Identification of novel PPARγ target genes by integrated analysis of ChIP-on-chip and microarray expression data during adipocyte differentiation

PPARγ (peroxisome proliferator-activated receptor gamma) acts as a key molecule of adipocyte differentiation, and transactivates multiple target genes involved in lipid metabolic pathways. Identification of PPARγ target genes will facilitate to predict the extent to which the drugs can affect and also to understand the molecular basis of lipid metabolism. Here, we have identified five target genes regulated directly by PPARγ during adipocyte differentiation in 3T3-L1 cells using integrated analyses of ChIP-on-chip and expression microarray. We have confirmed the direct PPARγ regulation of five genes by luciferase reporter assay in NIH-3T3 cells. Of these five genes Hp, Tmem143 and 1100001G20Rik are novel PPARγ targets. We have also detected PPREs (PPAR response elements) sequences in the promoter region of the five genes computationally. Unexpectedly, most of the PPREs detected proved to be atypical, suggesting the existence of more atypical PPREs than previously thought in the promoter region of PPARγ regulated genes.     

Cells lacking IKKα show nuclear cyclin D1 overexpression and a neoplastic phenotype: role of IKKα as a tumor suppressor

The catalytic subunits of IκB kinase (IKK) complex, IKKα and IKKβ, are involved in activation of NF-κB and in mediating a variety of other biological functions. Though these proteins have a high-sequence homology, IKKα exhibits different functional characteristics as compared with IKKβ. Earlier, we have shown that cyclin D1 is overexpressed and predominantly localized in the nucleus of IKKα(-/-) cells, indicating that IKKα regulates turnover and subcellular distribution of cyclin D1, which is mediated by IKKα-induced phosphorylation of cyclin D1. Because cyclin D nuclear localization is implicated in tumor development, we examined whether the absence of IKKα leads to tumor development as well. In the current study, we show that IKKα plays a critical role in tumorigenesis. Though IKKα(-/-) MEF cells show a slower anchorage-dependent growth, they are clonogenic in soft agar. These cells are tumorigenic in nude mice. Microarray analysis of IKKα(-/-) cells indicates a differential expression of genes involved in proliferation and apoptosis. Furthermore, analysis of microarray data of human lung cancer cell lines revealed decreased IKKα RNA expression level as compared with cell lines derived from normal bronchial epithelium. These results suggest that IKKα may function as a tumor suppressor gene. Absence of IKKα may induce tumorigenicity by nuclear localization of cyclin D1 and modulating the expression of genes involved in neoplastic transformation.     

Implication of the β2-microglobulin gene in the generation of tumor escape phenotypes

Classical MHC molecules present processed peptides from endogenous protein antigens on the cell surface, which allows CD8(+) cytotoxic T lymphocytes (CTLs) to recognize and respond to the abnormal antigen repertoire of hazardous cells, including tumor cells. The light chain, β2-microglobulin (β2m), is an essential constant component of all trimeric MHC class I molecules. There is convincing evidence that β2m deficiency generates immune escape phenotypes in different tumor entities, with an exceptionally high frequency in colorectal carcinoma (CRC) and melanoma. Damage of a single β2m gene by LOH on chromosome 15 may be sufficient to generate a tumor cell precommitted to escape. In addition, this genetic lesion is followed in some tumors by a mutation of the second gene (point mutation or insertion/deletion), which produces a tumor cell unable to express any HLA class I molecule. The pattern of mutations found in microsatellite unstable colorectal carcinoma (MSI-H CRC) and melanoma showed a striking similarity, namely the predominance of frameshift mutations in repetitive CT elements. This review emphasizes common but also distinct molecular mechanisms of β2m loss in both tumor types. It also summarizes recent studies that point to an acquired β2m deficiency in response to cancer immunotherapy, a barrier to successful vaccination or adoptive cellular therapy.     

N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1''s phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α''s intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1''s recognition of H3K9me-marked nucleosomes.