Binding Of Transcription Factor Complexes Research Articles

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus.

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

The human variome: genomic and epigenomic diversity

Work from the HUGO Pan-Asian Single Nucleotide Polymorphism (SNP) consortium has mapped human genetic diversity in different subpopulations in Asia (Abdulla et al, 2009). Now this highlights the question whether disease-SNP associations identified by genome-wide association studies (GWAS) in one subpopulation can be applied to other subpopulations or whether separate GWAS should be considered for different subpopulations because of their different SNP distributions (Haiman & Stram, 2010). DNA methylation is an epigenetic mark in the mammalian genome. ‘Epigenetic diversity’ encompasses variation in DNA methylation and like ‘genetic diversity’, ‘epigenetic diversity’ may also co-vary with phenotype (Feinberg et al, 2010). Mechanistically, disease-associated SNPs may be found in DNA regulatory regions where the genetic variation affects cognate binding of transcription factor complexes, effecting on the expression of disease-relevant genes (Harismendy et al, 2011). Similarly, variation in the methylation of a DNA regulatory region (either increased or decreased methylation) can also alter the binding of transcription factor complexes and regulate both distal and proximal gene expression (Choy et al, 2010; Jones & Takai, 2001; Phillips & Corces, 2009; Weaver et al, 2004). The availability of genome-wide maps of DNA methylation (Choy et al, 2010; Down et al, 2008; Lister et al, 2009) led us to examine whether a relationship exists between DNA methylation and regions of heterogeneous genetic variation. The Fixation Index (Fst) is a measure of fixation rate of SNP between two subpopulations. SNPs with high Fst are ones with more heterogeneity in a subpopulation compared to another. Using DNA methylation maps that were generated from individuals of European ancestry, we made the interesting discovery that high Fst-SNPs (Duan et al, 2008) (N = 8751) from European ancestry showed an aggregate methylation density pattern of modulation centred on SNPs compared to flanks of the SNPs (Fig 1). Methylation maps from Europeans showed density modulation only at sites of European high Fst-SNPs (i.e. high Fst-SNPs for European vs. African + European vs. Asian), but not sites of Asian or African high Fst-SNPs (i.e. high Fst-SNPs for Asian vs. European; or African vs. European, Fig 1). Similarly aggregate methylation density modulation was also absent in a negative control set of randomly selected SNPs (N = 8000). On the basis that increased DNA methylation may be a means of regulating transcription factor complex binding and therefore mark genomic regions that are important to control by methylation (Choy et al, 2010; Jones & Takai, 2001), our findings support the notion that sites of heterogeneous genetic variation in one subpopulation are functionally relevant to the corresponding subpopulation, but sites of heterogeneous genetic variation of a different subpopulation, are not. Figure 1 Methylation density across sites of SNPs This is consistent with the need to consider subpopulation differences in genetic variation when studying disease-SNP association. By extension, we propose that the epigenomes of subpopulations should also be considered as the International Human Epigenome Consortium gears up to generate 1000 reference epigenomes (Beck, 2010). Subpopulation differences will be important when drawing conclusions from associations between disease and the human variome.

Quantitative analysis of binding of transcription factor complex to biotinylated DNA probe by a streptavidin–agarose pulldown assay

Gene expression is regulated by a large complex of proteins that bind to the promoter/enhancer region of a gene. We determined whether a streptavidin–bead binding assay might be useful in detecting individual proteins in the complex comprising transactivators, coactivators, mediators, and general transcription factors. We used biotinylated cyclooxygenase-2 promoter probes as a model. Nuclear extracts obtained from human fibroblasts treated with or without an agonist were incubated with a 5 ′-biotinylated probe and streptavidin–agarose beads at room temperature for 1 h. After centrifugation, the pellet was washed and proteins in the complex were assessed by immunoblots. An array of transcription factors was detectable concurrently in the same batch of pellets at basal state. p300 and its associated factor PCAF levels but not Srb7, Med7, or TFII B were increased by phorbol ester or tumor necrosis factor α stimulation. Only trace of CREB-binding protein was detected. These results suggest that p300 and PCAF are the predominant coactivators for COX-2 promoter activation. Our findings indicate that the streptavidin–bead pulldown assay is valuable for determining the binding of a large number of transcription factors to promoter/enhancer and evaluating the relationship of protein binding with regulation of gene expression.

Curcumin alters EpRE and AP-1 binding complexes and elevates glutamate-cysteine ligase gene expression.

Dietary use of curcumin, the active component of tumeric, one of the most widely used spices, is linked to several beneficial health effects, although the underlying molecular mechanisms remain largely unknown. Correlations have been established between curcumin exposure and increases in enzymes for glutathione synthesis, particularly glutamate-cysteine ligase (GCL), and metabolism as well as glutathione content, suggesting the eliciting of an adaptive response to stress. In this study, using HBE1 cells, we found that the mechanism of curcumin-induced GCL elevation occurred via transcription of the two Gcl genes. Gcl transcription has been shown in several systems to be mediated through binding of transcription factor complexes to TRE and EpRE elements. Studies herein showed that curcumin caused modest but sustained increases in binding of proteins to DNA sequences for both cis elements but, more importantly, altered the compositions and nuclear content of proteins in these complexes. Curcumin exposure increased JunD and c-Jun content in AP-1 complexes and increased JunD while decreasing MafG/MafK in EpRE complexes. Thus, the beneficial effects elicited by curcumin appear to be due to changes in the pool of transcription factors that compose EpRE and AP-1 complexes, affecting gene expression of GCL and other phase II enzymes.