Multiple potential intragenic regulatory elements in the CFTR gene

The CFTR gene exhibits a complex pattern of expression that shows temporal and spatial regulation though the control mechanisms have not been fully elucidated. We have mapped DNase I hypersensitive sites (DHS) flanking the CFTR gene to identify potential regulatory elements. We previously characterized DHS at -79.5 and -20.9 kb with respect to the CFTR translational start site, DHS 3' to the gene at 4574 + 5.4-7.4 and 4574 + 15.6 kb, and a regulatory element in the first intron of the gene at 185 + 10 kb. We generated a cosmid contig to provide probes to evaluate the whole of the CFTR gene for DHS and have now mapped novel sites in introns 2, 3, 10, 16, 17a, 18, 20, and 21. These DHS show different patterns of cell-specific expression.     

Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.     

Integrative analysis of epigenetics data identifies gene-specific regulatory elements

Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water.     

Search for enhancers: teleost models in comparative genomic and transgenic analysis of cis regulatory elements

Homology searches between DNA sequences of evolutionary distant species (phylogenetic footprinting) offer a fast detection method for regulatory sequences. Because of the small size of their genomes, tetraodontid species such as the Japanese pufferfish and green spotted pufferfish have become attractive models for comparative genomics. A disadvantage of the tetraodontid species is, however, that they cannot be bred and manipulated routinely under laboratory conditions, so these species are less attractive for developmental and genetic analysis. In contrast, an increasing arsenal of transgene techniques with the developmental model species zebrafish and medaka are being used for functional analysis of cis regulatory sequences. The main disadvantage is the much larger genome. While comparison between many loci proved the suitability of phylogenetic footprinting using fish and mammalian sequences, fast rate of change in enhancer structure and gene duplication within teleosts may obscure detection of homologies. Here we discuss the contribution and potentials provided by different teleost models for the detection and functional analysis of conserved cis-regulatory elements.     

Comparative functional analysis of the cow and mouse myostatin genes reveals novel regulatory elements in their upstream promoter regions

Myostatin is a paracrine/autocrine factor that inhibits muscle growth, and mutations that affect myostatin activity or expression produce dramatic increases in muscle mass in several species. However, at present it is less clear whether differences in myostatin expression or activity exist between species with differing body sizes. Here we demonstrate that mouse muscle expresses far greater levels of myostatin mRNA than cow. In addition, activity of a 1200 bp mouse myostatin promoter construct was significantly greater than that of a 1200 bp cow myostatin promoter construct in C₂C₁₂ myotubes. In contrast, activity of reporter constructs flanked by one or both untranslated regions (UTRs) was not significantly different between the two species. Sequence analysis identified a number of promoter regions which differed between larger species (cow, pig, goat, sheep, human) and smaller (mouse, rat), including a TATA-box sequence, a CACCC box, two AT-rich regions (AT1 and AT2), and a palindromic sequence (PAL). We therefore used mutagenesis to alter the mouse sequence for each of these elements to that of the cow. Mutagenesis of the TATA, CACC, and AT1 sequences of the mouse to those of the cow significantly decreased activity of the mouse myostatin promoter compared to the wild type mouse promoter, while mutation of the AT2 and PAL sequences tended to increase promoter activity. Finally, the cow myostatin promoter was less responsive to FoxO signaling than the mouse myostatin promoter. Together these data support the hypothesis that differences in promoter activity between mouse and cow may contribute to differences in expression of the myostatin gene between these species.     

Genome‐wide association analysis identifies potential regulatory genes for eumelanin pigmentation in chicken plumage

Plumage color in chicken is determined by the proportion of eumelanin and pheomelanin pigmentation. As the main ingredient in plumage melanin, eumelanin plays a key role in the dark black, brown and grey coloration. However, very few studies have been performed to identify the related genes and mutations on a genome‐wide scale. Herein, a resource family consisting of one backcross population and two F2 cross populations between a black roster and Yukou Brown I parent stockbreed was constructed for identification of genes related to eumelanin pigmentation. Chickens with eumelanin in their plumage were classified as the case group, and the rest were considered the control group. A genome‐wide association study of this phenotype and genotypes using Affymetrix 600K HD SNP arrays in this F2 family revealed 13 significantly associated SNPs and in 10 separate genes on chromosomes 1, 2, 3 and 5. Based on previous studies in model species, we inferred that genes, including NUAK family kinase 1 (NUAK1) and sonic hedgehog (SHH), may play roles in the development of neural crest cells or melanoblasts during the embryonic period, which may also affect the eumelanin pigmentation. Our results facilitate the understanding of the genetic basis of eumelanin pigmentation in chicken plumage.     

A comparative genomic analysis of plant hormone related genes in different species

Plant hormones are small molecules that play important roles throughout the life span of a plant,known as auxin,gibberellin,cytokinin,abscisic acid,ethylene,jasmonic acid,salicylic acid,and brassinosteroid.Genetic and molecular studies in the model organism Arabidopsis thaliana have revealed the individual pathways of various plant hormone responses.In this study,we selected 479 genes that were convincingly associated with various hormone actions based on genetic evidence.By using these 479 genes as queries,a genome-wide search for their orthoiogues in several species(microorganisms,plants and animals) was performed.Meanwhile,a comparative analysis was conducted to evaluate their evolutionary relationship.Our analysis revealed that the metabolisms and functions of plant hormones are generally more sophisticated and diversified in higher plant species.In particular,we found that several phytohormone receptors and key signaling components were not present in lower plants or animals.Meanwhile,as the genome complexity increases,the orthologne genes tend to have more copies and probably gain more diverse functions.Our study attempts to introduce the classification and phylogenic analysis of phytohormone related genes,from metabolism enzymes to receptors and signaling components,in different species.     

A comparative genomic analysis of plant hormone related genes in different species

Plant hormones are small molecules that play important roles throughout the life span of a plant,known as auxin,gibberellin,cyto-kinin,abscisic acid,ethylene,jasmonic acid,salicylic acid,and brassinosteroid.Genetic and molecular studies in the model organism Arabidopsis thaliana have revealed the individual pathways of various plant hormone responses.In this study,we selected 479 genes that were convincingly associated with various hormone actions based on genetic evidence.By using these 479 genes as querie...     

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp.     

Analysis of 3D genomic interactions identifies candidate host genes that transposable elements potentially regulate

Background

The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.

Results

Using PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes.

Conclusions

4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.