Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

<p>Abstract</p> <p>Background</p> <p>Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development.</p> <p>Results</p> <p>A developmental study covering the first 12 days following birth was conducted on a <it>Dnmt3L </it>mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in <it>Dnmt3L</it>^-/-males, pointing to a mitotic defect. A temporal expression study showed that expression of <it>Dnmt3L </it>is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in <it>Dnmt3L</it>^+/+germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content.</p> <p>Conclusion</p> <p>Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed.</p

Subgroup-specific gene expression profiles and mixed epistasis in chronic lymphocytic leukemia

Understanding the molecular and phenotypic heterogeneity of cancer is a prerequisite for effective treatment. For chronic lymphocytic leukemia (CLL), recurrent genetic driver events have been extensively cataloged, but this does not suffice to explain the disease's diverse course. Here, we performed RNA sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major, orthogonal axes of gene expression variation: the first one represented the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, the three-group stratification of CLL by global DNA methylation. The second axis aligned with trisomy 12 status and affected chemokine, MAPK and mTOR signaling. We discovered non-additive effects (epistasis) of IGHV mutation status and trisomy 12 on multiple phenotypes, including the expression of 893 genes. Multiple types of epistasis were observed, including synergy, buffering, suppression and inversion, suggesting that molecular understanding of disease heterogeneity requires studying such genetic events not only individually but in combination. We detected strong differentially expressed gene signatures associated with major gene mutations and copy number aberrations including SF3B1, BRAF and TP53, as well as del(17)(p13), del(13)(q14) and del(11)(q22.3) beyond dosage effect. Our study reveals previously underappreciated gene expression signatures for the major molecular subtypes in CLL and the presence of epistasis between them

Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.

BackgroundRestriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."ResultsWe report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.ConclusionThe new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation

Critical Period of Nonpromoter DNA Methylation Acquisition during Prenatal Male Germ Cell Development

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.This work was supported by grants from the Congress Directed Medical Research Program (BC995364 and DAMD17-01-1-0241), Cure Cancer Australia Foundation, NHMRC Australia (projects 1047149, Fellowships 1058356, 481310, 1043400), the Australian Research Council Discovery Project (DP110102288), Princeton University, NIH grant 1R01GM110161-01 (AK), Sidney Kimmel Foundation for Cancer Research (AK), Burroughs Wellcome Foundation (AK), Banque Nationale de Paris-Paribas Australia and New Zealand, Mostyn Family Foundation, Cue Clothing Co., Estee Lauder Australia, RT Hall Trust and Fellowships (ECF-13-08 and ECF-16-022) from the National Breast Cancer Foundatio

ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer.

We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al

Drug-perturbation-based stratification of blood cancer

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.Peer reviewe

The association of academic tracking to depressive symptoms among adolescents in three Caribbean countries

<p>Abstract</p> <p>Background</p> <p>Students who are tracked into low performing schools or classrooms that limit their life chances may report increased depressive symptoms. Limited research has been conducted on academic tracking and its association with depressive symptoms among high school students in the Caribbean. This project examines levels of depressive symptoms among tenth grade students tracked within and between high schools in Jamaica, St. Vincent and St. Kitts and Nevis.</p> <p>Methods</p> <p>Students enrolled in grade ten of the 2006/2007 academic year in Jamaica, St. Kitts and Nevis and St. Vincent were administered the Beck Depression Inventory II (BDI-II). In Jamaica and St. Vincent, academic tracking was operationalized using data provided by the local Ministries of Education. These Ministries ranked ordered schools according to students' performance on Caribbean school leaving examinations. In St. Kitts and Nevis tracking was operationalized by classroom assignments within schools whereby students were grouped into classrooms according to their levels of academic achievement. Multiple regression analyses were conducted to examine the relationships between academic tracking and BDI-II depression scores.</p> <p>Results</p> <p>A wide cross-section of 4^thform students in each nation was sampled (n = 1738; 278 from Jamaica, 737 St. Kitts and Nevis, 716 from St. Vincent; 52% females, 46.2% males and 1.8% no gender reported; age 12 to 19 years, mean = 15.4 yrs, sd = .9 yr). Roughly half (53%) of the students reported some symptoms of depression with 19.2% reporting moderate and 10.7% reporting severe symptoms of depression. Students in Jamaica reported significantly higher depression scores than those in either St. Kitts and Nevis or St. Vincent (p < .01). Students assigned to a higher academic track reported significantly lower BDI-II scores than students who were assigned to the lower academic track (p < .01).</p> <p>Conclusions</p> <p>There appears to be an association between academic tracking and depressive symptoms that is differentially manifested across the islands of Jamaica, St. Kitts and Nevis and St. Vincent.</p