Allelic imbalance metre (Allim), a new tool for measuring allele‐specific gene expression with RNA‐seq data

Estimating differences in gene expression among alleles is of high interest for many areas in biology and medicine. Here, we present a user‐friendly software tool, Allim, to estimate allele‐specific gene expression. Because mapping bias is a major problem for reliable estimates of allele‐specific gene expression using RNA‐seq, Allim combines two different strategies to account for the mapping biases. In order to reduce the mapping bias, Allim first generates a polymorphism‐aware reference genome that accounts for the sequence variation between the alleles. Then, a sequence‐specific simulation tool estimates the residual mapping bias. Statistical tests for allelic imbalance are provided that can be used with the bias corrected RNA‐seq data.     

Prenatal exposure to dexamethasone in the mouse induces sex‐specific differences in placental gene expression

Prenatal stress during pregnancy leads to sex‐specific effects on fetal development and disease susceptibility over the life span; however, the origin of sex differences has not been identified. The placenta not only plays a key role in fetal growth and development throughout pregnancy, but also affects the fetal programming underlying subsequent adult health and accounts. Therefore, sex‐specific adaptation of the placenta may be central to the sex differences in fetal growth and survival. Here, we analyzed the effects of prenatal dexamethasone (Dex) on sex‐specific changes in placental gene expression using RNA‐Seq. Placental tissues from males and females were separated into two developmentally distinct fetal and maternal parts at E11.5 stage. The majority of genes in female placentas were downregulated by prenatal Dex, whereas those were mostly maintained or rather upregulated in male placentas. RNA‐Seq results were validated using independent biological replicates from the same stage and placental tissue samples from E18.5 by realtime PCR assays. Activation of various inflammatory response‐related genes, chemokines and their receptors, particularly in male placentas, strongly implies that prenatal Dex exposure causes sex‐specific physiological responses that can lead to inflammatory diseases involving vascular pathology.     

A simple method to score single nucleotide polymorphisms based on allele‐specific PCR and primer‐induced fragment‐length variation

We describe a simple protocol to genotype single nucleotide polymorphisms (SNPs), which combines allele‐specific polymerase chain reaction (PCR) with fragment‐length analysis. Three primers are used in the PCR: two allele‐specific forward primers with a length‐difference and one reverse primer. The forward primers induce a length‐difference between the SNP‐variants, which can be assessed with standard fragment‐length analyses. We designed primers for 21 SNPs, and codominance was achieved for 76% of these SNPs. An inexpensive and flexible laser‐detection scoring protocol can be achieved with multiplex scoring and by incorporating the M13(‐21) genotyping method.     

Genotyping of pantophysin I (Pan I) of Atlantic cod (Gadus morhua L.) by allele‐specific PCR

The two main allelic variants of the Atlantic cod (Gadus morhua L.) pantophysin I (Pan I) locus have different frequencies within different cod stocks. The Dra I polymorphism which distinguishes the two alleles can thus be used for discrimination of coastal and offshore cod populations. We present a new method for Pan I genotyping using fluorescent allele‐specific duplex polymerase chain reaction (PCR). This method is more rapid, reliable and cost‐effective than the previously published method and it is not affected by DNA source and quality. This improvement is important for studies demanding high throughput and accuracy of Pan I genotyping     

Genome‐wide characterization and expression analysis of genetic variants in sweet orange

Heterozyosity is an important feature of many plant genomes, and is related to heterosis. Sweet orange, a highly heterozygous species, is thought to have originated from an inter‐species hybrid between pummelo and mandarin. To investigate the heterozygosity of the sweet orange genome and examine how this heterozygosity affects gene expression, we characterized the genome of Valencia orange for single nucleotide variations (SNVs), small insertions and deletions (InDels) and structural variations (SVs), and determined their functional effects on protein‐coding genes and non‐coding sequences. Almost half of the genes containing large‐effect SNVs and InDels were expressed in a tissue‐specific manner. We identified 3542 large SVs (>50 bp), including deletions, insertions and inversions. Most of the 296 genes located in large‐deletion regions showed low expression levels. RNA‐Seq reads and DNA sequencing reads revealed that the alleles of 1062 genes were differentially expressed. In addition, we detected approximately 42 Mb of contigs that were not found in the reference genome of a haploid sweet orange by de novo assembly of unmapped reads, and annotated 134 protein‐coding genes within these contigs. We discuss how this heterozygosity affects the quality of genome assembly. This study advances our understanding of the genome architecture of sweet orange, and provides a global view of gene expression at heterozygous loci.     

Simple and rapid bioluminescent detection of two verotoxin genes using allele‐specific PCR of E. coli O157: H7

Allele‐specific PCR for E. coli O157 was conducted with primers specific to verotoxin genes, verotoxin 1 (VT1) and verotoxin 2 (VT2). VT is an important cause of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) worldwide. We developed a simple, rapid bioluminescent detection method for E. coli O157. The method is based on the determination of pyrophosphoric acid (PPi) released during allele‐specific PCR. Thus, released PPi is converted to ATP by ATP sulphurylase and the concentration of ATP is determined using the firefly luciferase reaction. As a result, VT1, VT2 and DNA with VT1/VT2 were clearly identified by this method. This protocol, which does not require expensive equipment, can be utilized to monitor the PCR product rapidly. Additionally, this methodology can be used as a high‐throughput approach for measuring PCR products. Copyright © 2003 John Wiley & Sons, Ltd.     

ChIP‐Seq of ERα and RNA polymerase II defines genes differentially responding to ligands

We used ChIP‐Seq to map ERα‐binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF‐7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identify 10 205 high confidence ERα‐binding sites in response to E2 of which 68% contain an estrogen response element (ERE) and only 7% contain a FOXA1 motif. Remarkably, 596 genes change significantly in RNAPII occupancy (59% up and 41% down) already after 1 h of E2 exposure. Although promoter proximal enrichment of RNAPII (PPEP) occurs frequently in MCF‐7 cells (17%), it is only observed on a minority of E2‐regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERα DNA binding and prevent RNAPII loading on the promoter and coding body on E2‐upregulated genes. Both ligands act differently on E2‐downregulated genes: tamoxifen acts as an agonist thus downregulating these genes, whereas fulvestrant antagonizes E2‐induced repression and often increases RNAPII occupancy. Furthermore, our data identify genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus (partial) antagonist loaded ERα acts mechanistically different on E2‐activated and E2‐repressed genes.     

Extensive allele‐level remodeling of histone methylation modification in reciprocal F1 hybrids of rice subspecies

Epigenetic mechanisms play a major role in heterosis, partly as a result of the remodeling of epigenetic modifications in F₁ hybrids. Based on chromatin immunoprecipitation‐sequencing (ChIP‐Seq) analyses, we show that at the allele level extensive histone methylation remodeling occurred for a subset of genomic loci in reciprocal F₁ hybrids of Oryza sativa (rice) cultivars Nipponbare and 93‐11, representing the two subspecies japonica and indica. Globally, the allele modification‐altered loci in leaf or root of the reciprocal F₁ hybrids involved ?12–43% or more of the genomic regions carrying either of two typical histone methylation markers, H3K4me3 (>21 000 genomic regions) and H3K27me3 (>11 000 genomic regions). Nevertheless, at the total modification level, the majority (from ?43 to >90%) of the modification‐altered alleles lay within the range of parental additivity in the hybrids because of concerted alteration in opposite directions, consistent with an overall attenuation of allelic differences in the modifications. Importantly, of the genomic regions that did show non‐additivity in total modification level by either marker in the two tissues of hybrids, >80% manifested transgressivity, which involved genes enriched in specific functional categories. Extensive allele‐level alteration of H3K4me3 alone was positively correlated with genome‐wide changes in allele‐level gene expression, whereas at the total level, both H3K4me3 and H3K27me3 remodeling, although affecting just a small number of genes, contributes to the overall non‐additive gene expression to variable extents, depending on tissue/marker combinations. Our results emphasize the importance of allele‐level analysis in hybrids to assess the remodeling of epigenetic modifications and their relation to changes in gene expression.