A human mitofusin 2 mutation can cause mitophagic cardiomyopathy.

Cardiac muscle has the highest mitochondrial density of any human tissue, but mitochondrial dysfunction is not a recognized cause of isolated cardiomyopathy. Here, we determined that the rare mitofusin (MFN) 2 R400Q mutation is 15-20× over-represented in clinical cardiomyopathy, whereas this specific mutation is not reported as a cause of MFN2 mutant-induced peripheral neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). Accordingly, we interrogated the enzymatic, biophysical, and functional characteristics of MFN2 Q400 versus wild-type and CMT2A-causing MFN2 mutants. All MFN2 mutants had impaired mitochondrial fusion, the canonical MFN2 function. Compared to MFN2 T105M that lacked catalytic GTPase activity and exhibited normal activation-induced changes in conformation, MFN2 R400Q and M376A had normal GTPase activity with impaired conformational shifting. MFN2 R400Q did not suppress mitochondrial motility, provoke mitochondrial depolarization, or dominantly suppress mitochondrial respiration like MFN2 T105M. By contrast to MFN2 T105M and M376A, MFN2 R400Q was uniquely defective in recruiting Parkin to mitochondria. CRISPR editing of the R400Q mutation into the mouse Mfn2 gene induced perinatal cardiomyopathy with no other organ involvement; knock-in of Mfn2 T105M or M376V did not affect the heart. RNA sequencing and metabolomics of cardiomyopathic Mfn2 Q/Q400 hearts revealed signature abnormalities recapitulating experimental mitophagic cardiomyopathy. Indeed, cultured cardiomyoblasts and in vivo cardiomyocytes expressing MFN2 Q400 had mitophagy defects with increased sensitivity to doxorubicin. MFN2 R400Q is the first known natural mitophagy-defective MFN2 mutant. Its unique profile of dysfunction evokes mitophagic cardiomyopathy, suggesting a mechanism for enrichment in clinical cardiomyopathy.

Mitochondria are organelles with an essential role in providing energy to the cells of the body. If damaged, they are repaired by fusing and exchanging contents with sister mitochondria in a process that requires mitofusin proteins. While mutations in the gene for mitofusin 2 have been linked to nerve damage, they do not appear to affect the heart ­ despite high concentrations of mitochondria in heart muscle cells. However, previous research showed that experimentally disrupting the programmed removal of mitochondria, a process also regulated by mitofusin 2, can cause heart muscle disease known as cardiomyopathy. This suggests that mutations affecting different mitofusin 2 roles might harm individual cell types in different ways. To investigate, Franco et al. carried out a genetic screen of people with cardiomyopathy, identifying a rare mitofusin 2 mutation, called R400Q, that was more common in this group. Experiments showed that R400Q caused cardiomyopathy in mice and affected mitochondrial repair and replacement, but not movement. By contrast, a mutation linked to Charcot-Marie-Tooth disease type 2A ­ which causes nerve damage ­ affected mitochondrial movement but not clearance, leading to nerve cell damage but not cardiomyopathy. This led Franco et al. to suggest that mitochondrial movement is central to nerve cell health, whereas mitochondrial repair and replacement plays an important role in cardiac development. Genetic cardiomyopathies affect around 1 in 500 people, but only half of the gene mutations responsible are known. These results suggest that mutations affecting mitochondrial quality control factors could be involved, highlighting a direction for future studies into modifiers of cardiomyopathy.

Breast Cancer Mutations HER2V777L and PIK3CAH1047R Activate the p21-CDK4/6-Cyclin D1 Axis to Drive Tumorigenesis and Drug Resistance.

In metastatic breast cancer, HER2-activating mutations frequently co-occur with mutations in PIK3CA, TP53, or CDH1. Of these co-occurring mutations, HER2 and PIK3CA are the most commonly comutated gene pair, with approximately 40% of HER2-mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we generated genetically engineered mice with the HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers showed accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancer cells were resistant to the pan-HER tyrosine kinase inhibitor, neratinib, but were effectively treated with neratinib plus the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan. Proteomic and RNA-seq analysis of HP breast cancers identified increased gene expression of cyclin D1 and p21WAF1/Cip1 and changes in cell-cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for treating HP breast cancers, with neratinib plus palbociclib showing a statistically significant reduction in development of mouse HP tumors as compared to either drug alone. The efficacy of both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations was validated using a human breast cancer patient-derived xenograft with very similar HER2 and PIK3CA mutations to the HP mice. Further, these two drug combinations effectively treated spontaneous lung metastasis in syngeneic mice transplanted with HP breast cancer organoids. This study provides valuable preclinical data to support the ongoing phase 1 clinical trials of these drug combinations in breast cancer. SIGNIFICANCE: In HER2-mutated breast cancer, PIK3CA mutation activates p21-CDK4/6-cyclin D1 signaling to drive resistance to HER2-targeted therapies, which can be overcome using CDK4/6 inhibitors.

A phase 1b study of venetoclax and alvocidib in patients with relapsed/refractory acute myeloid leukemia.

Relapsed/refractory (R/R) Acute Myeloid Leukemia (AML) is a genetically complex and heterogeneous disease with a poor prognosis and limited treatment options. Thus, there is an urgent need to develop therapeutic combinations to overcome drug resistance in AML. This open-label, multicenter, international, phase 1b study evaluated the safety, efficacy, and pharmacokinetics of venetoclax in combination with alvocidib in patients with R/R AML. Patients were treated with escalating doses of venetoclax (400, 600, and 800 mg QD, orally, days 1-28) and alvocidib (45 and 60 mg/m2 , intravenously, days 1-3) in 28-day cycles. The combination was found to be safe and tolerable, with no maximum tolerated dose reached. Drug-related Grade ≥3 adverse events were reported in 23 (65.7%) for venetoclax and 24 (68.6%) for alvocidib. No drug-related AEs were fatal. Gastrointestinal toxicities, including diarrhea, nausea, and vomiting were notable and frequent; otherwise, the toxicities reported were consistent with the safety profile of both agents. The response rate was modest (complete remission [CR] + incomplete CR [CRi], 11.4%; CR + CRi + partial response rate + morphologic leukemia-free state, 20%). There was no change in alvocidib pharmacokinetics with increasing doses of venetoclax. However, when venetoclax was administered with alvocidib, AUC24 and Cmax decreased by 18% and 19%, respectively. A recommended phase 2 dose was not established due to lack of meaningful increase in efficacy across all cohorts compared to what was previously observed with each agent alone. Future studies could consider the role of the sequence, dosing, and the use of a more selective MCL1 inhibitor for the R/R AML population.

A human STAT3 gain-of-function variant confers T cell dysregulation without predominant Treg dysfunction in mice.

Primary immune regulatory disorders (PIRD) represent a group of disorders characterized by immune dysregulation, presenting with a wide range of clinical disease, including autoimmunity, autoinflammation, or lymphoproliferation. Autosomal dominant germline gain-of-function (GOF) variants in STAT3 result in a PIRD with a broad clinical spectrum. Studies in patients have documented a decreased frequency of FOXP3+ Tregs and an increased frequency of Th17 cells in some patients with active disease. However, the mechanisms of disease pathogenesis in STAT3 GOF syndrome remain largely unknown, and treatment is challenging. We developed a knock-in mouse model harboring a de novo pathogenic human STAT3 variant (p.G421R) and found these mice developed T cell dysregulation, lymphoproliferation, and CD4+ Th1 cell skewing. Surprisingly, Treg numbers, phenotype, and function remained largely intact; however, mice had a selective deficiency in the generation of iTregs. In parallel, we performed single-cell RNA-Seq on T cells from STAT3 GOF patients. We demonstrate only minor changes in the Treg transcriptional signature and an expanded, effector CD8+ T cell population. Together, these findings suggest that Tregs are not the primary driver of disease and highlight the importance of preclinical models in the study of disease mechanisms in rare PIRD.

Determining subpopulation methylation profiles from bisulfite sequencing data of heterogeneous samples using DXM.

Epigenetic changes, such as aberrant DNA methylation, contribute to cancer clonal expansion and disease progression. However, identifying subpopulation-level changes in a heterogeneous sample remains challenging. Thus, we have developed a computational approach, DXM, to deconvolve the methylation profiles of major allelic subpopulations from the bisulfite sequencing data of a heterogeneous sample. DXM does not require prior knowledge of the number of subpopulations or types of cells to expect. We benchmark DXM's performance and demonstrate improvement over existing methods. We further experimentally validate DXM predicted allelic subpopulation-methylation profiles in four Diffuse Large B-Cell Lymphomas (DLBCLs). Lastly, as proof-of-concept, we apply DXM to a cohort of 31 DLBCLs and relate allelic subpopulation methylation profiles to relapse. We thus demonstrate that DXM can robustly find allelic subpopulation methylation profiles that may contribute to disease progression using bisulfite sequencing data of any heterogeneous sample.

Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.

The genetic modules that contribute to human evolution are poorly understood. Here we investigate positive selection in the Epidermal Differentiation Complex locus for skin barrier adaptation in diverse HapMap human populations (CEU, JPT/CHB, and YRI). Using Composite of Multiple Signals and iSAFE, we identify selective sweeps for LCE1A-SMCP and involucrin (IVL) haplotypes associated with human migration out-of-Africa, reaching near fixation in European populations. CEU-IVL is associated with increased IVL expression and a known epidermis-specific enhancer. CRISPR/Cas9 deletion of the orthologous mouse enhancer in vivo reveals a functional requirement for the enhancer to regulate Ivl expression in cis. Reporter assays confirm increased regulatory and additive enhancer effects of CEU-specific polymorphisms identified at predicted IRF1 and NFIC binding sites in the IVL enhancer (rs4845327) and its promoter (rs1854779). Together, our results identify a selective sweep for a cis regulatory module for CEU-IVL, highlighting human skin barrier evolution for increased IVL expression out-of-Africa.

Epigenomic regulation of human T-cell leukemia virus by chromatin-insulator CTCF.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes an aggressive T-cell malignancy and a variety of inflammatory conditions. The integrated provirus includes a single binding site for the epigenomic insulator, CCCTC-binding protein (CTCF), but its function remains unclear. In the current study, a mutant virus was examined that eliminates the CTCF-binding site. The mutation did not disrupt the kinetics and levels of virus gene expression, or establishment of or reactivation from latency. However, the mutation disrupted the epigenetic barrier function, resulting in enhanced DNA CpG methylation downstream of the CTCF binding site on both strands of the integrated provirus and H3K4Me3, H3K36Me3, and H3K27Me3 chromatin modifications both up- and downstream of the site. A majority of clonal cell lines infected with wild type HTLV-1 exhibited increased plus strand gene expression with CTCF knockdown, while expression in mutant HTLV-1 clonal lines was unaffected. These findings indicate that CTCF binding regulates HTLV-1 gene expression, DNA and histone methylation in an integration site dependent fashion.

Association of Circulating Sex Hormones With Inflammation and Disease Severity in Patients With COVID-19.

Importance: Male sex is a risk factor for developing severe COVID-19 illness. It is not known whether sex hormones contribute to this predisposition. Objective: To investigate the association of concentrations of serum testosterone, estradiol, and insulinlike growth factor 1 (IGF-1, concentrations of which are regulated by sex hormone signaling) with COVID-19 severity. Design, Setting, and Participants: This prospective cohort study was conducted using serum samples collected from consecutive patients who presented from March through May 2020 to the Barnes Jewish Hospital in St Louis, Missouri, with COVID-19 (diagnosed using nasopharyngeal swabs). Exposures: Testosterone, estradiol, and IGF-1 concentrations were measured at the time of presentation (ie, day 0) and at days 3, 7, 14, and 28 after admission (if the patient remained hospitalized). Main Outcomes and Measures: Baseline hormone concentrations were compared among patients who had severe COVID-19 vs those with milder COVID-19 illness. RNA sequencing was performed on circulating mononuclear cells to understand the mechanistic association of altered circulating hormone concentrations with cellular signaling pathways. Results: Among 152 patients (90 [59.2%] men; 62 [40.8%] women; mean [SD] age, 63 [16] years), 143 patients (94.1%) were hospitalized. Among 66 men with severe COVID-19, median [interquartile range] testosterone concentrations were lower at day 0 (53 [18 to 114] ng/dL vs 151 [95 to 217] ng/dL; P = .01) and day 3 (19 [6 to 68] ng/dL vs 111 [49 to 274] ng/dL; P = .006) compared with 24 men with milder disease. Testosterone concentrations were inversely associated with concentrations of interleukin 6 (ß = -0.43; 95% CI, -0.52 to -0.17; P < .001), C-reactive protein (ß = -0.38; 95% CI, -0.78 to -0.16; P = .004), interleukin 1 receptor antagonist (ß = -0.29; 95% CI, -0.64 to -0.06; P = .02), hepatocyte growth factor (ß = -0.46; 95% CI, -0.69 to -0.25; P < .001), and interferon γ-inducible protein 10 (ß = -0.32; 95% CI, -0.62 to -0.10; P = .007). Estradiol and IGF-1 concentrations were not associated with COVID-19 severity in men. Testosterone, estradiol, and IGF-1 concentrations were similar in women with and without severe COVID-19. Gene set enrichment analysis revealed upregulated hormone signaling pathways in CD14+CD16- (ie, classical) monocytes and CD14-CD16+ (ie, nonclassical) monocytes in male patients with COVID-19 who needed intensive care unit treatment vs those who did not. Conclusions and Relevance: In this single-center cohort study of patients with COVID-19, lower testosterone concentrations during hospitalization were associated with increased disease severity and inflammation in men. Hormone signaling pathways in monocytes did not parallel serum hormone concentrations, and further investigation is required to understand their pathophysiologic association with COVID-19.

Distinct genetic pathways define pre-malignant versus compensatory clonal hematopoiesis in Shwachman-Diamond syndrome.

To understand the mechanisms that mediate germline genetic leukemia predisposition, we studied the inherited ribosomopathy Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with high risk of myeloid malignancies at an early age. To define the mechanistic basis of clonal hematopoiesis in SDS, we investigate somatic mutations acquired by patients with SDS followed longitudinally. Here we report that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. We show that germline SBDS deficiency establishes a fitness constraint that drives selection of somatic clones via two distinct mechanisms with different clinical consequences. EIF6 inactivation mediates a compensatory pathway with limited leukemic potential by ameliorating the underlying SDS ribosome defect and enhancing clone fitness. TP53 mutations define a maladaptive pathway with enhanced leukemic potential by inactivating tumor suppressor checkpoints without correcting the ribosome defect. Subsequent development of leukemia was associated with acquisition of biallelic TP53 alterations. These results mechanistically link leukemia predisposition to germline genetic constraints on cellular fitness, and provide a rational framework for clinical surveillance strategies.

Integrated Analysis of DNA Methylation, Hydroxymethylation, and Gene Expression Data Using ME-Class2.

There is increasing interest in understanding the pathological role of DNA methylation changes in disease by profiling genome-wide methylation changes. This includes both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The typical profiling study is designed to measure 5mC and/or 5hmC levels alongside gene expression in a set of samples and controls to determine a list of candidate genes whose 5mC and/or 5hmC changes are associated with expression changes. We recently showed that ME-Class2 substantially outperforms other bioinformatic approaches at accurately identify genes with highly associated methylation and expression changes. ME-Class2 further illuminated how synergistic changes in 5mC and 5hmC potentially contribute to gene silencing and activation. Here we present a detailed protocol for using ME-Class2 to analyze genome-wide methylation (5mC and/or 5hmC) and expression data. Further, we provide advice about extending ME-Class2 to study the relationships between other epigenetic marks.

Methylation-directed glycosylation of chromatin factors represses retrotransposon promoters.

The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1ß), a scaffolding protein without intrinsic repressive or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed with O-linked ß-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer of N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed that O-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss of O-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal of O-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed that O-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.

ME-Class2 reveals context dependent regulatory roles for 5-hydroxymethylcytosine.

Since the discovery of 5-hydroxymethylcytosine (5hmC) as a prominent DNA modification found in mammalian genomes, an emergent question has been what role this mark plays in gene regulation. 5hmC is hypothesized to function as an intermediate in the demethylation of 5-methylcytosine (5mC) and in the reactivation of silenced promoters and enhancers. Further, weak positive correlations are observed between gene body 5hmC and gene expression. We previously demonstrated that ME-Class is an effective tool to understand relationships between whole-genome bisulfite sequencing data and expression. In this work, we present ME-Class2, a machine-learning based tool to perform integrative 5mCG, 5hmCG and expression analysis. Using ME-Class2 we analyze whole-genome single-base resolution 5mCG and 5hmCG datasets from 20 primary tissue and cell samples to reveal relationships between 5hmCG and expression. Our analysis indicates that conversion of 5mCG to 5hmCG within 2 kb of the transcription start site associates with distinct functions depending on the summed level of 5mCG + 5hmCG. Unchanged levels of 5mCG + 5hmCG (conversion from 5mCG to stable 5hmCG) associate with repression. Meanwhile, decreases in 5mCG + 5hmCG (5hmCG-mediated demethylation) associate with gene activation. Our results demonstrate that ME-Class2 will prove invaluable to interpret genome-wide 5mC and 5hmC datasets and guide mechanistic studies into the function of 5hmCG.

Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome.

Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear. To address this deficiency, we generated a viable animal model of pericentromeric hypomethylation through mutation of the ICF-gene ZBTB24. Deletion of zebrafish zbtb24 caused a progressive loss of 5mC at pericentromeres and ICF-like phenotypes. Hypomethylation of these repeats triggered derepression of pericentromeric transcripts and activation of an interferon-based innate immune response. Injection of pericentromeric RNA is sufficient to elicit this response in wild-type embryos, and mutation of the MDA5-MAVS dsRNA-sensing machinery blocks the response in mutants. These findings identify activation of the innate immune system as an early consequence of pericentromeric hypomethylation, implicating derepression of pericentromeric transcripts as a trigger of autoimmunity. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis.

Tumor cells disseminate early in tumor development making metastasis-prevention strategies difficult. Identifying proteins that promote the outgrowth of disseminated tumor cells may provide opportunities for novel therapeutic strategies. Despite multiple studies demonstrating that the mesenchymal-to-epithelial transition (MET) is critical for metastatic colonization, key regulators that initiate this transition remain unknown. We serially passaged lung metastases from a primary triple negative breast cancer xenograft to the mammary fat pads of recipient mice to enrich for gene expression changes that drive metastasis. An unbiased transcriptomic signature of potential metastatic drivers was generated, and a high throughput gain-of-function screen was performed in vivo to validate candidates. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver. CEACAM5 overproduction enriched for an epithelial gene expression pattern and facilitated tumor outgrowth at metastatic sites. Tissues from patients with metastatic breast cancer confirmed elevated levels of CEACAM5 in lung metastases relative to breast tumors, and an inverse correlation between CEACAM5 and the mesenchymal marker vimentin was demonstrated. Thus, CEACAM5 facilitates tumor outgrowth at metastatic sites by promoting MET, warranting its investigation as a therapeutic target and biomarker of aggressiveness in breast cancer.

Donor Lymphocyte Infusions Used to Treat Mixed-Chimeric and High-Risk Patient Populations in the Relapsed and Nonrelapsed Settings after Allogeneic Transplantation for Hematologic Malignancies Are Associated with High Five-Year Survival if Persistent Full Donor Chimerism Is Obtained or Maintained.

Mixed chimerism (MC), a persistent or increasing number of host cells after allogeneic hematopoietic stem cell transplantation (HSCT), is a predictor of disease relapse. Donor lymphocyte infusions (DLI) have the potential to enhance the graft-versus-malignancy (GVM) effect, reducing the risk of relapse in patients with MC. Hence, in addition to utilizing DLI in the relapsed setting, there is a motivation to pursue pre-emptive DLI for patients in complete remissions after HSCT. To assess the safety and efficacy of DLI, records of 86 patients who received DLI between 2003 and 2015 at a single institution were studied retrospectively. Patients who received DLI included 50 patients with relapsed/residual (RR) disease, 29 patients with emerging MC without detectable disease, and 7 patients in an "other" cohort who had neither RR disease nor emerging MC after HSCT. DLI were administered using a dose-escalation protocol. After DLI, 93% of MC patients converted to full donor chimerism (FDC). Nonrelapsed patients (MC and other) reported high overall survival (OS) at 1 and 5 years (83% at 1 year, 70% at 5 years for MC; 86% at 1 year, 69% at 5 years for other) and was statistically superior to 5-year OS for RR patients (nonrelapsed 69% versus RR 28%; P = .00032). Improved survival correlated with successful conversion to FDC after DLI for RR and MC cohorts: 71% 2-year OS for patients converted to FDC versus 13% for patients who failed to achieve FDC (P < .0001). DLI for nonrelapsed patients was associated with a superior 5-year progression-free survival (PFS) of 71% compared with 18% 5-year PFS in the RR group (P < .0001). Relapse/progressive disease was the most frequent cause of death (41%). Seven MC (24%), 2 other (29%), and 39 RR patients (78%) relapsed or did not respond after DLI. Overall, 6 patients (7%) died of graft-versus-host disease after DLI. Our results demonstrate a successful dose-escalation approach for nonrelapsed patients that correlated with high survival and a high rate of achieving FDC in MC and RR populations. DLI remain a viable option to boost the GVM effect in the relapsed setting and may pre-emptively protect against relapse in MC populations after HSCT.

DNA methylation and DNA methyltransferases.

The prevailing views as to the form, function, and regulation of genomic methylation patterns have their origin many years in the past, at a time when the structure of the mammalian genome was only dimly perceived, when the number of protein-encoding mammalian genes was believed to be at least five times greater than the actual number, and when it was not understood that only ~10% of the genome is under selective pressure and likely to have biological function. We use more recent findings from genome biology and whole-genome methylation profiling to provide a reappraisal of the shape of genomic methylation patterns and the nature of the changes that they undergo during gametogenesis and early development. We observe that the sequences that undergo deep changes in methylation status during early development are largely sequences without regulatory function. We also discuss recent findings that begin to explain the remarkable fidelity of maintenance methylation. Rather than a general overview of DNA methylation in mammals (which has been the subject of many reviews), we present a new analysis of the distribution of methylated CpG dinucleotides across the multiple sequence compartments that make up the mammalian genome, and we offer an updated interpretation of the nature of the changes in methylation patterns that occur in germ cells and early embryos. We discuss the cues that might designate specific sequences for demethylation or de novo methylation during development, and we summarize recent findings on mechanisms that maintain methylation patterns in mammalian genomes. We also describe the several human disorders, each very different from the other, that are caused by mutations in DNA methyltransferase genes.

Modeling complex patterns of differential DNA methylation that associate with gene expression changes.

Numerous genomic studies are underway to determine which genes are abnormally regulated by DNA methylation in disease. However, we have a poor understanding of how disease-specific methylation changes affect expression. We thus developed an integrative analysis tool, Methylation-based Gene Expression Classification (ME-Class), to explain specific variation in methylation that associates with expression change. This model captures the complexity of methylation changes around a gene promoter. Using 17 whole-genome bisulfite sequencing and RNA-seq datasets from different tissues from the Roadmap Epigenomics Project, ME-Class significantly outperforms standard methods using methylation to predict differential gene expression change. To demonstrate its utility, we used ME-Class to analyze 32 datasets from different hematopoietic cell types from the Blueprint Epigenome project. Expression-associated methylation changes were predominantly found when comparing cells from distantly related lineages, implying that changes in the cell's transcriptional program precede associated methylation changes. Training ME-Class on normal-tumor pairs from The Cancer Genome Atlas indicated that cancer-specific expression-associated methylation changes differ from tissue-specific changes. We further show that ME-Class can detect functionally relevant cancer-specific, expression-associated methylation changes that are reversed upon the removal of methylation. ME-Class is thus a powerful tool to identify genes that are dysregulated by DNA methylation in disease.

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis.

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system.

Abnormal X chromosome inactivation and sex-specific gene dysregulation after ablation of FBXL10.

BACKGROUND: Almost all CpG-rich promoters in the mammalian genome are bound by the multidomain FBXL10 protein (also known as KDM2B, JHDM1B, CXXC2, and NDY1). FBXL10 is expressed as two isoforms: FBXL10-1, a longer form that contains an N-terminal histone demethylase domain with C-terminal F-box, CXXC, PHD, RING, and leucine-rich repeat domains, and FBXL10-2, a shorter form that initiates at an alternative internal exon and which lacks the histone demethylase domain but retains all other annotated domains. Selective deletion of Fbxl10-1 had been reported to produce a low penetrance and variable phenotype; most of the mutant animals were essentially normal. We constructed mutant mouse strains that were either null for Fbxl10-2 but wild type for Fbxl10-1 or null for both Fbxl10-1 and Fbxl10-2. RESULTS: Deletion of Fbxl10-2 (in a manner that does not perturb expression of Fbxl10-1) produced a phenotype very different from the Fbxl10-1 mutant, with craniofacial abnormalities, neural tube defects, and increased lethality, especially in females. Mutants that lacked both FBXL10-1 and FBXL10-2 showed embryonic lethality and even more extreme sexual dimorphism, with more severe gene dysregulation in mutant female embryos. X-linked genes were most severely dysregulated, and there was marked overexpression of Xist in mutant females although genes that encode factors that bind to Xist RNA were globally downregulated in mutant female as compared to male embryos. CONCLUSIONS: FBXL10 is the first factor shown to be required both for the normal expression and function of the Xist gene and for normal expression of proteins that associate with Xist RNA; it is proposed that FBXL10 coordinates the expression of Xist RNA with proteins that associate with this RNA. The function of FBXL10 is largely independent of the histone demethylase activity of the long form of the protein.

Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation.

Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.