DARESOME enables concurrent profiling of multiple DNA modifications with restriction enzymes in single cells and cell-free DNA.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most abundant DNA modifications that have important roles in gene regulation. Detailed studies of these different epigenetic marks aimed at understanding their combined effects and dynamic interconversion are, however, hampered by the inability of current methods to simultaneously measure both modifications, particularly in samples with limited quantities. We present DNA analysis by restriction enzyme for simultaneous detection of multiple epigenomic states (DARESOME), an assay based on modification-sensitive restriction digest and sequential tag ligation that can concurrently perform quantitative profiling of unmodified cytosine, 5mC, and 5hmC in CCGG sites genome-wide. DARESOME reveals the opposing roles of 5mC and 5hmC in gene expression regulation as well as their interconversion during aging in mouse brain. Implementation of DARESOME in single cells demonstrates pronounced 5hmC strand bias that reflects the semiconservative replication of DNA. Last, we showed that DARESOME enables integrative genomic, 5mC, and 5hmC profiling of cell-free DNA that uncovered multiomics cancer signatures in liquid biopsy.

Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients.

Genome-wide analysis of cell-free DNA methylation profile is a promising approach for sensitive and specific detection of many cancers. However, scaling such assays for clinical translation is impractical because of the high cost of whole-genome bisulfite sequencing. We show that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors most of the cancer-specific methylation signature. Here, we report on the simple and effective heat enrichment of CpG-rich regions for bisulfite sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation and quantitative tracking of colorectal cancer patient's response to treatment at much reduced sequencing cost suitable for frequent monitoring. We also show tumor epigenetic subtyping using Heatrich-BS, which could enable patient stratification. Heatrich-BS holds great potential for highly scalable screening and monitoring of cancer using liquid biopsy.

Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Tissue-specific cell-free DNA degradation quantifies circulating tumor DNA burden.

Profiling of circulating tumor DNA (ctDNA) may offer a non-invasive approach to monitor disease progression. Here, we develop a quantitative method, exploiting local tissue-specific cell-free DNA (cfDNA) degradation patterns, that accurately estimates ctDNA burden independent of genomic aberrations. Nucleosome-dependent cfDNA degradation at promoters and first exon-intron junctions is strongly associated with differential transcriptional activity in tumors and blood. A quantitative model, based on just 6 regulatory regions, could accurately predict ctDNA levels in colorectal cancer patients. Strikingly, a model restricted to blood-specific regulatory regions could predict ctDNA levels across both colorectal and breast cancer patients. Using compact targeted sequencing (<25 kb) of predictive regions, we demonstrate how the approach could enable quantitative low-cost tracking of ctDNA dynamics and disease progression.

ENPP1 Mutation Causes Recessive Cole Disease by Altering Melanogenesis.

Cole disease is a genodermatosis of pigmentation following a strict dominant mode of inheritance. In this study, we investigated eight patients affected with an overlapping genodermatosis after recessive inheritance. The patients presented with hypo- and hyperpigmented macules over the body, resembling dyschromatosis universalis hereditaria in addition to punctuate palmoplantar keratosis. By homozygosity mapping and whole-exome sequencing, a biallelic p.Cys120Arg mutation in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was identified in all patients. We found that this mutation, like those causing dominant Cole disease, impairs homodimerization of the ENPP1 enzyme that is mediated by its two somatomedin-B-like domains. Histological analysis revealed structural and molecular changes in affected skin that were likely to originate from defective melanocytes because keratinocytes do not express ENPP1. Consistently, RNA-sequencing analysis of patient-derived primary melanocytes revealed alterations in melanocyte development and in pigmentation signaling pathways. We therefore conclude that germline ENPP1 cysteine-specific mutations, primarily affecting the melanocyte lineage, cause a clinical spectrum of dyschromatosis, in which the p.Cys120Arg allele represents a recessive and more severe form of Cole disease.

Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A.

Autophagy is a cellular catabolic process critical for cell viability and homoeostasis. Inhibition of mammalian target of rapamycin (mTOR) complex-1 (mTORC1) activates autophagy. A puzzling observation is that amino acid starvation triggers more rapid autophagy than pharmacological inhibition of mTORC1, although they both block mTORC1 activity with similar kinetics. Here we find that in addition to mTORC1 inactivation, starvation also causes an increase in phosphatase activity towards ULK1, an mTORC1 substrate whose dephosphorylation is required for autophagy induction. We identify the starvation-stimulated phosphatase for ULK1 as the PP2A-B55α complex. Treatment of cells with starvation but not mTORC1 inhibitors triggers dissociation of PP2A from its inhibitor Alpha4. Furthermore, pancreatic ductal adenocarcinoma cells, whose growth depends on high basal autophagy, possess stronger basal phosphatase activity towards ULK1 and require ULK1 for sustained anchorage-independent growth. Taken together, concurrent mTORC1 inactivation and PP2A-B55α stimulation fuel ULK1-dependent autophagy.

The ULK1 complex: sensing nutrient signals for autophagy activation.

The Atg1/ULK1 complex plays a central role in starvation-induced autophagy, integrating signals from upstream sensors such as MTOR and AMPK and transducing them to the downstream autophagy pathway. Much progress has been made in the last few years in understanding the mechanisms by which the complex is regulated through protein-protein interactions and post-translational modifications, providing insights into how the cell modulates autophagy, particularly in response to nutrient status. However, how the ULK1 complex transduces upstream signals to the downstream central autophagy pathway is still unclear. Although the protein kinase activity of ULK1 is required for its autophagic function, its protein substrate(s) responsible for autophagy activation has not been identified. Furthermore, examples of potential ULK1-independent autophagy have emerged, indicating that under certain specific contexts, the ULK1 complex might be dispensable for autophagy activation. This raises the question of how the autophagic machinery is activated independent of the ULK1 complex and what are the biological functions of such noncanonical autophagy pathways.

Epigenetic regulation by RARα maintains ligand-independent transcriptional activity.

Retinoic acid receptors (RARs) α, ß and γ are key regulators of embryonic development. Hematopoietic differentiation is regulated by RARα, and several types of leukemia show aberrant RARα activity. Through microarray expression analysis, we identified transcripts differentially expressed between F9 wild-type (Wt) and RARα knockout cells cultured in the absence or presence of the RAR-specific ligand all trans retinoic acid (RA). We validated the decreased Mest, Tex13, Gab1, Bcl11a, Tcfap2a and HMGcs1 transcript levels, and increased Slc38a4, Stmn2, RpL39l, Ref2L, Mobp and Rlf1 transcript levels in the RARa knockout cells. The decreased Mest and Tex13 transcript levels were associated with increased promoter CpG-island methylation and increased repressive histone modifications (H3K9me3) in RARα knockout cells. Increased Slc38a4 and Stmn2 transcript levels were associated with decreased promoter CpG-island methylation and increased permissive histone modifications (H3K9/K14ac, H3K4me3) in RARα knockout cells. We demonstrated specific association of RARα and RXRα with the Mest promoter. Importantly, stable expression of a dominant negative, oncogenic PML-RARα fusion protein in F9 Wt cells recapitulated the decreased Mest transcript levels observed in RARα knockout cells. We propose that RARα plays an important role in cellular memory and imprinting by regulating the CpG methylation status of specific promoter regions.

Migration of growth factor-stimulated epithelial and endothelial cells depends on EGFR transactivation by ADAM17.

The fibroblast growth factor receptor 2-IIIb (FGFR2b) and the vascular endothelial growth factor receptor 2 (VEGFR2) are tyrosine kinases that can promote cell migration and proliferation and have important roles in embryonic development and cancer. Here we show that FGF7/FGFR2b-dependent activation of epidermal growth factor receptor (EGFR)/ERK1/2 signalling and cell migration in epithelial cells require stimulation of the membrane-anchored metalloproteinase ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF). Moreover, VEGF-A/VEGFR2-induced migration of human umbilical vein endothelial cells also depends on EGFR/ERK1/2 signalling and shedding of the ADAM17 substrate HB-EGF. The pathway used by the FGF7/FGFR2b signalling axis to stimulate shedding of substrates of ADAM17, including ligands of the EGFR, involves Src, p38 mitogen-activated protein-kinase and PI3K, but does not require the cytoplasmic domain of ADAM17. Based on these findings, ADAM17 emerges as a central component in a triple membrane-spanning pathway between FGFR2b or VEGFR2 and EGFR/ERK1/2 that is required for cell migration in keratinocytes and presumably also in endothelial cells.

Innate inflammatory responses of human decidual cells to periodontopathic bacteria.

OBJECTIVE: The purpose of this study was to test the hypothesis that periodontopathic bacteria exert potent proinflammatory effects in human decidua. STUDY DESIGN: The immunostimulatory effects of Gram-positive and negative periodontopathic bacteria and their lipopolysaccharides were tested in human decidual cell cultures in comparison with Escherichia coli. Cytokine production was measured by enzyme-linked immunosorbent assay; inflammatory gene expression was measured by oligonucleotide arrays and quantitative real time-polymerase chain reaction. RESULTS: All bacteria that were tested elicited an inflammatory response, although concentration-dependence and efficacy varied considerably with organism and culture. Lipopolysaccharides were more potent stimuli than intact bacterial cells, although bacteria exerted greater effects at high concentrations. Of 112 genes on the arrays, 18 genes were stimulated significantly by one or more lipopolysaccharide preparation. CONCLUSION: The ability of periodontopathic bacteria to stimulate a decidual inflammatory response is highly variable and partly dependent on the presence and structure of constituent lipopolysaccharides. This adds to our understanding of the causal association between periodontal disease and preterm birth.

Processing of autophagic protein LC3 by the 20S proteasome.

Ubiquitin-proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin-like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3-processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP- and ubiquitin-independent, and requires both the N-terminal helices and the ubiquitin fold of LC3; addition of the N-terminal helices of LC3 to the N terminus of ubiquitin renders ubiquitin susceptible to 20S proteasomal activity. Further, the 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupts the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3-binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting relevance of the controversial ubiquitin-independent proteolytic activity of the 20S proteasome.