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PubMed2026

Co-regulation of HIV control and cytomegalovirus pp65-specific IL-1β and TNF-α responses by genetic variants in the MHC region.

The spontaneous control of HIV infection in the absence of antiretroviral therapy, termed HIV control, is associated with genetic variation in the Major Histocompatibility Complex (MHC) locus. These variants are known to influence the immune response to HIV itself. However, people living with HIV are often co-infected with other pathogens that can also elicit immune responses, which might also be regulated by these variants. Here, we assessed whether genetic variants associated with HIV control influence cytokine responses to various co-pathogens. HIV-control-associated single nucleotide polymorphisms (SNPs) were enriched among variants regulating TNF-α and IL-1β production upon CMV pp65 peptide pool stimulation. The top enriched SNPs, rs1128175-A and rs2853971-A, were linked to lower odds of HIV control and increased cytokine responses to CMV. These SNPs were in linkage disequilibrium (LD) with classical HLA alleles HLA-B*07:02 and HLA-C*07:02. Intracellular cytokine staining showed CMV serostatus-dependent production of TNF-α by monocytes and CD8 T cells. The rs1128175-A/rs2853971-A/HLA-B*07:02/HLA-C*07:02 haplotype was associated with increased IFN-γ production by CD8 T cells upon CMV pp65 peptide pool stimulation, indicating an effect on memory responses. Quantitative trait locus (QTL) mapping showed that rs1128175 and rs2853971 influence HLA-B and HLA-C expression, DNA methylation levels and cell-type-specific cis-effects on chromatin accessibility, as well as CD8 T cell subset abundance. These QTL associations suggest that variants associated with poor HIV control are linked to heightened pro-inflammatory responses to CMV pp65 through effects on antigen presentation, epigenetic modifications, gene expression and immune cell repertoire, potentially negatively affecting HIV control status.

Humans↗
PubMed2001

Direct analysis of chromosome methylation.

DNA methylation is a possible candidate for a genomic imprinting marker in mammals. This epigenetic modification of DNA satisfies several essential criteria for the identification of the parental origin of individual alleles and larger portions of the genome: DNA methylation is stably propagated in somatic cells during cell division, it is reversible, it may inactivate the target sequence, and male and female gametes have different methylation patterns (reviewed in ref. 1).

Animals↗
PubMed2001

Probing chromatin structure with nuclease sensitivity assays.

To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). For example, the differentially methylated 5 -portion of the human SNRPN gene-a sequence element that controls imprinting in the Prader-Willi and Angelman syndromes' domain on chromosome 15q11- q13-has strong DNase-I hypersensitive sites on the unmethylated paternal chromosome (4). A differentially methylated region that regulates the imprinting of H19 and that of the neighboring insulin-like growth factor-2 gene on mouse chromosome 7 was also found to have parental chromosome-specific hypersensitive sites (5,6). The precise nature of the allelic nuclease hypersensitivity in these and other imprinted loci remains to be determined in more detail, for example, by applying complementary chromatin methodologies (7,8). However, it is commonly observed that a nuclease hypersensitive site corresponds to a small region where nucleosomes are absent or partially disrupted.

Animals↗
PubMed2004

Real-time PCR-based assay for quantitative determination of methylation status.

The best studied epigenetic modification in mammals is the methylation of cytosine. During the development and progression of malignant neoplasia, a global hypomethylation is often accompanied by a locus-specific increase in methylation. Also, during normal development specific alterations in DNA methylation patterns take place. In recent years it has become clear that in many situations only quantitative changes in methylation levels occur and that the pure qualitative detection of cytosine methylation misses important biological and pathophysiological information. Therefore, several protocols were developed for the quantitative detection of cytosine methylation. Here, we describe a real-time polymerase chain reaction-based assay for the sensitive and precise quantification of methylated and unmethylated alleles after bisulfite treatment of genomic DNA. In addition to providing quantitative methylation data, this methodology is suitable for high-throughput analysis.

Animals↗
PubMed2004

DMB (DNMT-magnetic beads) assay: measuring DNA methyltransferase activity in vitro.

DNA methylation is an epigenetic modification of DNA that leads to heritable alterations in transcriptional regulation and conformational changes in chromatin structure of higher eukaryotes. Mammalian DNA methyltransferases, which are the enzymes responsible for DNA methylation, have attracted the attention of both basic and clinical researchers because they appear to participate in embryogenesis and carcinogenesis via chromatin modification. DNA methyltransferase catalyzes the transfer of a methyl group into DNA strands. Since traditional assays for DNA methyltransferase activity in vitro have insufficient reproducibility, there is a need in the art for more sensitive and quantitative methods for measuring enzymatic activity. We report a novel assay system, in which the activity of a DNA methyltransferase is measured as the incorporation of tritium into biotinylated DNA oligonucleotides. The DNA is immobilized onto magnetic beads with streptavidin covalently attached to the bead surface. The radioactive DNA can easily be separated from the unreacted radioactive substrate using a magnet. The radioactivity is counted by the liquid scintillation system. This DMB assay is simple and easy, has very low background, and, most importantly, is highly reproducible for the precise enzymatic analysis of any DNA methyltransferase in vitro.

Animals↗
PubMed2006

Nuclear reprogramming: an overview.

Nuclear reprogramming is an intriguing phenomenon, in which specialized somatic cells reacquire pluripotency through the global resetting of epigenetic modifications without changes occurring to their nuclear DNA information. The nuclear reprogramming activity retained by unfertilized eggs and embryonic stem cells is readily observable through the techniques of nuclear transplantation and cell fusion, respectively. Furthermore, researches involving somatic stem cells have provided evidence that spontaneous cell fusion functions, at least in part, in maintaining the homeostasis of various tissues through the generation of replacement cells in vivo. Novel approaches by application of the nuclear reprogramming activity may open new avenues producing genetically matched personalized pluripotential stem cells derived from patient somatic cells.

Animals↗
PubMed2005

Role of IL-6 in the neural stem cell differentiation.

In the developing brain neurons, astrocytes, and oligodendrocytes are differentiated from common multipotent progenitors called neural stem cells. We have examined the effect of cytokines on mouse neuroepithelial cells that are known to contain neural stem cells. Cytokines belonging to the interleukin (IL)-6 family and those classified into the bone morphogenetic protein (BMP) family act in synergy on neuroepithelial cells to induce astrocyte differentiation. Cooperation of these two types of cytokines is explained by the formulation of a complex between their respective downstream transcription factors (signal tranducer and activator of transcription [STAT]-3 and Smad1), bridged by a transcriptional coactivator (p300). Whereas BMPs family cytokines are involved in astrocyte differentiation, they inhibit neuronal differentiation. This appears to change the fate of neural progenitor cells from neurogenesis to astrocytogenesis. Interestingly, during brain development, neuronal differentiation starts at a very early stage and continues throughout development, whereas astrocytes appear just before term. We have shown that astrocyte differentiation is largely dependent on IL-6 family cytokine-mediated STAT3 activation and that there exists a STAT3 binding element in the promoter region of the gene for the astrocyte marker glial fibrillary acidic protein. A cytosine residue in this element is highly methylated in neuroepithelial cells in a mid-gestational stage but becomes demethylated in accordance with brain development. Because this methylation inhibits STAT3 binding, we suggest that DNA methylation is a critical determinant in the developmental stage-dependent regulation of astrocytogeneis. In conclusion, fate of neural stem cells during development is regulated by cell-intrinsic programs, such as epigenetic modification (including DNA methylation), and signaling crosstalk of cell-external mediators (including IL-6 family cytokines).

Animals↗
PubMed2003

DNA methylation in haematological malignancies: the role of decitabine.

Normal cell development and function is dependent upon controlled gene expression. DNA methylation is an epigenetic modification that can play an important role in the control of gene expression. DNA methylation at cytosine residues in gene promoter CpG sequences is known to inhibit gene transcription. Inappropriate inhibition of the transcription of tumour suppressor genes, genes that inhibit angiogenesis and metastasis and genes involved in DNA repair by uncontrolled methylation, can lead to unregulated growth and proliferation of a cell and carcinogenesis. Promoter hypermethylation affecting the p16 gene, resulting in gene silencing, has been shown to occur in many human solid tumours and a 'hypermethylation profile' in some leukaemias has been defined. The molecular mechanisms by which aberrant DNA methylation takes place during carcinogenesis are still not clear. However, the large number of target genes (involved in tumorigenesis) that are silenced by aberrant methylation suggests that inhibition of this process may have potential as cancer therapy. Decitabine (NSC-127716, Dacogen; SuperGen) is a potent and specific hypomethylating agent and an inhibitor of the DNA methyltransferase activity that mediates DNA methylation. Decitabine has been shown to have a broad range of antineoplastic activity in preclinical studies. This agent has exhibited significant activity in the treatment of patients with myelodysplastic syndrome, chronic myeloid leukaemia and acute myeloid leukaemia, although clinical Phase I and II studies with solid tumours have not been very promising. Phase II and III studies are currently ongoing to evaluate decitabine, both alone and in combination, in various stages of these haematological malignancies.

Animals↗
PubMed2003

Disruption of imprinting in cloned mouse fetuses from embryonic stem cells.

Cloned mice typically display abnormal development, such as overgrowth of fetuses and placentae. Quantitative expression analysis of eight imprinted genes (H19, Igf2, Igf2r, Air, Peg1/Mest, Peg3, Nuronatin (Nnat) and Ndn) and an alternate transcript of Igf2 (P0) in embryonic stem cloned fetuses and placentae at days 9.5, 12.5 and 17.5 after mating was carried out by real time PCR to investigate whether epigenetic modification of imprinted genes is responsible for overgrowth of the fetus and placental hypertrophy. In addition, the methylation pattern through the bisulphite sequencing method in differentially methylated regions of H19 and Igf2r was examined in day 9.5 fetuses and placentae. The results showed clearly that the expression of H19 gene decreased in cloned fetuses at days 12.5 and 17.5 after mating and in placentae at day 17.5 after mating, and Igf2 was also repressed in fetuses at days 9.5 and 12.5 after mating and in placentae at day 17.5 after mating. In contrast, the transcription of P0, which is a placental-specific transcript variant of Igf2, increased at more than four times the control in cloned placenta at day 12.5 after mating. Day 9.5 fetuses that have developed normally revealed only hypermethylated alleles in the H19 differently methylated region (DMR), and both hyper- and hypomethylated alleles in the Igf2r DMR2. These results show that inappropriate reprogramming in some imprinted genes affects the development of cloned embryos, and that aberrant P0 Igf2 transcription in particular may cause the overgrowth of cloned fetuses and placentae.

Animals↗
PubMed2006

Nuclear transfer reprogramming does not improve the low developmental potency of embryonic stem cells induced by long-term culture.

Epigenetic states of embryonic stem (ES) cells are easily altered by long-term cultivation and lose their developmental potential. To rescue this reduced developmental capacity, nuclear transfer (NT) of ES cells was carried out, and original ES and ES cells from cloned blastocysts (ntES) cells established after NT were compared with in vitro differentiation ability and developmental potential by embryoid body formation and tetraploid aggregation respectively. In the establishment of ntES cell lines, the oocytes fused with the ES cell were activated, and further cultured to cloned blastocysts. When in vitro differentiation ability was examined between original and ntES cell lines derived from ES cells with extensive passages (ES-ep), the day of appearance of simple embryoid body, cystic embryoid body, and spontaneous beating was almost similar. The developmental rates of ES-ep cells, that aggregated with tetraploid embryos to term, ranged from 3 to 6%. Moreover, the majority of live pups died soon after birth. In the ntES cell lines derived from ES-ep cells, developmental rates ranged from 0 to 5%. Those pups also died soon after birth, similar to the ES-ep-derived pups. These results suggest that profound epigenetic modifications of ES cells were retained in the re-established cell lines by NT.

Animals↗
PubMed2004

Alterations of common chromosome fragile sites in hematopoietic malignancies.

Conditions of replication stress affect expression of all common fragile regions, including FRA3B (chromosome 3p14.2), FRA16D (16q23), FRA6E (6q26), FRA7G (7q31.2), and FRAXB (Xp22.3), and a number of cancer cell lines exhibit homozygous deletions in 2 or more common fragile regions. In 1996 the fragile histidine triad (FHIT) gene was isolated from the region encompassing the most active fragile FRA3B locus, and recently the WW domain-containing oxidoreductase gene (WWOX) was identified at FRA16D. These 2 fragile genes are altered or deleted in various epithelial tumors and exhibit tumor suppressor function. Aberration or absence of WWOX expression recently was detected in primary hematopoietic malignancies. The aberration resulted not only from genomic deletions but also possibly from epigenetic modifications associated with expression of fragility. Thus chromosomal aberrations at common fragile sites, in addition to the well-defined hallmark leukemia chromosome translocations, are involved in clinicopathological outcomes of hematopoietic malignancies.

Acid Anhydride Hydrolases↗
PubMed2005

Variable reprogramming of the pluripotent stem cell marker Oct4 in mouse clones: distinct developmental potentials in different culture environments.

A prevailing view of cloning by somatic-cell nuclear transfer is that reprogramming of gene expression occurs during the first few hours after injection of the nucleus into an oocyte, that the process is stochastic, and that the type of reprogramming needed for cloning success is foreign and unlikely to be readily achieved in the ooplasm. Here, we present evidence that the release of reprogramming capacity is contingent on the culture environment of the clone while the contribution of aneuploidy to altered gene expression is marginal. In particular, the rate of blastocyst formation in clones and the regional distribution of mRNA for the pluripotent stem cell marker Oct4 in clonal blastocysts was highly dependent on the culture environment after cumulus cell nuclear transfer, unlike that in genetically equivalent zygotes. Epigenetic modifications of genetically identical somatic nuclei continue after the first cell division of the clones and are amenable to a degree of experimental control, and their development to the blastocyst stage and appropriate expression of Oct4 predict further outcome, such as derivation of embryonic stem (ES) cells, but not fetal development. This observation indicates that development to the blastocyst stage is not equivalent to full reprogramming and lends support to the novel concept that ES cells are not the equivalent of the inner cell mass, hence the discrepancy between ES cell derivability and fetal development of clones.

Animals↗
PubMed2005

DNA methylation is required for silencing of ant4, an adenine nucleotide translocase selectively expressed in mouse embryonic stem cells and germ cells.

The capacity for cellular differentiation is governed not only by the repertoire of available transcription factors but by the accessibility of cis-regulatory elements. Studying changes in epigenetic modifications during stem cell differentiation will help us understand how cells maintain or lose differentiation potential. We investigated changes in DNA methylation during the transition of pluripotent embryonic stem cells (ESCs) into differentiated cell types. Using a methylation-sensitive restriction fingerprinting method, we identified a novel adenine nucleotide (ADP/ATP) translocase gene, Ant4, that was selectively hypomethylated and expressed in undifferentiated mouse ESCs. In contrast to other pluripotent stem cell-specific genes such as Oct-4 and Nanog, the Ant4 gene was readily derepressed in differentiated cells after 5-aza-2'-deoxycytidine treatment. Moreover, expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b was essential for repression and DNA methylation of the Ant4 gene during ESC differentiation. Although the deduced amino acid sequence of Ant4 is highly homologous to the previously identified Ant isoforms, the expression of Ant4 was uniquely restricted to developing gametes in adult mice, and its promoter hypomethylation was observed only in testis. Additionally, Ant4 was expressed in primordial germ cells. These data indicate that Ant4 is a pluripotent stem cell- and germ cell-specific isoform of adenine nucleotide translocase in mouse and that DNA methylation plays a primary role in its transcriptional silencing in somatic cells.

Animals↗
PubMed2006

The emerging roles of DNA methylation in the clinical management of prostate cancer.

Aberrant DNA methylation is one of the hallmarks of carcinogenesis and has been recognized in cancer cells for more than 20 years. The role of DNA methylation in malignant transformation of the prostate has been intensely studied, from its contribution to the early stages of tumour development to the advanced stages of androgen independence. The most significant advances have involved the discovery of numerous targets such as GSTP1, Ras-association domain family 1A (RASSF1A) and retinoic acid receptor beta2 (RARbeta2) that become inactivated through promoter hypermethylation during the course of disease initiation and progression. This has provided the basis for translational research into methylation biomarkers for early detection and prognosis of prostate cancer. Investigations into the causes of these methylation events have yielded little definitive data. Aberrant hypomethylation and how it impacts upon prostate cancer has been less well studied. Herein we discuss the major developments in the fields of prostate cancer and DNA methylation, and how this epigenetic modification can be harnessed to address some of the key issues impeding the successful clinical management of prostate cancer.

Biomarkers, Tumor↗
PubMed2006

The Sycp1-Cre transgenic mouse and male germ cell inhibition of NF-kappa b.

Successful spermatogenesis requires autocrine, paracrine, and endocrine signaling throughout the testes. The seminiferous tubules contain somatic Sertoli cells in tight association with numerous germ cell populations. To address the in vivo biologic roles of genes during spermatogenesis, spatial and temporal restriction of gene inhibition is a useful approach. To this end, Cre-LoxP technology can produce cell-specific knockdowns of genes, allowing dissection of the underlying processes that manifest as functional deficits in whole animals. Here we report the use of the synaptonemal complex protein 1-Cre (Sycp1-Cre) to create germ cell-specific nuclear factor kappaB knockdown mice through floxed IkappaB kinasebeta. We observed a LoxP gene recombination rate of approximately 43% using Sycp1-Cre, as determined by offspring genotype. In addition, we confirm that, with multiple generations, the LoxP sites fail to recombine due to epigenetic modification. This detailed examination of the meiotic Sycp1-Cre recombinase activity highlights the obstacles to germ cell-specific gene inhibition through Cre/LoxP technology in the testis. Taken together, these data demonstrate a need for early spermatogonial expression of Cre recombinase, as an alternative to meiotic Cre expression, for the creation of germ cell-specific knockout mice.

Animals↗
PubMedDate not supplied

Role of the cytosine DNA-methyltransferase and p16INK4a genes in the development of mouse lung tumors.

CpG island methylation is an epigenetic modification of DNA associated with the silencing of gene transcription. The p16INK4a (p16) tumor suppressor gene is inactivated in human non-small cell lung cancers (NSCLCs) by either homozygous deletion or aberrant methylation. Inactivation of tumor suppressor genes by methylation has been linked in part to altered activity of the cytosine DNA-methyltransferase (DNA-MTase), the enzyme that catalyzes DNA methylation at CpG sites. The purpose of these studies was to define the role of DNA-MTase and p16 in the development of murine lung cancer. DNA-MTase activity was determined in alveolar type II and Clara cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity leading to an increase in overall DNA methylation was found only in alveolar type II cells, the target for murine adenocarcinomas. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. In addition, enzyme activity increased incrementally during lung cancer progression. Expression of p16 was detected in all primary lung tumors from A/J mice; however, levels of expression differed by up to 15-fold between tumors. The apparent low levels of expression seen in approximately half of the tumors was not attributed to methylation of the p16 gene. In contrast to the detection of p16 expression in primary tumors, this gene was deleted in four tumor-derived cell lines induced in the A/J mouse by NNK. The results from these studies indicate that the modulation of DNA-MTase activity was cell specific, segregated with susceptibility, and occurred early in neoplastic evolution. Thus, the marked increase in enzyme activity detected in alveolar type II cells after carcinogen treatment could be a major factor contributing to the high susceptibility for chemical-induced neoplasia associated with the A/J mouse strain. The inactivation of the p16 gene in murine cancers induced by NNK most likely arises as a late event via homozygous deletion.

Adenocarcinoma↗
PubMed1997

Multiplicity of antioxidant enzyme catalase in mouse liver cells.

Multiplicity of catalase activity has been observed in crude homogenates from the tissue and cell lines of mouse liver by ethanol/Triton X-100/heat treatment. The five enzymatically active catalase bands were designated as CAT1, CAT2, CAT3, CAT4, and CAT5 with a nondenatured molecular mass of 270kDa, 258kDa, 229kDa, 210kDa, or 197kDa, respectively. Cultured mouse liver cell lines, mouse liver tissue homogenate, and pure mouse liver catalase showed only one catalase band (CAT1) after ethanol/Triton X-100 treatment at 4 degrees C for 72 hr. The same treatment but incubated at 37 degrees C for 72 hr yielded three bands (CAT2, CAT4, CAT5) in normal cell line, only one band (CAT5) in MNNG-transformed and SV40-transformed cells, two bands (CAT1, CAT4) in mouse liver tissue homogenates, and two bands (CAT1, CAT3) in pure mouse liver catalase. These five catalase bands were further biochemically characterized. The CAT1, CAT2, and CAT3 are sensitive to heat (68 degrees C, 1 min), while CAT4 and CAT5 are rather heat resistant. The sensitivity to catalase inhibitors, such as aminotriazole, azide, or cyanide varies among the isoforms. Protease inhibitors could prevent the formation of CAT3 and CAT4, but not CAT5. Treatment with protease, however, removed all forms of catalase except CAT5. We conclude from this study that the appearance of different catalase bands is likely due to epigenetic modification of the protein, particularly proteolysis. The lowered catalase activity in transformed cells might also be attributable to the loss of two catalase isoforms.

Animals↗
PubMed2026

Cell-of-origin Discovery in Infant Leukemia through Integration of 3D Models and Patient Transcriptomic Data.

Pediatric hematological malignancies remain challenging to investigate and model due to the age group-specificity of certain genetic abnormalities. In utero origin has been demonstrated for a subset of pediatric leukemias, placing their respective cell of origin (CoO) during embryonic development. We recently reported a 3D hemogenic gastruloid (haemGx) model of embryonic blood formation derived from mouse embryonic stem cells, resolving the spatio-temporal complexity of developmental hematopoiesis. Importantly, it allows genetic engineering to introduce disease-relevant mutations. Using haemGx, we modeled the most common acute myeloid leukemia exclusive to infants (infAML), subtype t(7;12)(q36;p13), which arises in utero and is characterized by MNX1 overexpression. Here, we detail a method to define susceptibility to specific mutations that integrate phenotypic and transcriptional changes in the haemGx system and compares them with patient data. By proxy of our MNX1-overexpression haemGx, we show a pipeline from cell engineering to downstream analyses of leukemogenic potential. In particular, we focus on the clinical relevance of the model by integrating single-cell and/or bulk RNA sequencing from the haemGx platform with patient data to extract cellular composition and temporal placement of the putative CoO. This method is adaptable to the introduction of other oncogenic mutations, chromosomal rearrangements, or epigenetic modifications, as well as to chemical perturbations, including drug vulnerability and growth factor dependence. This flexibility allows for broad application across diverse disease contexts, enabling mechanistic dissection of how specific alterations disrupt early developmental trajectories with clinical relevance.

Humans↗