Genome-Wide Characterization of Endogenous Retroviruses in the Bat Myotis lucifugus Reveals Recent and Diverse Infections

Bats are increasingly recognized as reservoir species for a variety of zoonotic viruses that pose severe threats to human health. While many RNA viruses have been identified in bats, little is known about bat retroviruses. Endogenous retroviruses (ERVs) represent genomic fossils of past retroviral infections and, thus, can inform us on the diversity and history of retroviruses that have infected a species lineage. Here, we took advantage of the availability of a high-quality genome assembly for the little brown bat, Myotis lucifugus, to systematically identify and analyze ERVs in this species. We mined an initial set of 362 potentially complete proviruses from the three main classes of ERVs, which were further resolved into 13 major families and 86 subfamilies by phylogenetic analysis. Consensus or representative sequences for each of the 86 subfamilies were then merged to the Repbase collection of known ERV/long terminal repeat (LTR) elements to annotate the retroviral complement of the bat genome. The results show that nearly 5% of the genome assembly is occupied by ERV-derived sequences, a quantity comparable to findings for other eutherian mammals. About one-fourth of these sequences belong to subfamilies newly identified in this study. Using two independent methods, intraelement LTR divergence and analysis of orthologous loci in two other bat species, we found that the vast majority of the potentially complete proviruses identified in M. lucifugus were integrated in the last ∼25 million years. All three major ERV classes include recently integrated proviruses, suggesting that a wide diversity of retroviruses is still circulating in Myotis bats.     

Identification of a Class of Chromatin Boundary Elements

Boundary elements are thought to define the ends of functionally independent domains of genetic activity. An assay for boundary activity based on this concept measures the ability to insulate a bracketed, chromosomally integrated reporter gene from position effects. Despite their presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. The Drosophila protein BEAF binds the scs′ boundary element of the 87A7 hsp70 locus and roughly half of polytene chromosome interband loci. To see if these sites represent a class of boundary elements that have BEAF in common, we have isolated and studied several genomic BEAF binding sites as candidate boundary elements (cBEs). BEAF binds with high affinity to clustered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to confer position-independent expression on a mini-white reporter gene in transgenic flies. Furthermore, point mutations in CGATA motifs that eliminate binding by BEAF also eliminate the ability to confer position-independent expression. Taken together, these findings suggest that clustered CGATA motifs are a hallmark of a BEAF-utilizing class of boundary elements found at many loci. This is the first example of a class of boundary elements that share a sequence motif and a binding protein.Chromatin appears to be partitioned into chromosomal domains that are operationally defined by bracketing DNA regions called boundary elements or insulators (10; see reference 34 for a review). Boundary elements are presumably necessary to curtail the potentially promiscuous behavior of enhancers, limiting their action to the domain in which they reside. The biological activity of a boundary element is experimentally measured by either position-independent expression or enhancer-blocking assays. If this view of chromosomal organization is correct, boundary elements play a very important functional role. Yet only a few examples have been identified, and each is so far a unique case, as they do not appear to have notable sequence homologies or to have binding activities in common.The best-characterized boundary elements are the scs and scs′ regions found to bracket the 87A7 hsp70 heat shock puff of Drosophila melanogaster polytene chromosomes (33) and a 340-bp fragment from the gypsy retrotransposon (11). The scs/scs′ and the gypsy-derived elements have a boundary function in both of the assays mentioned above. They confer position-independent expression on a bracketed reporter gene by insulating the transgene from both activating and repressive effects at the site of chromosomal integration, and they block communication between a specific enhancer and promoter when interposed (20, 21, 31). It is important to note that boundary elements do not inactivate promoters or enhancers; they only block communication when interposed (2, 3, 21, 32). For instance, if an enhancer and boundary element are located between two divergently transcribed promoters, the enhancer cannot activate the promoter with the intervening boundary element but can activate the other promoter. Thus, the positional functioning of boundary elements is distinct from the bidirectional repressive effect of silencer elements.The boundary activity of the gypsy-derived element is known to be mediated by the binding of the zinc finger protein su(Hw) to its reiterated binding sites (31). The su(Hw) protein has been studied in some detail, and regions involved in DNA binding, enhancer blocking, and interactions with mod(mdg4) have been identified (8, 13, 22). Interactions between the mod(mdg4) gene product and the su(Hw) protein are necessary for boundary function (9). In addition to loss of enhancer blocking, it has been suggested that some mod(mdg4) mutations lead to an unmasked activity that represses certain promoters (3).To address the boundary activity of scs′ at a biochemical level, we previously characterized two cDNAs encoding the related scs′ boundary element-associated factors BEAF-32A and -32B (14, 38). The BEAF activity in Drosophila nuclear extracts appears to be composed predominantly of trimers of one 32A and two 32B subunits. Interactions between BEAF subunits results in cooperative binding to the three CGATA motifs of the high-affinity binding site in scs′ which, in turn, facilitates binding to the lower-affinity binding site located some 200 bp away (14).Evidence of a role for BEAF in boundary activity derives from an enhancer-blocking assay in Drosophila D1 cells: seven tandem copies of a 48-bp oligonucleotide containing the scs′ high-affinity binding site had enhancer-blocking activity (although less than that obtained by using scs′), while point mutations that eliminated BEAF binding further reduced this activity (38). We immunolocalized BEAF to numerous interbands and puff boundaries on polytene chromosomes, suggesting the existence of a common class of boundary elements in Drosophila and that the band-interband structure of polytene chromosomes could be related to the localization of boundary elements.In this study, we isolated some of these genomic BEAF binding sites and used transgenic flies to demonstrate that the newly isolated sequences tested represent boundary elements. The only homology found between these candidate boundary elements (cBEs) and scs′ are clusters of CGATA motifs. Despite the varied spacing and orientations of the motifs in the different clusters, BEAF interacts with all of the clusters. We also used transgenic flies to directly establish the functional importance of BEAF binding sites by mutagenesis of CGATA motifs. This strongly indicates that the hundreds of BEAF binding sites in the Drosophila genome represent an abundant class of boundary elements, providing the first example of a class of binding elements that share a sequence motif and a binding protein.     

Identification of Genome-Wide Copy Number Variations among Diverse Pig Breeds Using SNP Genotyping Arrays

Copy number variations (CNVs) are important forms of genetic variation complementary to SNPs, and can be considered as promising markers for some phenotypic and economically important traits or diseases susceptibility in domestic animals. In the present study, we performed a genome-wide CNV identification in 14 individuals selected from diverse populations, including six types of Chinese indigenous breeds, one Asian wild boar population, as well as three modern commercial foreign breeds. We identified 63 CNVRs in total, which covered 9.98 Mb of polymorphic sequence and corresponded to 0.36% of the genome sequence. The length of these CNVRs ranged from 3.20 to 827.21 kb, with an average of 158.37 kb and a median of 97.85 kb. Functional annotation revealed these identified CNVR have important molecular function, and may play an important role in exploring the genetic basis of phenotypic variability and disease susceptibility among pigs. Additionally, to confirm these potential CNVRs, we performed qPCR for 12 randomly selected CNVRs and 8 of them (66.67%) were confirmed successfully. CNVs detected in diverse populations herein are essential complementary to the CNV map in the pig genome, which provide an important resource for studies of genomic variation and the association between various economically important traits and CNVs.     

Systems-Based Analyses of Brain Regions Functionally Impacted in Parkinson's Disease Reveals Underlying Causal Mechanisms

Detailed analysis of disease-affected tissue provides insight into molecular mechanisms contributing to pathogenesis. Substantia nigra, striatum, and cortex are functionally connected with increasing degrees of alpha-synuclein pathology in Parkinson''s disease. We undertook functional and causal pathway analysis of gene expression and proteomic alterations in these three regions, and the data revealed pathways that correlated with disease progression. In addition, microarray and RNAseq experiments revealed previously unidentified causal changes related to oligodendrocyte function and synaptic vesicle release, and these and other changes were reflected across all brain regions. Importantly, subsets of these changes were replicated in Parkinson''s disease blood; suggesting peripheral tissue may provide important avenues for understanding and measuring disease status and progression. Proteomic assessment revealed alterations in mitochondria and vesicular transport proteins that preceded gene expression changes indicating defects in translation and/or protein turnover. Our combined approach of proteomics, RNAseq and microarray analyses provides a comprehensive view of the molecular changes that accompany functional loss and alpha-synuclein pathology in Parkinson''s disease, and may be instrumental to understand, diagnose and follow Parkinson''s disease progression.     

HiCORE: Hi-C Analysis for Identification of Core Chromatin Looping Regions with Higher Resolution

Genome-wide chromosome conformation capture (3C)-based high-throughput sequencing (Hi-C) has enabled identification of genome-wide chromatin loops. Because the Hi-C map with restriction fragment resolution is intrinsically associated with sparsity and stochastic noise, Hi-C data are usually binned at particular intervals; however, the binning method has limited reliability, especially at high resolution. Here, we describe a new method called HiCORE, which provides simple pipelines and algorithms to overcome the limitations of single-layered binning and predict core chromatin regions with three-dimensional physical interactions. In this approach, multiple layers of binning with slightly shifted genome coverage are generated, and interacting bins at each layer are integrated to infer narrower regions of chromatin interactions. HiCORE predicts chromatin looping regions with higher resolution, both in human and Arabidopsis genomes, and contributes to the identification of the precise positions of potential genomic elements in an unbiased manner.     

Enhancing Genome-Wide Copy Number Variation Identification by High Density Array CGH Using Diverse Resources of Pig Breeds

Copy number variations (CNVs) are important forms of genomic variation, and have attracted extensive attentions in humans as well as domestic animals. In the study, using a custom-designed 2.1 M array comparative genomic hybridization (aCGH), genome-wide CNVs were identified among 12 individuals from diverse pig breeds, including one Asian wild population, six Chinese indigenous breeds and two modern commercial breeds (Yorkshire and Landrace), with one individual of the other modern commercial breed, Duroc, as the reference. A total of 1,344 CNV regions (CNVRs) were identified, covering 47.79 Mb (∼1.70%) of the pig genome. The length of these CNVRs ranged from 3.37 Kb to 1,319.0 Kb with a mean of 35.56 Kb and a median of 11.11 Kb. Compared with similar studies reported, most of the CNVRs (74.18%) were firstly identified in present study. In order to confirm these CNVRs, 21 CNVRs were randomly chosen to be validated by quantitative real time PCR (qPCR) and a high rate (85.71%) of confirmation was obtained. Functional annotation of CNVRs suggested that the identified CNVRs have important function, and may play an important role in phenotypic and production traits difference among various breeds. Our results are essential complementary to the CNV map in the pig genome, which will provide abundant genetic markers to investigate association studies between various phenotypes and CNVs in pigs.