Identification of additional body weight QTLs in the Berlin Fat Mouse BFMI861 lines using time series data.

The Berlin Fat Mouse Inbred line (BFMI) is a model for obesity and metabolic syndrome. The sublines BFMI861-S1 and BFMI861-S2 differ in weight despite high genetic similarity and a shared obesity-related locus. This study focused on identifying additional body weight quantitative trait loci (QTLs) by analyzing weekly weight measurements in a male population of the advanced intercross line BFMI861-S1 x BFMI861-S2. QTL analysis, utilizing 200 selectively genotyped mice (GigaMUGA) and 197 males genotyped for top SNPs, revealed a genome-wide significant QTL on Chr 15 (68.46 to 81.40 Mb) for body weight between weeks 9 to 20. Notably, this QTL disappeared (weeks 21 to 23) and reappeared (weeks 24 and 25) coinciding with a diet change. Additionally, a significant body weight QTL on Chr 16 (3.89 to 22.79 Mb) was identified from weeks 6 to 25. Candidate genes, including Gpt, Cbx6, Apol6, Apol8, Sun2 (Chr 15) and Trap1, Rrn3, Mapk1 (Chr 16), were prioritized. This study unveiled two additional body weight QTLs, one of which is novel and responsive to diet changes. These findings illuminate genomic regions influencing weight in BFMI and emphasize the utility of time series data in uncovering novel genetic factors.

Unmapped short reads from whole-genome sequencing indicate potential infectious pathogens in german black Pied cattle.

When resequencing animal genomes, some short reads cannot be mapped to the reference genome and are usually discarded. In this study, unmapped reads from 302 German Black Pied cattle were analyzed to identify potential pathogenic DNA. These unmapped reads were assembled and blasted against NCBI's database to identify bacterial and viral sequences. The results provided evidence for the presence of pathogens. We found sequences of Bovine parvovirus 3 and Mycoplasma species. These findings emphasize the information content of unmapped reads for gaining insight into bacterial and viral infections, which is important for veterinarians and epidemiologists.

Whole-Genome Sequencing Data Reveal New Loci Affecting Milk Production in German Black Pied Cattle (DSN).

German Black Pied (DSN) is considered an ancestral population of the Holstein breed. The goal of the current study was to fine-map genomic loci for milk production traits and to provide sequence variants for selection. We studied genome-wide associations for milk-production traits in 2160 DSN cows. Using 11.7 million variants from whole-genome sequencing of 304 representative DSN cattle, we identified 1980 associated variants (-log10(p) ≥ 7.1) in 13 genomic loci on 9 chromosomes. The highest significance was found for the MGST1 region affecting milk fat content (-log10(p) = 11.93, MAF = 0.23, substitution effect of the minor allele (ßMA) = -0.151%). Different from Holstein, DGAT1 was fixed (0.97) for the alanine protein variant for high milk and protein yield. A key gene affecting protein content was CSN1S1 (-log10(p) = 8.47, MAF = 049, ßMA = -0.055%) and the GNG2 region (-log10(p) = 10.48, MAF = 0.34, ßMA = 0.054%). Additionally, we suggest the importance of FGF12 for protein and fat yield, HTR3C for milk yield, TLE4 for milk and protein yield, and TNKS for milk and fat yield. Selection for favored alleles can improve milk yield and composition. With respect to maintaining the dual-purpose type of DSN, unfavored linkage to genes affecting muscularity has to be investigated carefully, before the milk-associated variants can be applied for selection in the small population.

Effects of DGAT1 on milk performance in Sudanese Butana × Holstein crossbred cattle.

The improvement of milk production of indigenous Sudanese cattle such as Bos indicus Butana and its cross with Holstein is a major goal of the Sudanese government to ensure sufficient healthy nutrition in the country. In this study, we investigated the K232A polymorphism of diacylglycerol acyltransferase (DGAT1), a well-known modulator of milk production in other breeds. We determined allele frequencies and the allele effects on milk production. Therefore, 93 purebred Butana and 203 Butana × Holstein crossbred cattle were genotyped using competitive allele-specific PCR assays. Association analysis was performed using a linear mixed model in R. In purebred Butana cattle, the lysine DGAT1 protein variant K232, which is found to be associated with higher fat and protein contents, as well as higher fat yield was highly frequent at 0.929, while its frequency in Butana × Holstein crossbred cattle was 0.394. Significant effects were found on milk yield (P = 7.6 × 10-20), fat yield (P = 2.2 × 10-17), protein yield (P = 2.0 × 10-19) and lactose yield (P = 4.0 × 10-18) in crossbred cattle. As expected, the protein variant K232 was disadvantageous since it was decreasing milk, protein, and lactose yields by 1.741 kg, 0.063 kg and 0.084 kg, respectively. No significant effects were found for milk fat, protein, and lactose contents. The high frequency of the lysine DGAT1 protein variant K232 in Butana cattle could contribute to their high milk fat content in combination with low milk yield. In Butana × Holstein crossbred cattle, the DGAT1 marker can be used for effective selection and thus genetic improvement of milk production.

Validation of somatic cell score-associated SNPs from Holstein cattle in Sudanese Butana and Butana × Holstein crossbred cattle.

The Bos indicus zebu cattle Butana is the most commonly used indigenous dairy cattle breed in Sudan. In the last years, high-yielding Holstein dairy cattle were introgressed into Butana cattle to improve their milk yield and simultaneously keep their good adaption to extreme environmental conditions. With the focus on the improvement of milk production, other problems arose such as an increased susceptibility to mastitis. Thus, genetic selection for mastitis resistance should be considered to maintain healthy and productive cows. In this study, we tested 10 single nucleotide polymorphisms (SNPs) which had been associated with somatic cell score (SCS) in Holstein cattle for association with SCS in 37 purebred Butana and 203 Butana × Holstein crossbred cattle from Sudan. Animals were genotyped by competitive allele-specific PCR assays and association analysis was performed using a linear mixed model. All 10 SNPs were segregating in the crossbred Butana × Holstein populations, but only 8 SNPs in Sudanese purebred Butana cattle. The SNP on chromosome 13 was suggestively associated with SCS in the Butana × Holstein crossbred population (rs109441194, 13:79,365,467, PBF = 0.054) and the SNP on chromosome 19 was significantly associated with SCS in both populations (rs41257403, 19:50,027,458, Butana: PBF = 0.003, Butana × Holstein: PBF = 6.2 × 10-16). The minor allele of both SNPs showed an increase in SCS. Therefore, selection against the disadvantageous minor allele could be used for genetic improvement of mastitis resistance in the studied populations. However, investigations in a bigger population and across the whole genome are needed to identify additional genomic loci.

Transmission distortion and genetic incompatibilities between alleles in a multigenerational mouse advanced intercross line.

While direct additive and dominance effects on complex traits have been mapped repeatedly, additional genetic factors contributing to the heterogeneity of complex traits have been scarcely investigated. To assess genetic background effects, we investigated transmission ratio distortions (TRDs) of alleles from parent to offspring using an advanced intercross line (AIL) of an initial cross between the mouse inbred strains C57BL/6NCrl (B6N) and BFMI860-12 [Berlin Fat Mouse Inbred (BFMI)]. A total of 341 males of generation 28 and their respective 61 parents and 66 grandparents were genotyped using Mega Mouse Universal Genotyping Arrays. TRDs were investigated using allele transmission asymmetry tests, and pathway overrepresentation analysis was performed. Sequencing data were used to test for overrepresentation of nonsynonymous SNPs (nsSNPs) in TRD regions. Genetic incompatibilities were tested using the Bateson-Dobzhansky-Muller two-locus model. A total of 62 TRD regions were detected, many in close proximity to the telocentric centromere. TRD regions contained 44.5% more nsSNPs than randomly selected regions (182 vs 125.9 ± 17.0, P < 1 × 10-4). Testing for genetic incompatibilities between TRD regions identified 29 genome-wide significant incompatibilities between TRD regions [P(BF) < 0.05]. Pathway overrepresentation analysis of genes in TRD regions showed that DNA methylation, epigenetic regulation of RNA, and meiotic/meiosis regulation pathways were affected independent of the parental origin of the TRD. Paternal BFMI TRD regions showed overrepresentation in the small interfering RNA biogenesis and in the metabolism of lipids and lipoproteins. Maternal B6N TRD regions harbored genes involved in meiotic recombination, cell death, and apoptosis pathways. The analysis of genes in TRD regions suggests the potential distortion of protein-protein interactions influencing obesity and diabetic retinopathy as a result of disadvantageous combinations of allelic variants in Aass, Pgx6, and Nme8. Using an AIL significantly improves the resolution at which we can investigate TRD. Our analysis implicates distortion of protein-protein interactions as well as meiotic drive as the underlying mechanisms leading to the observed TRD in our AIL. Furthermore, genes with large amounts of nsSNPs located in TRD regions are more likely to be involved in pathways that are related to the phenotypic differences between the parental strains. Genes in these TRD regions provide new targets for investigating genetic adaptation, protein-protein interactions, and determinants of complex traits such as obesity.

Genomic diversity and relationship analyses of endangered German Black Pied cattle (DSN) to 68 other taurine breeds based on whole-genome sequencing.

German Black Pied cattle (Deutsches Schwarzbuntes Niederungsrind, DSN) are an endangered dual-purpose cattle breed originating from the North Sea region. The population comprises about 2,500 cattle and is considered one of the ancestral populations of the modern Holstein breed. The current study aimed at defining the breeds closest related to DSN cattle, characterizing their genomic diversity and inbreeding. In addition, the detection of selection signatures between DSN and Holstein was a goal. Relationship analyses using fixation index (FST), phylogenetic, and admixture analyses were performed between DSN and 68 other breeds from the 1000 Bull Genomes Project. Nucleotide diversity, observed heterozygosity, and expected heterozygosity were calculated as metrics for genomic diversity. Inbreeding was measured as excess of homozygosity (FHom) and genomic inbreeding (FRoH) through runs of homozygosity (RoHs). Region-wide FST and cross-population-extended haplotype homozygosity (XP-EHH) between DSN and Holstein were used to detect selection signatures between the two breeds, and RoH islands were used to detect selection signatures within DSN and Holstein. DSN showed a close genetic relationship with breeds from the Netherlands, Belgium, Northern Germany, and Scandinavia, such as Dutch Friesian Red, Dutch Improved Red, Belgian Red White Campine, Red White Dual Purpose, Modern Angler, Modern Danish Red, and Holstein. The nucleotide diversity in DSN (0.151%) was higher than in Holstein (0.147%) and other breeds, e.g., Norwegian Red (0.149%), Red White Dual Purpose (0.149%), Swedish Red (0.149%), Hereford (0.145%), Angus (0.143%), and Jersey (0.136%). The FHom and FRoH values in DSN were among the lowest. Regions with high FST between DSN and Holstein, significant XP-EHH regions, and RoH islands detected in both breeds harbor candidate genes that were previously reported for milk, meat, fertility, production, and health traits, including one QTL detected in DSN for endoparasite infection resistance. The selection signatures between DSN and Holstein provide evidence of regions responsible for the dual-purpose properties of DSN and the milk type of Holstein. Despite the small population size, DSN has a high level of diversity and low inbreeding. FST supports its relatedness to breeds from the same geographic origin and provides information on potential gene pools that could be used to maintain diversity in DSN.

Design and performance of a bovine 200 k SNP chip developed for endangered German Black Pied cattle (DSN).

BACKGROUND: German Black Pied cattle (DSN) are an endangered dual-purpose breed which was largely replaced by Holstein cattle due to their lower milk yield. DSN cattle are kept as a genetic reserve with a current herd size of around 2500 animals. The ability to track sequence variants specific to DSN could help to support the conservation of DSN's genetic diversity and to provide avenues for genetic improvement. RESULTS: Whole-genome sequencing data of 304 DSN cattle were used to design a customized DSN200k SNP chip harboring 182,154 variants (173,569 SNPs and 8585 indels) based on ten selection categories. We included variants of interest to DSN such as DSN unique variants and variants from previous association studies in DSN, but also variants of general interest such as variants with predicted consequences of high, moderate, or low impact on the transcripts and SNPs from the Illumina BovineSNP50 BeadChip. Further, the selection of variants based on haplotype blocks ensured that the whole-genome was uniformly covered with an average variant distance of 14.4 kb on autosomes. Using 300 DSN and 162 animals from other cattle breeds including Holstein, endangered local cattle populations, and also a Bos indicus breed, performance of the SNP chip was evaluated. Altogether, 171,978 (94.31%) of the variants were successfully called in at least one of the analyzed breeds. In DSN, the number of successfully called variants was 166,563 (91.44%) while 156,684 (86.02%) were segregating at a minor allele frequency > 1%. The concordance rate between technical replicates was 99.83 ± 0.19%. CONCLUSION: The DSN200k SNP chip was proved useful for DSN and other Bos taurus as well as one Bos indicus breed. It is suitable for genetic diversity management and marker-assisted selection of DSN animals. Moreover, variants that were segregating in other breeds can be used for the design of breed-specific customized SNP chips. This will be of great value in the application of conservation programs for endangered local populations in the future.

Genome-Wide Association Study Using Whole-Genome Sequence Data for Fertility, Health Indicator, and Endoparasite Infection Traits in German Black Pied Cattle.

This genome-wide association study (GWAS) aimed to identify sequence variants (SVs) and candidate genes associated with fertility and health in endangered German Black Pied cattle (DSN) based on whole-genome sequence (WGS) data. We used 304 sequenced DSN cattle for the imputation of 1797 genotyped DSN to WGS. The final dataset included 11,413,456 SVs of 1886 cows. Cow traits were calving-to-first service interval (CTFS), non-return after 56 days (NR56), somatic cell score (SCS), fat-to-protein ratio (FPR), and three pre-corrected endoparasite infection traits. We identified 40 SVs above the genome-wide significance and suggestive threshold associated with CTFS and NR56, and three important potential candidate genes (ARHGAP21, MARCH11, and ZNF462). For SCS, most associations were observed on BTA 25. The GWAS revealed 61 SVs, a cluster of 10 candidate genes on BTA 13, and 7 pathways for FPR, including key mediators involved in milk fat synthesis. The strongest associations for gastrointestinal nematode and Dictyocaulus viviparus infections were detected on BTA 8 and 24, respectively. For Fasciola hepatica infections, the strongest associated SVs were located on BTA 4 and 7. We detected 200 genes for endoparasite infection traits, related to 16 pathways involved in host immune response during infection.

Genomic Loci Affecting Milk Production in German Black Pied Cattle (DSN).

German Black Pied cattle (DSN) is an endangered population of about 2,550 dual-purpose cattle in Germany. Having a milk yield of about 2,500 kg less than the predominant dairy breed Holstein, the preservation of DSN is supported by the German government and the EU. The identification of the genomic loci affecting milk production in DSN can provide a basis for selection decisions for genetic improvement of DSN in order to increase market chances through the improvement of milk yield. A genome-wide association analysis of 30 milk traits was conducted in different lactation periods and numbers. Association using multiple linear regression models in R was performed on 1,490 DSN cattle genotyped with BovineSNP50 SNP-chip. 41 significant and 20 suggestive SNPs affecting milk production traits in DSN were identified, as well as 15 additional SNPs for protein content which are less reliable due to high inflation. The most significant effects on milk yield in DSN were detected on chromosomes 1, 6, and 20. The region on chromosome 6 was located nearby the casein gene cluster and the corresponding haplotype overlapped the CSN3 gene (casein kappa). Associations for fat and protein yield and content were also detected. High correlation between traits of the same lactation period or number led to some SNPs being significant for multiple investigated traits. Half of all identified SNPs have been reported in other studies, previously. 15 SNPs were associated with the same traits in other breeds. The other associated SNPs have been reported previously for traits such as exterior, health, meat and carcass, production, and reproduction traits. No association could be detected between DGAT1 and other known milk genes with milk production traits despite the close relationship between DSN and Holstein. The results of this study confirmed that many SNPs identified in other breeds as associated with milk traits also affect milk traits in dual-purpose DSN cattle and can be used for further genetic analysis to identify genes and causal variants that affect milk production in DSN cattle.

A genome-wide association study for clinical mastitis in the dual-purpose German Black Pied cattle breed.

The dual-purpose German Black Pied Cattle (DSN) has become an endangered breed of approximately 2,550 registered cows in Germany. The breed is genetically related to Holstein-Friesian cattle because the old DSN breed contributed to the selection of the modern Holstein dairy cow. In dairy farms, breeders aim to improve animal health and well-being by reducing the number of mastitis cases, which would also reduce milk losses and treatment costs. On the genomic level, no markers associated with clinical mastitis have been reported in DSN. Therefore, we performed a genome-wide association study on 1,062 DSN cows using a univariate linear mixed model that included a relatedness matrix to correct for population stratification. Although the statistical power was limited by the small population size, 3 markers were significantly associated, and 2 additional markers showed a suggestive association with clinical mastitis. Those markers accounted for 1 to 3% of the variance of clinical mastitis in the examined DSN population. One marker was found in the intragenic region of NEURL1 on BTA26, and the other 4 markers in intergenic regions on BTA3, BTA6, and BTA9. Further analyses identified 23 positional candidate genes. Among them is BMPR1B, which has been previously associated with clinical mastitis in other dairy cattle breeds. The markers presented here can be used for selection for mastitis-resistant animals in the endangered DSN population, and can broadly contribute to a better understanding of mastitis determinants in dairy cattle breeds.

DNA Sequence Variants and Protein Haplotypes of Casein Genes in German Black Pied Cattle (DSN).

Casein proteins were repeatedly examined for protein polymorphisms and frequencies in diverse cattle breeds. The occurrence of casein variants in Holstein Friesian, the leading dairy breed worldwide, is well known. The frequencies of different casein variants in Holstein are likely affected by selection for high milk yield. Compared to Holstein, only little is known about casein variants and their frequencies in German Black Pied cattle ("Deutsches Schwarzbuntes Niederungsrind," DSN). The DSN population was a main genetic contributor to the current high-yielding Holstein population. The goal of this study was to investigate casein (protein) variants and casein haplotypes in DSN based on the DNA sequence level and to compare these with data from Holstein and other breeds. In the investigated DSN population, we found no variation in the alpha-casein genes CSN1S1 and CSN1S2 and detected only the CSN1S1*B and CSN1S2*A protein variants. For CSN2 and CSN3 genes, non-synonymous single nucleotide polymorphisms leading to three different ß and κ protein variants were found, respectively. For ß-casein protein variants A 1 , A 2 , and I were detected, with CSN2*A 1 (82.7%) showing the highest frequency. For κ-casein protein variants A, B, and E were detected in DSN, with the highest frequency of CSN3*A (83.3%). Accordingly, the casein protein haplotype CSN1S1*B-CSN2*A 1 -CSN1S2*A-CSN3*A (order of genes on BTA6) is the most frequent haplotype in DSN cattle.

Genome-wide associations and functional gene analyses for endoparasite resistance in an endangered population of native German Black Pied cattle.

BACKGROUND: Gastrointestinal nematodes (GIN), liver flukes (Fasciola hepatica) and bovine lungworms (Dictyocaulus viviparus) are the most important parasitic agents in pastured dairy cattle. Endoparasite infections are associated with reduced milk production and detrimental impacts on female fertility, contributing to economic losses in affected farms. In quantitative-genetic studies, the heritabilities for GIN and F. hepatica were moderate, encouraging studies on genomic scales. Genome-wide association studies (GWAS) based on dense single nucleotide polymorphism (SNP) marker panels allow exploration of the underlying genomic architecture of complex disease traits. The current GWAS combined the identification of potential candidate genes with pathway analyses to obtain deeper insights into bovine immune response and the mechanisms of resistance against endoparasite infections. RESULTS: A 2-step approach was applied to infer genome-wide associations in an endangered dual-purpose cattle subpopulation [Deutsches Schwarzbuntes Niederungsrind (DSN)] with a limited number of phenotypic records. First, endoparasite traits from a population of 1166 Black and White dairy cows [including Holstein Friesian (HF) and DSN] naturally infected with GIN, F. hepatica and D. viviparus were precorrected for fixed effects using linear mixed models. Afterwards, the precorrected phenotypes were the dependent traits (rFEC-GIN, rFEC-FH, and rFLC-DV) in GWAS based on 423,654 SNPs from 148 DSN cows. We identified 44 SNPs above the genome-wide significance threshold (pBonf = 4.47 × 10- 7), and 145 associations surpassed the chromosome-wide significance threshold (range: 7.47 × 10- 6 on BTA 1 to 2.18 × 10- 5 on BTA 28). The associated SNPs identified were annotated to 23 candidate genes. The DAVID analysis inferred four pathways as being related to immune response mechanisms or involved in host-parasite interactions. SNP effect correlations considering specific chromosome segments indicate that breeding for resistance to GIN or F. hepatica as measured by fecal egg counts is genetically associated with a higher risk for udder infections. CONCLUSIONS: We detected a large number of loci with small to moderate effects for endoparasite resistance. The potential candidate genes regulating resistance identified were pathogen-specific. Genetic antagonistic associations between disease resistance and productivity were specific for specific chromosome segments. The 2-step approach was a valid methodological approach to infer genetic mechanisms in an endangered breed with a limited number of phenotypic records.

Finding the Optimal Imputation Strategy for Small Cattle Populations.

The imputation from lower density SNP chip genotypes to whole-genome sequence level is an established approach to generate high density genotypes for many individuals. Imputation accuracy is dependent on many factors and for small cattle populations such as the endangered German Black Pied cattle (DSN), determining the optimal imputation strategy is especially challenging since only a low number of high density genotypes is available. In this paper, the accuracy of imputation was explored with regard to (1) phasing of the target population and the reference panel for imputation, (2) comparison of a 1-step imputation approach, where 50 k genotypes are directly imputed to sequence level, to a 2-step imputation approach that used an intermediate step imputing first to 700 k and subsequently to sequence level, (3) the software tools Beagle and Minimac, and (4) the size and composition of the reference panel for imputation. Analyses were performed for 30 DSN and 30 Holstein Frisian cattle available from the 1000 Bull Genomes Project. Imputation accuracy was assessed using a leave-one-out cross validation procedure. We observed that phasing of the target populations and the reference panels affects the imputation accuracy significantly. Minimac reached higher accuracy when imputing using small reference panels, while Beagle performed better with larger reference panels. In contrast to previous research, we found that when a low number of animals is available at the intermediate imputation step, the 1-step imputation approach yielded higher imputation accuracy compared to a 2-step imputation. Overall, the size of the reference panel for imputation is the most important factor leading to higher imputation accuracy, although using a larger reference panel consisting of a related but different breed (Holstein Frisian) significantly reduced imputation accuracy. Our findings provide specific recommendations for populations with a limited number of high density genotyped or sequenced animals available such as DSN. The overall recommendation when imputing a small population are to (1) use a large reference panel of the same breed, (2) use a large reference panel consisting of diverse breeds, or (3) when a large reference panel is not available, we recommend using a smaller same breed reference panel without including a different related breed.

High dosage of zinc modulates T-cells in a time-dependent manner within porcine gut-associated lymphatic tissue.

Zn serves as a powerful feed additive to reduce post-weaning diarrhoea in pigs. However, the mechanisms responsible for Zn-associated effects on the adaptive immune responses following feeding of a very high dosage of Zn remain elusive. In this study, we examined the T-cell response in gut-associated lymphatic tissues of seventy-two weaned piglets. Piglets received diets with 57 mg Zn/kg (low Zn concentration, LZn), 164 mg Zn/kg (medium Zn concentration, MZn) or 2425 mg Zn/kg (high Zn concentration, HZn) mg Zn/kg feed for 1, 2 or 4 weeks. We observed that feeding the HZn diet for 1 week increased the level of activated T-helper cells (CD4+ and CD8α dim) compared with feeding MZn and LZn (P<0·05). In addition, we observed higher transcript amounts of interferon γ and T-box 21 (TBET) in the HZn group compared with the MZn and LZn groups (P<0·05). A gene set enrichment analysis revealed an over-representation of genes associated with 'cytokine signalling in immune system'. Remarkably, feeding of a very high Zn dosage led to a switch in the immune response after 2 weeks. We detected higher relative cell counts of CD4+CD25high regulatory T-helper cells (P<0·05) and a higher expression of forkhead box P3 (FOXP3) transcripts (P<0·05). After 4 weeks of feeding a high-dosage Zn diet, the relative CD4+ T-cell count (P<0·05) and the relative CD8ß + T-cell count (P<0·1) were reduced compared with the MZn group. We hypothesise that after 1 week the cellular T-helper 1 response is switched on and after 2 weeks it is switched off, leading to decreased numbers of T-cells.

Towards understanding the crosstalk between protein post-translational modifications: Homo- and heterotypic PTM pair distances on protein surfaces are not random.

Post-translational modifications (PTMs) represent an important regulatory layer influencing the structure and function of proteins. With broader availability of experimental information on the occurrences of different PTM types, the investigation of a potential "crosstalk" between different PTM types and combinatorial effects have moved into the research focus. Hypothesizing that relevant interferences between different PTM types and sites may become apparent when investigating their mutual physical distances, we performed a systematic survey of pairwise homo- and heterotypic distances of seven frequent PTM types considering their sequence and spatial distances in resolved protein structures. We found that actual PTM site distance distributions differ from random distributions with most PTM type pairs exhibiting larger than expected distances with the exception of homotypic phosphorylation site distances and distances between phosphorylation and ubiquitination sites that were found to be closer than expected by chance. Random reference distributions considering canonical acceptor amino acid residues only were found to be shifted to larger distances compared to distances between any amino acid residue type indicating an underlying tendency of PTM-amenable residue types to be further apart than randomly expected. Distance distributions based on sequence separations were found largely consistent with their spatial counterparts suggesting a primary role of sequence-based pairwise PTM-location encoding rather than folding-mediated effects. Our analysis provides a systematic and comprehensive overview of the characteristics of pairwise PTM site distances on proteins and reveals that, predominantly, PTM sites tend to avoid close proximity with the potential implication that an independent attachment or removal of PTMs remains possible. Proteins 2016; 85:78-92. © 2016 Wiley Periodicals, Inc.

The Identification of Cis-Regulatory Sequence Motifs in Gene Promoters Based on SNP Information.

Conservation of particular molecular sequence motifs throughout evolution is a strong indicator of their functional relevance as selective pressure likely prevented the accumulation of mutations. Known as "phylogenetic footprinting", this rationale has been exploited for the identification of novel functional motifs using sequence information from sequence alignments of diverse species, in particular transcription factor binding site motifs in aligned gene promoter sequences of orthologous genes. With the rapid advances of sequencing technologies, whole genome sequence information is accumulating not only across different species, but increasingly for variants of the same species exhibiting relatively little sequence variability, primarily present as single nucleotide polymorphisms (SNPs). Here, we lay out the basic strategy for the identification of functional cis-regulatory motifs in gene promoter regions based on SNP information.

Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity.

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites.

Characterization and identification of cis-regulatory elements in Arabidopsis based on single-nucleotide polymorphism information.

Identifying regulatory elements and revealing their role in gene expression regulation remains a central goal of plant genome research. We exploited the detailed genomic sequencing information of a large number of Arabidopsis (Arabidopsis thaliana) accessions to characterize known and to identify novel cis-regulatory elements in gene promoter regions of Arabidopsis by relying on conservation as the hallmark signal of functional relevance. Based on the genomic layout and the obtained density profiles of single-nucleotide polymorphisms (SNPs) in sequence regions upstream of transcription start sites, the average length of promoter regions in Arabidopsis could be established at 500 bp. Genes associated with high degrees of variability of their respective upstream regions are preferentially involved in environmental response and signaling processes, while low levels of promoter SNP density are common among housekeeping genes. Known cis-elements were found to exhibit a decreased SNP density than sequence regions not associated with known motifs. For 15 known cis-element motifs, strong positional preferences relative to the transcription start site were detected based on their promoter SNP density profiles. Five novel candidate cis-element motifs were identified as consensus motifs of 17 sequence hexamers exhibiting increased sequence conservation combined with evidence of positional preferences, annotation information, and functional relevance for inducing correlated gene expression. Our study demonstrates that the currently available resolution of SNP data offers novel ways for the identification of functional genomic elements and the characterization of gene promoter sequences.