Samuel G. Mackintosh

DNA methylation on N6-adenine in mammalian embryonic stem cells

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N6-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N6-methyladenine. An increase of N6-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N6-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N6-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N6-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N6-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.

Microalbuminuria in Type 1 Diabetes Is Associated With Enhanced Excretion of the Endocytic Multiligand Receptors Megalin and Cubilin

OBJECTIVE Proteinuria is the hallmark of diabetic nephropathy; yet, glomerular histology does not fully explain mechanisms contributing to proteinuria. Our objective was to identify proteins in the urine of individuals with type 1 diabetes and microalbuminuria that might implicate a mechanistic pathway operative in proteinuria. RESEARCH DESIGN AND METHODS Using a GeLC/MS platform proteomics approach, we compared the urine proteome from 12 healthy nondiabetic individuals, 12 subjects with type 1 diabetes yet normal urinary albumin excretion rates, and 12 subjects with type 1 diabetes and microalbuminuria (type 1 diabetes + microalbuminuria). RESULTS The abundance of megalin and cubilin, two multiligand receptors expressed in kidney proximal tubule cells and involved with the reuptake of filtered albumin and megalin/cubilin ligands, was significantly increased in type 1 diabetes + microalbuminuria urine, compared with both nonalbuminuric groups. CONCLUSIONS Aberrant shedding of megalin and cubilin could contribute to albuminuria in diabetes and to deficiency states of important vitamins and hormones.

Structural and Biological Identification of Residues on the Surface of NS3 Helicase Required for Optimal Replication of the Hepatitis C Virus

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a protein-protein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only ∼2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication.

DNA unwinding and protein displacement by superfamily 1 and superfamily 2 helicases

DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. A comprehensive description of these essential biochemical processes requires detailed understanding of helicase mechanisms. These enzymes are ubiquitous, having been identified in viruses, prokaryotes and eukaryotes. Disease states, such as xeroderma pigmentosum, Cockaynes syndrome, Blooms syndrome and Werners syndrome, have been linked to defects in specific genes coding for DNA helicases. Helicases have been placed into different subfamilies based on sequence comparison. The largest subgroups are termed superfamily 1 and superfamily 2. A proposed mechanism for helicases in these classes has been described in terms of an ‘inchworm model’. The inchworm model includes conformational changes driven by ATP binding and hydrolysis that allow unidirectional translocation along DNA. A monomeric form of the enzyme is proposed to have two DNA-binding sites that enable sequential steps of DNA binding and release. Significant differences exist between helicases in important aspects of the models such as the oligomerization state of the enzyme with some helicases functioning as monomers, some as dimers and others as higher-order oligomers.

Characterization of Staphylococcus aureus SarA Binding Sites

The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.

NS3 helicase from the hepatitis C virus can function as a monomer or oligomer depending on enzyme and substrate concentrations

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.

A CRISPR-based approach for proteomic analysis of a single genomic locus

Any given chromosomal activity (e.g., transcription) is governed predominantly by the local epiproteome. However, defining local epiproteomes has been limited by a lack of effective technologies to isolate discrete sections of chromatin and to identify with precision specific proteins and histone posttranslational modifications (PTMs). We report the use of the Cas9 and guide RNA (gRNA) components of the CRISPR system for gRNA-directed purification of a discrete section of chromatin. Quantitative mass spectrometry provides for unambiguous identification of proteins and histone PTMs specifically associated with the enriched chromatin. This CRISPR-based Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) approach revealed changes in the local epiproteome of a promoter during activation of transcription. CRISPR-ChAP-MS thus has broad applications for discovering molecular components and dynamic regulation of any in vivo activity at a given chromosomal location.

Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis

Despite the well-established role of heterochromatin in protecting chromosomal integrity during meiosis and mitosis, the contribution and extent of heterochromatic histone posttranslational modifications (PTMs) remain poorly defined. Here, we gained novel functional insight about heterochromatic PTMs by analyzing histone H3 purified from the heterochromatic germline micronucleus of the model organism Tetrahymena thermophila. Mass spectrometric sequencing of micronuclear H3 identified H3K23 trimethylation (H3K23me3), a previously uncharacterized PTM. H3K23me3 became particularly enriched during meiotic leptotene and zygotene in germline chromatin of Tetrahymena and C. elegans. Loss of H3K23me3 in Tetrahymena through deletion of the methyltransferase Ezl3p caused mislocalization of meiosis-induced DNA double-strand breaks (DSBs) to heterochromatin, and a decrease in progeny viability. These results show that an evolutionarily conserved developmental pathway regulates H3K23me3 during meiosis, and our studies in Tetrahymena suggest this pathway may function to protect heterochromatin from DSBs. DOI: http://dx.doi.org/10.7554/eLife.02996.001

Mapping the local protein interactome of the NuA3 histone acetyltransferase

Protein–protein interactions modulate cellular functions ranging from the activity of enzymes to signal transduction cascades. A technology termed transient isotopic differentiation of interactions as random or targeted (transient I‐DIRT) is described for the identification of stable and transient protein–protein interactions in vivo. The procedure combines mild in vivo chemical cross‐linking and non‐stringent affinity purification to isolate low abundance chromatin‐associated protein complexes. Using isotopic labeling and mass spectrometric readout, purified proteins are categorized with respect to the protein ‘bait’ as stable, transient, or contaminant. Here we characterize the local interactome of the chromatin‐associated NuA3 histone lysine‐acetyltransferase protein complex. We describe transient associations with the yFACT nucleosome assembly complex, RSC chromatin remodeling complex and a nucleosome assembly protein. These novel, physical associations with yFACT, RSC, and Nap1 provide insight into the mechanism of NuA3‐associated transcription and chromatin regulation.

A quantitative proteomic analysis of FFPE melanoma.

To the Editor, Currently, melanoma is diagnosed based on microscopic features, and some of these attributes, including tumor thickness, ulceration, mitotic index, and extent of lymph node involvement, have prognostic significance (1). Patients with melanoma detected at an early stage undergo surgery to remove the primary tumor, but some patients progress to advanced stage disease despite treatment (2). Thus, there is a major need for the identification of prognostic biomarkers of melanoma. Unfortunately, biomarker studies using frozen tissue from primary human tumors are problematic, due to the inherent instability and tissue heterogeneity of the samples. In contrast, formalin-fixed paraffin embedded (FFPE) tissue is very stable and can be coupled with laser microdissection for targeted sample isolation. However, harvesting enough cells and extracting the cross-linked proteins has been challenging. We describe an approach that successfully extracts sufficient amounts of protein from FFPE tissue for mass spectrometric analysis and for free quantification of identified proteins. Similar approaches have proven successful for the analysis of other FFPE samples (3,4,5). Our approach is the first described for the comprehensive analysis of melanoma and melanocytic nevi using a coupled method with gel electrophoresis and spectral counting. For this proof-of-principle analysis, FFPE patient samples were collected from a single melanocytic nevus and single example of metastatic melanoma. Approximately 100,000 cells of melanocytic nevus and metastatic melanoma lesions were harvested with a Leica AS LMD laser microdissector. Proteins were uncross-linked and extracted with the Liquid Tissue MS Protein Prep Kit (Expression Pathology). Equal amounts of the proteins were split into 3 gel lanes and were analyzed by Coomassie/SDS-PAGE, which revealed the extraction of micrograms of protein (Figure 1). Each gel lane was sliced into 17 bands of 2 mm each and digested with trypsin. Tryptic peptides from the 102 gel bands were analyzed by LC-MS/MS with a Thermo LTQ-XL mass spectrometer coupled to an Eksigent nanoLC-2D (6). We identified a total of 888 proteins (0.45% false discovery rate using a decoy database from 56,013 spectra). Relaxing the stringency of the protein identification to a false discovery rate of 1.7% provided for the identification of 1,167 unique proteins from 88,180 spectra. Figure 1 Technical triplicate analyses of metastatic melanoma and nevus samples demonstrate the reproducibility of the quantitative mass spectrometric approach for the analysis of FFPE tissue samples In order to determine whether a protein was differentially expressed between nevus and metastatic melanoma samples, a label-free approach based on spectral counting was used (7,8,9,10). Spectral count is the number of tandem mass spectra assigned to a given protein in all bands from a single gel lane. To determine the relative amount of a protein in a given gel lane, a normalized spectral abundance factor (NSAF) was calculated (7). The NSAF was calculated as follows: (NSAF)k=(SpCL)k∑i=1N(SpCL)i where k is a given protein, SpC is the spectral count, L is the length of the protein, and N is all proteins identified in the gel lane. Plotting the frequency distribution of ln(NSAF) values clearly showed that the data followed a normal distribution as indicated by the fitting of a Gaussian curve with an R2 value of 0.99 (Figure 2A). In accordance to t-test, there were 390 proteins out of 888 total proteins that were found differentially expressed (p<0.05) between metastatic melanoma and nevus lesions. The distribution of the p-values from the t-test was then divided into bins of size 0.025 and the number of proteins for each bin plotted in a bar graph (Figure 2B). The 32 most significant proteins, according to the lowest p-value from the t-test and adjusted with Bonferroni correction, were visually inspected by hierarchical clustering (Figure 2C). Two proteins of particular interest, silver and fatty acid synthase (SILV and FASN), were found in the top 10 most significant proteins and appeared as up-regulated in metastatic melanoma as compared to melanocytic nevus. SILV and FASN both have been shown to be up-regulated in cancers, which makes them appropriate validation tools for this proof-of-principle study (11,12,13). Figure 2 Label-free quantification of proteins identified from FFPE nevus and metastatic melanoma tissues For validation, melanocytic nevus and melanoma samples were stained with either SILV or FASN antibodies and the samples were scored based on the intensity of the staining and the percentage of extent (Figures 3 & 4). The intensity of staining was scored as nil (0), low (1), medium (2), or high (3). The percentage of melanocyte or melanoma cell staining was scored as 0 (no staining), 1 ( 50% staining). The intensity was multiplied by the percentage of extent and the following product is then categorized as such: 0–1 is 0; 2–3 is 1+; 4–5 is 2+; 6–9 is 3+. In general, most of benign lesions (benign nevi or dysplastic nevi) have low or no expression (0 or 1+) of SILV or FASN. Many of the melanomas have higher expression (2+ or 3+) of SILV or FAS. Table 1 shows the number of cases with each score for SILV and FASN indicating higher staining in melanoma compared to benign. Both SILV and FASN were found to be significantly different by Chi square analysis between melanoma and benign with p-values of >0.0001 and 0.0015, respectively. Figure 3 SILV is up-regulated in melanoma Figure 4 Fatty acid synthase is up-regulated in melanoma Table 1 Scoring of SILV and FASN staining In conclusion, we present an unbiased, high throughput and quantitative approach for the identification of proteins that are differentially expressed in metastatic melanoma. Using quantitative label-free mass spectrometry of laser microdissected samples, we have identified 390 proteins differentially expressed in melanoma. Two of these proteins, silver and fatty acid synthase, were validated as being up-regulated in melanoma. Our proof-of-principle analysis lays the foundation for an extensive examination of archived human melanoma tissues for the discovery of biomarkers that will help clinicians with diagnosis, prognosis and treatment of this cancer.