Parental modifiers,antisense transcripts and loss of imprinting

The kinship theory of genomic imprinting has explained parent-specific gene expression as the outcome of an evolutionary conflict between the two alleles at a diploid locus of an offspring over how much to demand from parents. Previous models have predicted that maternally derived (madumnal) alleles will be silent at demand-enhancing loci, while paternally derived (padumnal) alleles will be silent at demand-suppressing loci, but these models have not considered the evolution of trans-acting modifiers that are expressed in parents and influence imprinted expression in offspring. We show that such modifiers will sometimes be selected to reactivate the silent padumnal allele at a demand-suppressing locus but will not be selected to reactivate the silent madumnal allele at a demand-enhancing locus. Therefore, imprinting of demand-suppressing loci is predicted to be less evolutionarily stable than imprinting of demand-enhancing loci.     

Paternal imprinting of the SLC22A1LS gene located in the human chromosome segment 11p15.5

Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive. TGFBR1*6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1*6A does not contribute to the development of prostate cancer.     

Promoter characterization and regulation of expression of an imprinted gene SLC22A18AS

The SLC22A18/SLC22A18AS genes are a sense-antisense pair located at human chromosome segment 11p15.5. These genes are paternally imprinted: paternal alleles are silenced and maternal alleles are expressed. Although SLC22A18 is a well-characterized gene, very little is known regarding its antisense partner SLC22A18AS. We therefore sought to identify the potential cis-regulating elements including the promoter of this gene, differentially methylated regions (DMRs) and the translation of its putative ORF. Dual promoters (P1 and P2) were identified for this gene and both are devoid of consensus TATA and CCAAT boxes. However, the P1 promoter harbors a putative Sp1 binding site. Sp1 binds to the P1 promoter in vivo and positively regulates its activity. Promoter and CpG II island regions showed heavy methylation of CpG sites, but no DMRs were observed. Treatment of a non-SLC22A18AS expressing HuH7 cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored expression of this gene. The histone deacetylase (HDAC) inhibitor Trichostatin-A, on the other hand, failed to induce its expression. We suggest that the expression of this gene is methylation-dependent, but histone acetylation-independent. This gene was found to be translated with a cytoplasmic localization. The present data will help to understand the regulation of this gene and its role in tumorigenesis.     

Expression and analysis of two novel rat organic cation transporter homologs, SLC22A17 and SLC22A23

The organic cation transporter (OCT, SLC22) family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs, toxins, and endogenous metabolites in a variety of tissues. Many of the OCTs have been previously characterized, but there are a number of orphan genes whose functions remain unknown. In this study, two novel rat SLC22 genes, SLC22A17 (BOCT1) and SLC22A23 (BOCT2), were cloned and characterized. Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues. BOCT1 was strongly expressed in brain, primary neurons and brain endothelial cells, with highest expression in choroid plexus. BOCT2 was also abundantly expressed in brain, as well as in liver. To characterize the products of these genes, BOCT1 cDNA was isolated from a rat blood-brain barrier cDNA library, and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques. Plasmids expressing BOCT1 and BOCT2 were transfected into HEK-293 cells, as were control cDNAs for OCT1 and OCTN2. Recombinant cell surface protein was verified by western blot and fluorescence microscopy. Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays. The OCT1- and OCTN2-expressing cells transported the canonical substrates, 1-methyl-4-phenyl-pyridinium (MPP(+)) and carnitine, respectively. However, BOCT1 and BOCT2-expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates. These novel family members have a nonconserved amino terminus, relative to other OCTs, that may preclude typical SLC22 transport function.     

MicroRNA-26b is underexpressed in human breast cancer and induces cell apoptosis by targeting SLC7A11

MicroRNAs are widely dysregulated in various cancers and integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. Here, we show that miR-26b, which is down-regulated in human breast cancer specimens and cell lines, impairs viability and triggers apoptosis of human breast cancer MCF7 cells. SLC7A11 is identified as a direct target of miR-26b and its expression is remarkably increased in both breast cancer cell lines and clinical samples. Furthermore, SLC7A11 silence mimics miR-26b-aroused viability impairment and apoptosis in MCF7 cells. Our studies reveal a protective role of miR-26b in the molecular etiology of human breast cancer by promoting apoptosis.     

Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats

Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.