Keijo Viiri

DNA-Binding and -Bending Activities of SAP30L and SAP30 Are Mediated by a Zinc-Dependent Module and Monophosphoinositides

ABSTRACT Deacetylation of histones is carried out by a corepressor complex in which Sin3A is an essential scaffold protein. Two proteins in this complex, the Sin3A-associated proteins SAP30L and SAP30, have previously been suggested to function as linker molecules between various corepressors. In this report, we demonstrate new functions for human SAP30L and SAP30 by showing that they can associate directly with core histones as well as naked DNA. A zinc-coordinating structure is necessary for DNA binding, one consequence of which is bending of the DNA. We provide evidence that a sequence motif previously shown to be a nuclear localization signal is also a phosphatidylinositol (PI)-binding element and that binding of specific nuclear monophosphoinositides regulates DNA binding and chromatin association of SAP30L. PI binding also decreases the repression activity of SAP30L and affects its translocation from the nucleus to the cytoplasm. Our results suggest that SAP30L and SAP30 play active roles in recruitment of deacetylating enzymes to nucleosomes, and mediate key protein-protein and protein-DNA interactions involved in chromatin remodeling and transcription.

Myosin IXB gene region and gluten intolerance: linkage to coeliac disease and a putative dermatitis herpetiformis association

Background: Coeliac disease is caused by dietary gluten, which triggers chronic inflammation of the small intestine in genetically predisposed individuals. In one quarter of the patients the disease manifests in the skin as dermatitis herpetiformis. Recently, a novel candidate gene, myosin IXB on chromosome 19p13, was shown to be associated with coeliac disease in the Dutch and Spanish populations. The same gene has previously been associated with inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis risk, making myosin IXB a potential shared risk factor in these inflammatory disorders. Methods: In this study, previously reported myosin IXB variants were tested for genetic linkage and association with coeliac disease in 495 Hungarian and Finnish families and in an additional 270 patients and controls. Results and conclusion: The results show significant linkage (logarithm of odds (LOD) 3.76, p = 0.00002) to 19p13 which supports the presence of a genuine risk factor for coeliac disease in this locus. Myosin IXB variants were not associated with coeliac disease in this study; however, weak evidence of association with dermatitis herpetiformis was found. The association could not explain the strong linkage seen in both phenotypes, indicating that the role of other neighbouring genes in the region cannot be excluded. Therefore, more detailed genetic and functional studies are required to characterise the role of the myosin IXB gene in both coeliac disease and dermatitis herpetiformis.

The Dnmt3L ADD Domain Controls Cytosine Methylation Establishment during Spermatogenesis

Summary A critical aspect of mammalian gametogenesis is the reprogramming of genomic DNA methylation. The catalytically inactive adaptor Dnmt3L is essential to ensuring this occurs correctly, but the mechanism by which it functions is unclear. Using gene targeting to engineer a single-amino-acid mutation, we show that the Dnmt3L histone H3 binding domain (ADD) is necessary for spermatogenesis. Genome-wide single-base-resolution DNA methylome analysis of mutant germ cells revealed overall reductions in CG methylation at repetitive sequences and non-promoter CpG islands. Strikingly, we also observe an even more severe loss of non-CG methylation, suggesting an unexpected role for the ADD in this process. These epigenetic deficiencies were coupled with defects in spermatogonia, with mutant cells displaying marked changes in gene expression and reactivation of retrotransposons. Our results demonstrate that the Dnmt3L ADD is necessary for Dnmt3L function and full reproductive fitness.

Extracellular transglutaminase 2 has a role in cell adhesion, whereas intracellular transglutaminase 2 is involved in regulation of endothelial cell proliferation and apoptosis

Objective:  Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell–matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi.

Phosphoinositides as Regulators of Protein-Chromatin Interactions

Phosphoinositides can promote or reduce the binding of proteins with roles in transcription to chromatin. The molecular function of phospholipids in the nucleus has been only partially elucidated. The upsurge of epigenetic research has contributed to increased interest in nuclear phospholipids, such as phosphoinositides, and their involvement in gene transcription. However, the mechanisms by which phosphoinositides regulate transcription is still unknown at the molecular level. Certain phosphoinositide species can regulate protein-chromatin and protein–nucleic acid interactions, and specific nuclear target proteins link nuclear signaling lipids to gene expression. We propose that a phosphoinositide-mediated detachment of proteins from chromatin is a general biological mechanism that partly underlies the signaling effects of nuclear phosphoinositides.

TGF-β induces the expression of SAP30L, a novel nuclear protein

BackgroundWe have previously set up an in vitro mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. In this model the fibroblast-induced epithelial cell differentiation from secretory crypt cells to absorptive enterocytes is mediated via transforming growth factor-β (TGF-β), the major inhibitory regulator of epithelial cell proliferation known to induce differentiation in intestinal epithelial cells. The aim of this study was to identify novel genes whose products would play a role in this TGF-β-induced differentiation.ResultsDifferential display analysis resulted in the identification of a novel TGF-β upregulated mRNA species, the Sin3-associated protein 30-like, SAP30L. The mRNA is expressed in several human tissues and codes for a nuclear protein of 183 amino acids 70% identical with Sin3 associated protein 30 (SAP30). The predicted nuclear localization signal of SAP30L is sufficient for nuclear transport of the protein although mutating it does not completely remove SAP30L from the nuclei. In the nuclei SAP30L concentrates in small bodies which were shown by immunohistochemistry to colocalize with PML bodies only partially.ConclusionsBy reason of its nuclear localization and close homology to SAP30 we believe that SAP30L might have a role in recruiting the Sin3-histone deacetylase complex to specific corepressor complexes in response to TGF-β, leading to the silencing of proliferation-driving genes in the differentiating intestinal epithelial cells.

Interactions of Functional Apolipoprotein E Gene Promoter Polymorphisms With Smoking on Aortic Atherosclerosis

Background—Apolipoprotein E gene (APOE) interacts with environmental factors in defining risk for atherosclerosis. We studied whether the APOE ϵ2/ϵ3/ϵ4 genotype or APOE promoter polymorphisms −219G/T and +113G/C might interact with smoking on the development of fatty streaks. We also studied the previously unknown effects of +113G/C on transcriptional activity. Methods and Results—The fatty streak areas of aorta were measured morphometrically in subjects of the Helsinki Sudden Death Study. Within APOE ϵ3/ϵ3 subjects, there was a strong interaction between smoking and both −219G/T (P=0.009) and +113G/C (P=0.003) promoter polymorphisms on abdominal aorta fatty streak area: the −219T- and +113C-allele carriers had larger lesion areas compared with G/G (12.7% versus 5.9%, P=0.007; 12.9% versus 6.3%, P=0.010, respectively) within nonsmokers. Within smokers, the associations were inverse. Moreover, smoking increased the fatty streak area within −219G/G or +113G/G genotypes and −219G/+113G/ϵ3 haplotype carriers. Functional studies in reporter assay showed that in comparison with the +113G allele, the +113C allele had higher transcriptional activity and bound transcription factors from liver cell nuclear extract with significantly lower affinity. Conclusions—In middle-aged Finnish men with APOE ϵ3/ϵ3 genotype, the APOE promoter polymorphisms −219G/T and +113G/C interact with smoking in modulating aortic atherosclerosis. The +113G/C polymorphism has an effect on transcriptional activity.

Interactions of Functional APOE Promoter Polymorphisms with Smoking on Aortic Atherosclerosis

Background—Apolipoprotein E gene (APOE) interacts with environmental factors in defining risk for atherosclerosis. We studied whether the APOE ϵ2/ϵ3/ϵ4 genotype or APOE promoter polymorphisms −219G/T and +113G/C might interact with smoking on the development of fatty streaks. We also studied the previously unknown effects of +113G/C on transcriptional activity. Methods and Results—The fatty streak areas of aorta were measured morphometrically in subjects of the Helsinki Sudden Death Study. Within APOE ϵ3/ϵ3 subjects, there was a strong interaction between smoking and both −219G/T (P=0.009) and +113G/C (P=0.003) promoter polymorphisms on abdominal aorta fatty streak area: the −219T- and +113C-allele carriers had larger lesion areas compared with G/G (12.7% versus 5.9%, P=0.007; 12.9% versus 6.3%, P=0.010, respectively) within nonsmokers. Within smokers, the associations were inverse. Moreover, smoking increased the fatty streak area within −219G/G or +113G/G genotypes and −219G/+113G/ϵ3 haplotype carriers. Functional studies in reporter assay showed that in comparison with the +113G allele, the +113C allele had higher transcriptional activity and bound transcription factors from liver cell nuclear extract with significantly lower affinity. Conclusions—In middle-aged Finnish men with APOE ϵ3/ϵ3 genotype, the APOE promoter polymorphisms −219G/T and +113G/C interact with smoking in modulating aortic atherosclerosis. The +113G/C polymorphism has an effect on transcriptional activity.

Phylogenetic analysis of the SAP30 family of transcriptional regulators reveals functional divergence in the domain that binds the nuclear matrix.

BackgroundDeacetylation of histones plays a fundamental role in gene silencing, and this is mediated by a corepressor complex containing Sin3 as an essential scaffold protein. In this report we examine the evolution of two proteins in this complex, the Sin3-associated proteins SAP30L and SAP30, by using an archive of protein sequences from 62 species.ResultsOur analysis indicates that in tetrapods SAP30L is more similar than SAP30 to the ancestral protein, and the two copies in this group originated by gene duplication which occurred after the divergence of Actinopterygii and Sarcopterygii about 450 million years ago (Mya). The phylogenetic analysis and biochemical experiments suggest that SAP30 has diverged functionally from the ancestral SAP30L by accumulating mutations that have caused attenuation of one of the original functions, association with the nuclear matrix. This function is mediated by a nuclear matrix association sequence, which consists of a conserved motif in the C-terminus and the adjacent nucleolar localization signal (NoLS).ConclusionThese results add further insight into the evolution and function of proteins of the SAP30 family, which share many characteristic with nuclear scaffolding proteins that are intimately involved in regulation of gene expression. Furthermore, SAP30L seems essential to eukaryotic biology, as it is found in animals, plants, fungi, as well as some taxa of unicellular eukaryotes.

Alternative mRNA splicing of SAP30L regulates its transcriptional repression activity

Covalent modification of histones regulates chromatin structure and gene expression. Sin3A mediates the association of histone deacetylase enzymes with a large number of sequence‐specific transcriptional repressors. In this study we characterized three novel transcripts of SAP30L, a recently identified Sin3A‐associated protein. These splice variants show significant differences in transcriptional repression capabilities and associating histone deacetylase activities. Furthermore, they differ in binding to Sin3A and in subcellular localization when transiently transfected. These data suggest that the transcriptional repression of a Sin3A corepressor complex can be regulated not only by sequence‐specific transcriptional repressors, but also by modification of associated proteins, such as SAP30L.