Modulation of the activity of histone lysine methyltransferases and demethylases by curcumin analog in leukaemia cells.

Curcumin is a known epigenetic modifier that demonstrated antitumor effect in different types of cancer. The poor solubility and metabolic stability are major drawbacks that limit its development as an antitumor agent. Dimethoxycurcumin (DMC) is a more soluble and stable curcumin analog. In this study, we compared the effect of both drugs on a variety of histone posttranslational modifications and on the activity of histone lysine methyltransferase (HKMTs) and demethylase (HKDMTs) enzymes that target the H3K4, H3K9 and H3K27 epigenetic marks. Mass spectrometry was used to quantitate the changes in 95 histone posttranslational modifications induced by curcumin or DMC. The effect of both drugs on the enzymatic activity of HKMTs and HKDMs was measured using an antibody-based assay. Mass spectrometry analysis showed that curcumin and DMC modulated several histone modifications. Histone changes were not limited to lysine methylation and acetylation but included arginine and glutamine methylation. Only few histone modifications were similarly changed by both drugs. On the contrary, the effect of both drugs on the activity of HKMTs and HKDMs was very similar. Curcumin and DMC inhibited the HKMTs enzymes that target the H3K4, H3K9 and H3K27 marks and increased the activity of the HKDMs enzymes LSD1, JARID and JMJD2. In conclusion, we identified novel enzymatic targets for both curcumin and DMC that support their use and development as epigenetic modifiers in cancer treatment. The multiple targets modulated by both drugs could provide a therapeutic advantage by overcoming drug resistance development.

Differential Histone Posttranslational Modifications Induced by DNA Hypomethylating Agents.

INTRODUCTION: The prototype DNA hypomethylating agents 5-azacytidine (5AC) and decitabine (DAC) are currently FDA-approved for treatment of blood and bone marrow disorders like myelodysplastic syndrome. 5AC and DAC are considered similar drugs and were shown to induce histone modifications that modulate gene expression. The aim of this study is to compare the effect of both drugs on histone acetylation and methylation at multiple histone amino acids residues. METHODS: Mass spectrometry was used to compare the effect of both drugs on 95 different histone posttranslational modifications (PTMs) in leukemia cells. ChIP-Seq analysis was used to compare the impact of both drugs on the genome-wide acetylation of the H3K9 mark using primary leukemia cells from six de-identified AML patients. RESULTS: Both DAC and 5AC induced histone PTMs in different histone isoforms like H1.4, H2A, H3, H3.1, and H4. Changes in both histone methylation and acetylation were observed with both drugs; however, there were distinct differences in the histone modifications induced by the two drugs. Since both drugs were shown to increase the activity of the HDAC SIRT6 previously, we tested the effect of 5AC on the acetylation of H3K9, the physiological substrate SIRT6, using ChIP-Seq analysis and compared it to the previously published DAC-induced changes. Significant H3K9 acetylation changes (P< .05) were detected at 925 genes after 5AC treatment vs only 182 genes after DAC treatment. Nevertheless, the gene set modified by 5AC was different from that modified by DAC with only ten similar genes modulated by both drugs. CONCLUSION: Despite similarity in chemical structure and DNA hypomethylating activity, 5AC and DAC induced widely different histone PTMs and considering them interchangeable should be carefully evaluated. The mechanism of these histone PTM changes is not clear and may involve modulation of the activity or the expression of the enzymes inducing histone PTMs.

Identification of the inhibitor of growth protein 4 (ING4) as a potential target in prostate cancer therapy.

INhibitor of Growth protein 4 (ING4) is a potential chromatin modifier that has been implicated in several cancer-related processes. However, the role of ING4 in prostate cancer (PC) is largely unknown. This study aimed to assess ING4's role in global transcriptional regulation in PC cells to identify potential cellular processes associated with ING4 loss. RNA-Seq using next-generation sequencing (NGS) was used to identify altered genes in LNCaP PC cells following ING4 depletion. Ingenuity pathways analysis (IPA®) was applied to the data to highlight candidates, ING4-regulated pathways, networks and cellular processes. Selected genes were validated using RT-qPCR. RNA-Seq of LNCaP cells revealed a total of 159 differentially expressed genes (fold change ≥ 1.5 or ≤ - 1.5, FDR ≤ 0.05) following ING4 knockdown. RT-qPCR used to validate the expression level of selected genes was in agreement with RNA-Seq results. Key genes, unique pathways, and biological networks were identified using IPA® analysis. This is the first report of global gene regulation in PC cells by ING4. The resultant differential expression profile revealed the potential role of ING4 in PC pathogenesis possibly through modulation of key genes, pathways and biological networks that are central drivers of the disease. Collectively, these findings shed light on a novel transcriptional regulator of PC that ultimately may influence the disease progression and as a potential target in the disease therapy.

Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits.

Transcriptome Analysis of Invasive Plants in Response to Mineral Toxicity of Reclaimed Coal-Mine Soil in the Appalachian Region.

Efficient postmining reclamation requires successful revegetation. By using RNA sequencing, we evaluated the growth response of two invasive plants, goutweed (Aegopodium podagraria L.) and mugwort (Artemisia vulgaris), grown in two Appalachian acid-mine soils (MS-I and -II, pH ∼ 4.6). Although deficient in macronutrients, both soils contained high levels of plant-available Al, Fe and Mn. Both plant types showed toxicity tolerance, but metal accumulation differed by plant and site. With MS-I, Al accumulation was greater for mugwort than goutweed (385 ± 47 vs 2151 ± 251 µg g-1). Al concentration was similar between mine sites, but its accumulation in mugwort was greater with MS-I than MS-II, with no difference in accumulation by site for goutweed. An in situ approach revealed deregulation of multiple factors such as transporters, transcription factors, and metal chelators for metal uptake or exclusion. The two plant systems showed common gene expression patterns for different pathways. Both plant systems appeared to have few common heavy-metal pathway regulators addressing mineral toxicity/deficiency in both mine sites, which implies adaptability of invasive plants for efficient growth at mine sites with toxic waste. Functional genomics can be used to screen for plant adaptability, especially for reclamation and phytoremediation of contaminated soils and waters.

Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon.

BACKGROUND: A large single nucleotide polymorphism (SNP) dataset was used to analyze genome-wide diversity in a diverse collection of watermelon cultivars representing globally cultivated, watermelon genetic diversity. The marker density required for conducting successful association mapping depends on the extent of linkage disequilibrium (LD) within a population. Use of genotyping by sequencing reveals large numbers of SNPs that in turn generate opportunities in genome-wide association mapping and marker-assisted selection, even in crops such as watermelon for which few genomic resources are available. In this paper, we used genome-wide genetic diversity to study LD, selective sweeps, and pairwise FST distributions among worldwide cultivated watermelons to track signals of domestication. RESULTS: We examined 183 Citrullus lanatus var. lanatus accessions representing domesticated watermelon and generated a set of 11,485 SNP markers using genotyping by sequencing. With a diverse panel of worldwide cultivated watermelons, we identified a set of 5,254 SNPs with a minor allele frequency of ≥ 0.05, distributed across the genome. All ancestries were traced to Africa and an admixture of various ancestries constituted secondary gene pools across various continents. A sliding window analysis using pairwise FST values was used to resolve selective sweeps. We identified strong selection on chromosomes 3 and 9 that might have contributed to the domestication process. Pairwise analysis of adjacent SNPs within a chromosome as well as within a haplotype allowed us to estimate genome-wide LD decay. LD was also detected within individual genes on various chromosomes. Principal component and ancestry analyses were used to account for population structure in a genome-wide association study. We further mapped important genes for soluble solid content using a mixed linear model. CONCLUSIONS: Information concerning the SNP resources, population structure, and LD developed in this study will help in identifying agronomically important candidate genes from the genomic regions underlying selection and for mapping quantitative trait loci using a genome-wide association study in sweet watermelon.

Linkage disequilibrium and population-structure analysis among Capsicum annuum L. cultivars for use in association mapping.

Knowledge of population structure and linkage disequilibrium among the worldwide collections of peppers currently classified as hot, mild, sweet and ornamental types is indispensable for applying association mapping and genomic selection to improve pepper. The current study aimed to resolve the genetic diversity and relatedness of Capsicum annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes in samples collected in 2011 and 2012. The physical distance covered by the entire set of SSRs used was 2,265.9 Mb from the 3.48-Gb hot-pepper genome size. The model-based program STRUCTURE was used to infer five clusters, which was further confirmed by classical molecular-genetic diversity analysis. Mean heterozygosity of various loci was estimated to be 0.15. Linkage disequilibrium (LD) was used to identify 17 LD blocks across various chromosomes with sizes from 0.154 Kb to 126.28 Mb. CAMS-142 of chromosome 1 was significantly associated with both capsaicin (CA) and dihydrocapsaicin (DCA) levels. Further, CAMS-142 was located in an LD block of 98.18 Mb. CAMS-142 amplified bands of 244, 268, 283 and 326 bp. Alleles 268 and 283 bp had positive effects on both CA and DCA levels, with an average R(2) of 12.15 % (CA) and 12.3 % (DCA). Eight markers from seven different chromosomes were significantly associated with fruit weight, contributing an average effect of 15 %. CAMS-199, HpmsE082 and CAMS-190 are the three major quantitative trait loci located on chromosomes 8, 9, and 10, respectively, and were associated with fruit weight in samples from both years of the study. This research demonstrates the effectiveness of using genome-wide SSR-based markers to assess features of LD and genetic diversity within C. annuum.

iCn3D: From Web-Based 3D Viewer to Structural Analysis Tool in Batch Mode.

iCn3D was initially developed as a web-based 3D molecular viewer. It then evolved from visualization into a full-featured interactive structural analysis software. It became a collaborative research instrument through the sharing of permanent, shortened URLs that encapsulate not only annotated visual molecular scenes, but also all underlying data and analysis scripts in a FAIR manner. More recently, with the growth of structural databases, the need to analyze large structural datasets systematically led us to use Python scripts and convert the code to be used in Node. js scripts. We showed a few examples of Python scripts at https://github.com/ncbi/icn3d/tree/master/icn3dpython to export secondary structures or PNG images from iCn3D. Users just need to replace the URL in the Python scripts to export other annotations from iCn3D. Furthermore, any interactive iCn3D feature can be converted into a Node. js script to be run in batch mode, enabling an interactive analysis performed on one or a handful of protein complexes to be scaled up to analysis features of large ensembles of structures. Currently available Node. js analysis scripts examples are available at https://github.com/ncbi/icn3d/tree/master/icn3dnode. This development will enable ensemble analyses on growing structural databases such as AlphaFold or RoseTTAFold on one hand and Electron Microscopy on the other. In this paper, we also review new features such as DelPhi electrostatic potential, 3D view of mutations, alignment of multiple chains, assembly of multiple structures by realignment, dynamic symmetry calculation, 2D cartoons at different levels, interactive contact maps, and use of iCn3D in Jupyter Notebook as described at https://pypi.org/project/icn3dpy.

Dynamic changes in genome-wide histone H3 lysine 4 methylation patterns in response to dehydration stress in Arabidopsis thaliana.

BACKGROUND: The molecular mechanisms of genome reprogramming during transcriptional responses to stress are associated with specific chromatin modifications. Available data, however, describe histone modifications only at individual plant genes induced by stress. We have no knowledge of chromatin modifications taking place at genes whose transcription has been down-regulated or on the genome-wide chromatin modification patterns that occur during the plant's response to dehydration stress. RESULTS: Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq) we established the whole-genome distribution patterns of histone H3 lysine 4 mono-, di-, and tri-methylation (H3K4me1, H3K4me2, and H3K4me3, respectively) in Arabidopsis thaliana during watered and dehydration stress conditions. In contrast to the relatively even distribution of H3 throughout the genome, the H3K4me1, H3K4me2, and H3K4me3 marks are predominantly located on genes. About 90% of annotated genes carry one or more of the H3K4 methylation marks. The H3K4me1 and H3K4me2 marks are more widely distributed (80% and 84%, respectively) than the H3K4me3 marks (62%), but the H3K4me2 and H3K4me1 levels changed only modestly during dehydration stress. By contrast, the H3K4me3 abundance changed robustly when transcripts levels from responding genes increased or decreased. In contrast to the prominent H3K4me3 peaks present at the 5'-ends of most transcribed genes, genes inducible by dehydration and ABA displayed atypically broader H3K4me3 distribution profiles that were present before and after the stress. CONCLUSIONS: A higher number (90%) of annotated Arabidopsis genes carry one or more types of H3K4me marks than previously reported. During the response to dehydration stress the changes in H3K4me1, H3K4me2, and H3K4me3 patterns show different dynamics and specific patterns at up-regulated, down-regulated, and unaffected genes. The different behavior of each methylation mark during the response process illustrates that they have distinct roles in the transcriptional response of implicated genes. The broad H3K4me3 distribution profiles on nucleosomes of stress-induced genes uncovered a specific chromatin pattern associated with many of the genes involved in the dehydration stress response.

Author Correction: Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

Serotonergic system, cognition, and BPSD in Alzheimer's disease.

Behavioral and Psychological Symptoms of Dementia (BPSD), present in almost 90% of patients with Alzheimer's Disease (AD), cause extensive impairment leading to reduced independence and inability to complete activities of daily living. Though BPSD includes a wide range of symptoms, such as agitation, aggression, disinhibition, anxiety, depression, apathy, delusions, and hallucinations. Certain BPSD in AD co-present and can be clustered into distinct domains based on their frequency of co-occurrence. As these BPSD are so pervasive in any stages of AD, the disease may be better characterized as a disorder of heterogeneous degenerative symptoms across a number of symptom domains, with the most prominent domain comprising memory and cognitive deficits. Importantly, there are no FDA-approved drugs to treat these BPSD, and new approaches must be considered to develop effective treatments for AD patients. The biogenic monoamine 5-hydroxytryptamine (5-HT), or serotonin, works as both a neurotransmitter and neuromodulator, which has been tied to cognitive decline and multiple BPSD domains. This review summarizes the evidence for specific serotonergic system alterations across some of the well-studied cognitive, behavioral, and psychiatric domains. Though differences in overall serotonergic transmission occur in AD, circuit-specific alterations in individual 5-HT receptors (5-HTRs) are likely linked to the heterogeneous presentation of BPSD in AD.

Comparative docking studies of CYP1b1 and its PCG-associated mutant forms.

Molecular docking has been used to compare and contrast the binding modes of oestradiol with the wild-type and some disease-associated mutant forms of the human CYP1b1 protein.The receptor structures used for docking were derived from molecular dynamics simulations of homology-modelled structures. Earlier studies involving molecular dynamics and principal component analysis indicated that mutations could have a disruptive effect on function,by destabilizing the native properties of the functionally important regions, especially those of the haem-binding and substrate-binding regions,which constitute the site of catalytic activity of the enzyme.In order to gain more insights into the possible differences in substrate-binding and catalysis between the wild-type and mutant proteins,molecular docking studies were carried out. Mutants showed altered protein -ligand interactions compared with the wild-type as a consequence of changes in the geometry of the substrate-binding region and in the position of haem relative to the active site. An important difference in ligand -protein interactions between the wild-type and mutants is the presence of stacking interaction with phenyl residues in the wild-type,which is either completely absent or considerably weaker in mutants.The present study revealed essential differences in the interactions between ligand and protein in wild-type and disease mutants,and helped in understanding the deleterious nature of disease mutations at the level of molecular function.

Metagenome Sequences of Sediment from a Recovering Industrialized Appalachian River in West Virginia.

We sequenced two metagenomes from upper sediment layers (0 to 5 and 6 to 10 cm) from the Kanawha River, West Virginia. The watershed includes inputs from the forested Appalachian Mountains, surface coal mining, municipal residues, and extensive chemical manufacturing. The dominant bacterial phyla were Proteobacteria, Bacteriodetes, Firmicutes, Actinobacteria, and Chloroflexi Xenobiotic degradation pathways were present.

Metagenome Sequences of a Thermophilic Anaerobic Digester Adapted to a Low C/N Ratio, High-Ammonia Feedstock (Poultry Litter).

We sequenced the metagenome of a pilot-scale thermophilic digester with long-term, stable performance on poultry litter feedstock which has a very low C/N ratio, a high ammonia level, and high lignocellulose content. Firmicutes were the dominant phylum (68.9%). Other abundant phyla included Bacteroidetes, Euryarchaeota, and Thermotogae This microbiome represents a hydrogenotrophic methanogenic community with high diversity.

Genome-wide Diversity and Association Mapping for Capsaicinoids and Fruit Weight in Capsicum annuum L.

Accumulated capsaicinoid content and increased fruit size are traits resulting from Capsicum annuum domestication. In this study, we used a diverse collection of C. annuum to generate 66,960 SNPs using genotyping by sequencing. The study identified 1189 haplotypes containing 3413 SNPs. Length of individual linkage disequilibrium (LD) blocks varied along chromosomes, with regions of high and low LD interspersed with an average LD of 139 kb. Principal component analysis (PCA), Bayesian model based population structure analysis and an Euclidean tree built based on identity by state (IBS) indices revealed that the clustering pattern of diverse accessions are in agreement with capsaicin content (CA) and fruit weight (FW) classifications indicating the importance of these traits in shaping modern pepper genome. PCA and IBS were used in a mixed linear model of capsaicin and dihydrocapsaicin content and fruit weight to reduce spurious associations because of confounding effects of subpopulations in genome-wide association study (GWAS). Our GWAS results showed SNPs in Ankyrin-like protein, IKI3 family protein, ABC transporter G family and pentatricopeptide repeat protein are the major markers for capsaicinoids and of 16 SNPs strongly associated with FW in both years of the study, 7 are located in known fruit weight controlling genes.

Lysine biotinylation and methionine oxidation in the heat shock protein HSP60 synergize in the elimination of reactive oxygen species in human cell cultures.

Previous studies suggest that the number of proteins containing covalently bound biotin is larger than previously thought. Here, we report the identity of some of these proteins. Using mass spectrometry, we discovered 108 novel biotinylation sites in the human embryonic kidney HEK293 cell proteome; members of the heat shock protein (HSP) superfamily were overrepresented among the novel biotinylated proteins. About half of the biotinylated proteins also displayed various degrees of methionine oxidation, which is known to play an important role in the defense against reactive oxygen species; for biotinylated HSPs, the percent of methionine sulfoxidation approached 100%. Protein structure analysis suggests that methionine sulfoxides localize in close physical proximity to the biotinylated lysines on the protein surface. Mass spectrometric analysis revealed that between one and five of the methionine residues in the C-terminal KEEKDPGMGAMGGMGGGMGGGMF motif are oxidized in HSP60. The likelihood of methionine sulfoxidation is higher if one of the adjacent lysine residues is biotinylated. Knockdown of HSP60 caused a 60% increase in the level of reactive oxygen species in fibroblasts cultured in biotin-sufficient medium. When HEK293 cells were transferred from biotin-sufficient medium to biotin-free medium, the level of reactive oxygen species increased by >9 times compared with baseline controls and a time-response relationship was evident. High levels of methionine sulfoxidation coincided with cell cycle arrest in the G0/G1 and S phases in biotin-depleted cells. We conclude that biotinylation of lysines synergizes with sulfoxidation of methionines in heat shock proteins such as HSP60 in the defense against reactive oxygen species.

Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene.

Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

Enrichment of meiotic recombination hotspot sequences by avidin capture technology.

About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5'-CCNCCNTNNCCNC-3'. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single-stranded DNA, denatured double-stranded DNA, and large fragments (>3000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.

Effects of single-nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis.

Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.