1 Y495C mutation.

Functional Analysis of DNMT1 SNPs (rs2228611 and rs2114724) Associated with Schizophrenia.

A recent study showed the association of minor alleles of rs2228611 (T allele) and rs2114724 (T allele) of DNMT1 with schizophrenia (SZ) and suggested their effects on splicing of the transcripts. We performed a replication study using 310 controls and 304 SZ patients and confirmed the association of the homozygous minor allele genotypes with SZ (P = 0.04 for rs2114724 and P = 0.007 for rs2228611). This significant association persisted after Bonferroni correction when the previously published data of 301 controls and 325 patients were also considered (P ≤ 0.0002). In addition, we found that the proportion of male patients with homozygous minor alleles at rs2114724 was significantly higher than that of females (P = 0.002). When haplotype analysis of both loci was performed, we observed a significant association of T/T-T/T and T/T-C/T (P = 0.04) haplotypes with SZ. To gain insights into the functional effects of the two SNPs on the levels of DNMT1 transcripts, quantitative real-time PCR experiments were performed using peripheral blood monocytes from 10 individuals each with T/T-T/T (homozygous minor allele), C/T-C/T (heterozygous), and C/C-C/C (homozygous major allele) haplotypes. Independently, the levels of DNMT1 protein were also compared in three individuals each by immunofluorescence. These results suggest that neither DNMT1 transcript nor the protein levels were significantly different in the peripheral blood monocytes among the individuals studied for the three groups. Taken together, our results confirm that the two minor alleles in homozygosity are associated with SZ but with no discernible effects on transcript or protein levels of DNMT1 in the peripheral blood monocytes of the small number of samples tested.

Reproducible differentiation and characterization of neurons from mouse embryonic stem cells.

Investigation on the effects of disease-associated mutations on neurodevelopment is an essential approach to understand the molecular basis of neurological disorders and can be achieved by generating suitable animal models. However, some of the mutations preclude development of animal models, leaving cell-based models as the only options. Mouse embryonic stem cells (mESCs) are attractive because of the well-established technologies for introducing disease-associated mutations and the feasibility of investigating the abnormalities during different stages of neurogenesis. Importantly, such transgenic mESCs enable large-scale screening and identification of the most promising small molecules and/or drug candidates before undertaking expensive animal studies. Although neuronal differentiation from mESCs is one of the earliest methods to be developed, we observed that the published as well as publicly available methods did not yield neurons consistently. Here, we describe a 16-day differentiation protocol that consistently induced differentiation of mESCs into neurons. This step-wise protocol enables monitoring of the neuronal differentiation process at different stages as well as characterization using the markers for immature and mature neurons by using immunocytochemistry and quantitative real-time PCRs.•Development of a method for differentiating mouse ES cells into neurons.•Differentiating the mouse ES cells into embryoid bodies prior to induction of neuronal differentiation results in better neuron formation.

Genome-wide methylation data from R1 (wild-type) and the transgenic Dnmt1Tet/Tet mouse embryonic stem cells overexpressing DNA methyltransferase 1 (DNMT1).

Defects in epigenetic mechanisms are well-recognized in multiple neurodevelopmental disorders including Schizophrenia (SZ). In addition to aberrant epigenetic marks, dysregulated epigenetic machinery was also identified among the contributory factors in SZ patients. Among these, overexpression of DNA methyltransferase 1 (DNMT1) was the first to be identified. In this context, Dnmt1tet/tet (Tet/Tet), a mouse embryonic stem cell (ESC) line that overexpresses DNMT1 in ESCs and neurons, was developed to study abnormal neurogenesis. In an attempt to understand whether DNMT1 overexpression is associated with aberrant DNA methylation, we compared the genome-wide methylation levels of R1 (wild-type) and Tet/Tet ESCs and their neuronal derivatives by RRBS. The RRBS data (GSE152817) showed an average mappability of ∼59% and an average coverage of 40X per locus. The data was processed to determine the methylation percentages of target genes and was visualized using the UCSC genome browser. The observed methylation differences were validated by Combined Bisulfite Restriction Analysis (COBRA). The methylome data described here can be used to study the relationship between DNMT1 overexpression, alterations in methylation levels and dysregulation of SZ-associated genes.

Analysis of transcript levels of a few schizophrenia candidate genes in neurons from a transgenic mouse embryonic stem cell model overexpressing DNMT1.

Overexpression of DNA Methyltransferase I (DNMT1) is considered as one of the etiological factors for schizophrenia (SZ). However, information on genes subjected to dysregulation because of DNMT1 overexpression is limited. To test whether a larger group of SZ-associated genes are affected, we selected 15 genes reported to be dysregulated in patients (Gad1, Reln, Ank3, Cacna1c, Dkk3, As3mt, Ppp1r11, Smad5, Syn1, Wnt1, Pdgfra, Gsk3b, Cxcl12, Tcf4 and Fez1). Transcript levels of these genes were compared between neurons derived from Dnmt1tet/tet (Tet/Tet) mouse embryonic stem cells (ESCs) that overexpress DNMT1 with R1 (wild-type) neurons. Transcript levels of thirteen genes were significantly altered in Tet/Tet neurons of which, the dysregulation patterns of 11 were similar to patients. Transcript levels of eight out of these eleven were also significantly altered in Tet/Tet ESCs, but the dysregulation patterns of only five were similar to neurons. Comparative analyses among ESCs, embryoid bodies and neurons divided the 15 genes into four distinct groups with a majority showing developmental stage-specific patterns of dysregulation. Reduced Representational Bisulfite Sequencing data from neurons did not show any altered promoter DNA methylation for the dysregulated genes. Doxycycline treatment of Tet/Tet ESCs that eliminated DNMT1, reversed the direction of dysregulation of only four genes (Gad1, Dkk3, As3mt and Syn1). These results suggest that 1. Increased DNMT1 affected the levels of a majority of the transcripts studied, 2. Dysregulation appears to be independent of promoter methylation, 3. Effects of increased DNMT1 levels were reversible for only a subset of the genes studied, and 4. Increased DNMT1 levels may affect transcript levels of multiple schizophrenia-associated genes.

Selected renal cells modulate disease progression in rodent models of chronic kidney disease via NF-κB and TGF-ß1 pathways.

AIM: Identification of mechanistic pathways for selected renal cell (SRC) therapeutic bioactivity in rodent models of chronic kidney disease. MATERIALS & METHODS: In vivo and in vitro functional bioassays applied to investigate regenerative outcomes associated with delivery of SRC to diseased rodent kidney. RESULTS: In vivo, SRC reduces chronic infiltration by monocytes/macrophages. SRC attenuates NF-κB and PAI-1 responses while simultaneously promoting host tubular cell expansion through trophic cues. In vitro, SRC-derived conditioned media attenuates TNF-α-induced NF-κB response, TGF-ß-mediated PAI-1 response and increases expression of transcripts associated with cell cycle regulation. Observed bioactive responses were from vesicle and nonvesicle-associated factors, including specific miRNAs. CONCLUSION: We identify a paracrine mechanism for SRC immunomodulatory and trophic cues on host renal tissues, catalyzing long-term functional benefits in vivo.

A population of selected renal cells augments renal function and extends survival in the ZSF1 model of progressive diabetic nephropathy.

New treatment paradigms that slow or reverse progression of chronic kidney disease (CKD) are needed to relieve significant patient and healthcare burdens. We have shown that a population of selected renal cells (SRCs) stabilized disease progression in a mass reduction model of CKD. Here, we further define the cellular composition of SRCs and apply this novel therapeutic approach to the ZSF1 rat, a model of severe progressive nephropathy secondary to diabetes, obesity, dyslipidemia, and hypertension. Injection of syngeneic SRCs into the ZSF1 renal cortex elicited a regenerative response that significantly improved survival and stabilized disease progression to renal structure and function beyond 1 year posttreatment. Functional improvements included normalization of multiple nephron structures and functions including glomerular filtration, tubular protein handling, electrolyte balance, and the ability to concentrate urine. Improvements to blood pressure, including reduced levels of circulating renin, were also observed. These functional improvements following SRC treatment were accompanied by significant reductions in glomerular sclerosis, tubular degeneration, and interstitial inflammation and fibrosis. Collectively, these data support the utility of a novel renal cell-based approach for slowing renal disease progression associated with diabetic nephropathy in the setting of metabolic syndrome, one of the most common causes of end-stage renal disease.

Molecular characterization of the regenerative response induced by intrarenal transplantation of selected renal cells in a rodent model of chronic kidney disease.

Dedifferentiation and proliferation of resident tubular epithelial cells is a mechanism of action potentially contributing to repair and regeneration in kidneys presenting with ischemic or chronic disease. To more efficiently develop cell and tissue engineering technologies for the kidney, we have developed molecular assays to evaluate the acquisition of a pluripotent state associated with stem/progenitor cell phenotype during induction of a regenerative response within the kidneys of rats with chronic kidney disease (CKD) following therapeutic intervention. Intrarenal delivery of selected bioactive renal cells leads to significant upregulation of pluripotency-associated SOX2 mRNA within the diseased kidney tissue from 1 to 24 weeks after treatment. The overall regenerative response index was assessed by quantitative composite expression of CD24, NODAL and LEFTY1 proteins, which were induced within 1 week of cell treatment and peaked at 12 weeks after treatment, reaching statistical significance (p < 0.05) compared to untreated CKD controls. Molecular assays that incorporate the assessment of SOX2 and the regenerative response index may prove to be valuable tools for the detection and monitoring of the tissue response after the delivery of regenerative treatments for CKD, thereby significantly shortening the developmental timelines associated with such therapies.

Isolation, characterization, and expansion methods for defined primary renal cell populations from rodent, canine, and human normal and diseased kidneys.

Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.

Tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in rodent model of chronic kidney disease.

Established chronic kidney disease (CKD) may be identified by severely impaired renal filtration that ultimately leads to the need for dialysis or kidney transplant. Dialysis addresses only some of the sequelae of CKD, and a significant gap persists between patients needing transplant and available organs, providing impetus for development of new CKD treatment modalities. Some postulate that CKD develops from a progressive imbalance between tissue damage and the kidney's intrinsic repair and regeneration processes. In this study we evaluated the effect of kidney cells, delivered orthotopically by intraparenchymal injection to rodents 4-7 wk after CKD was established by two-step 5/6 renal mass reduction (NX), on the regeneration of kidney function and architecture as assessed by physiological, tissue, and molecular markers. A proof of concept for the model, cell delivery, and systemic effect was demonstrated with a heterogeneous population of renal cells (UNFX) that contained cells from all major compartments of the kidney. Tubular cells are known contributors to kidney regeneration in situ following acute injury. Initially tested as a control, a tubular cell-enriched subpopulation of UNFX (B2) surprisingly outperformed UNFX. Two independent studies (3 and 6 mo in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and blood urea nitrogen). The specificity of B2 effects was verified by direct comparison to cell-free vehicle controls and an equivalent dose of non-B2 cells. Quantitative histological evaluation of kidneys at 6 mo after treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor (TGF)-ß1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.

The crystal structure of TREX1 explains the 3' nucleotide specificity and reveals a polyproline II helix for protein partnering.

The TREX1 enzyme processes DNA ends as the major 3' --> 5' exonuclease activity in human cells. Mutations in the TREX1 gene are an underlying cause of the neurological brain disease Aicardi-Goutières syndrome implicating TREX1 dysfunction in an aberrant immune response. TREX1 action during apoptosis likely prevents autoimmune reaction to DNA that would otherwise persist. To understand the impact of TREX1 mutations identified in patients with Aicardi-Goutières syndrome on structure and activity we determined the x-ray crystal structure of the dimeric mouse TREX1 protein in substrate and product complexes containing single-stranded DNA and deoxyadenosine monophosphate, respectively. The structures show the specific interactions between the bound nucleotides and the residues lining the binding pocket of the 3' terminal nucleotide within the enzyme active site that account for specificity, and provide the molecular basis for understanding mutations that lead to disease. Three mutant forms of TREX1 protein identified in patients with Aicardi-Goutières syndrome were prepared and the measured activities show that these specific mutations reduce enzyme activity by 4-35,000-fold. The structure also reveals an 8-amino acid polyproline II helix within the TREX1 enzyme that suggests a mechanism for interactions of this exonuclease with other protein complexes.

Initial cleavage of the human immunodeficiency virus type 1 GagPol precursor by its activated protease occurs by an intramolecular mechanism.

Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.

Mutagenesis of the dimer interface residues of tethered and untethered HIV-1 protease result in differential activity and suggest multiple mechanisms of compensation.

As is the case for all retroviruses, the protease of HIV-1 is only functional as a homodimer; dimerization of two protease monomers results in the formation of the enzyme active site. This dimer structure is supported primarily by interactions between the first four amino-terminal and the last four carboxy-terminal amino acids. These eight amino acids form a beta-sheet in which hydrophobic residues are oriented towards the core of the molecule and polar residues are directed towards the solvent. Although the structure of the dimer interface has been determined, the forces that support dimerization have not been fully characterized. Here, we describe a tethered construct in which two protease monomers are joined by a 5 amino acid linker. We evaluate the relative role of each dimer interface residue in functional homo- and heterodimers. Our studies indicate that the hydrophobic residues of the dimer interface are particularly important in maintaining enzyme activity and that enzyme activity is more sensitive to substitutions of the C-terminal amino acids. Further, we demonstrate that the presence of the tether is able to compensate for mutations within the dimer interface that inactivate the enzyme.