The impact of cis-acting polymorphisms on the human phenotype

Cis-acting polymorphisms that affect gene expression are now known to be frequent, although the extent and mechanisms by which such variation affects the human phenotype are, as yet, only poorly understood. Key signatures of cis-acting variation are differences in gene expression that are tightly associated with regulatory SNPs or expression Quantitative Trait Loci (eQTL) and an imbalance of allelic expression (AEI) in heterozygous samples. Such cis-acting sequence differences appear often to have been under selection within and between populations and are also thought to be important in speciation. Here we describe the example of lactase persistence. In medical research, variants that affect regulation in cis have been implicated in both monogenic and polygenic disorders, and in the metabolism of drugs. In this review we suggest that by further understanding common regulatory variations and how they interact with other genetic and environmental variables it will be possible to gain insight into important mechanisms behind complex disease, with the potential to lead to new methods of diagnosis and treatments.     

Effects of cis and trans regulatory variations on the expression divergence of heat shock response genes between yeast strains

Phenotypic variation among individuals in a population can be due to DNA sequence variation in protein coding regions or in regulatory elements. Recently, many studies have indicated that mutations in regulatory elements may be the major cause of phenotypic evolution. However, the mechanisms for evolutionary changes in gene expression are still not well understood. Here, we studied the relative roles of cis and trans regulatory changes in Saccharomyces cerevisiae cells to cope with heat stress. It has been found that the expression level of ~ 300 genes was induced at least two fold and that of ~ 500 genes was repressed at least two fold in response to heat shock. From the former set of genes, we randomly selected 65 genes that showed polymorphism(s) between the BY and RM strains for pyrosequencing analysis to explore the relative contributions of cis and trans regulatory variations to the expression divergence between BY and RM. Our data indicated that the expression divergence between BY and RM was mainly due to trans regulatory variations under either the normal condition or the heat stress condition. However, the relative contribution of trans regulatory variation was decreased from 76.9% to 61.5% after the heat shock stress. These results indicated that the cis regulatory variation may play an important role in the adaption to heat stress. In our data, 43.1% (28 genes) of the 65 genes showed the same trend of cis or trans variation effect after the heat shock stress, 35.4% (23 genes) showed an increased cis variation effect and 21.5% (14 genes) showed an increased trans variation effect after the heat shock stress. Thus, our data give insights into the relative roles of cis and trans variations in response to heat shock in yeast.     

Robustness under Functional Constraint: The Genetic Network for Temporal Expression in Drosophila Neurogenesis

Precise temporal coordination of gene expression is crucial for many developmental processes. One central question in developmental biology is how such coordinated expression patterns are robustly controlled. During embryonic development of the Drosophila central nervous system, neural stem cells called neuroblasts express a group of genes in a definite order, which leads to the diversity of cell types. We produced all possible regulatory networks of these genes and examined their expression dynamics numerically. From the analysis, we identified requisite regulations and predicted an unknown factor to reproduce known expression profiles caused by loss-of-function or overexpression of the genes in vivo, as well as in the wild type. Following this, we evaluated the stability of the actual Drosophila network for sequential expression. This network shows the highest robustness against parameter variations and gene expression fluctuations among the possible networks that reproduce the expression profiles. We propose a regulatory module composed of three types of regulations that is responsible for precise sequential expression. This study suggests that the Drosophila network for sequential expression has evolved to generate the robust temporal expression for neuronal specification.     

Comprehensive analysis of gene-environmental interactions with temporal gene expression profiles in Pseudomonas aeruginosa

To explore gene-environment interactions, based on temporal gene expression information, we analyzed gene and treatment information intensively and inferred interaction networks accordingly. The main idea is that gene expression reflects the response of genes to environmental factors, assuming that variations of gene expression occur under different conditions. Then we classified experimental conditions into several subgroups based on the similarity of temporal gene expression profiles. This procedure is useful because it allows us to combine diverse gene expression data as they become available, and, especially, allowing us to lay the regulatory relationships on a concrete biological basis. By estimating the activation points, we can visualize the gene behavior, and obtain a consensus gene activation order, and hence describe conditional regulatory relationships. The estimation of activation points and building of synthetic genetic networks may result in important new insights in the ongoing endeavor to understand the complex network of gene regulation.     

Modifier effects between regulatory and protein-coding variation

Genome-wide associations have shown a lot of promise in dissecting the genetics of complex traits in humans with single variants, yet a large fraction of the genetic effects is still unaccounted for. Analyzing genetic interactions between variants (epistasis) is one of the potential ways forward. We investigated the abundance and functional impact of a specific type of epistasis, namely the interaction between regulatory and protein-coding variants. Using genotype and gene expression data from the 210 unrelated individuals of the original four HapMap populations, we have explored the combined effects of regulatory and protein-coding single nucleotide polymorphisms (SNPs). We predict that about 18% (1,502 out of 8,233 nsSNPs) of protein-coding variants are differentially expressed among individuals and demonstrate that regulatory variants can modify the functional effect of a coding variant in cis. Furthermore, we show that such interactions in cis can affect the expression of downstream targets of the gene containing the protein-coding SNP. In this way, a cis interaction between regulatory and protein-coding variants has a trans impact on gene expression. Given the abundance of both types of variants in human populations, we propose that joint consideration of regulatory and protein-coding variants may reveal additional genetic effects underlying complex traits and disease and may shed light on causes of differential penetrance of known disease variants.     

GAPDH is not regulated in human glioblastoma under hypoxic conditions

Background

Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results

We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion

GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.