Metric for Measuring the Effectiveness of Clustering of DNA Microarray Expression

BACKGROUND: The recent advancement of microarray technology with lower noise and better affordability makes it possible to determine expression of several thousand genes simultaneously. The differentially expressed genes are filtered first and then clustered based on the expression profiles of the genes. A large number of clustering algorithms and distance measuring matrices are proposed in the literature. The popular ones among them include hierarchal clustering and k-means clustering. These algorithms have often used the Euclidian distance or Pearson correlation distance. The biologists or the practitioners are often confused as to which algorithm to use since there is no clear winner among algorithms or among distance measuring metrics. Several validation indices have been proposed in the literature and these are based directly or indirectly on distances; hence a method that uses any of these indices does not relate to any biological features such as biological processes or molecular functions. RESULTS: In this paper we have proposed a metric to measure the effectiveness of clustering algorithms of genes by computing inter-cluster cohesiveness and as well as the intra-cluster separation with respect to biological features such as biological processes or molecular functions. We have applied this metric to the clusters on the data set that we have created as part of a larger study to determine the cancer suppressive mechanism of a class of chemicals called retinoids. We have considered hierarchal and k-means clustering with Euclidian and Pearson correlation distances. Our results show that genes of similar expression profiles are more likely to be closely related to biological processes than they are to molecular functions. The findings have been supported by many works in the area of gene clustering. CONCLUSION: The best clustering algorithm of genes must achieve cohesiveness within a cluster with respect to some biological features, and as well as maximum separation between clusters in terms of the distribution of genes of a behavioral group across clusters. We claim that our proposed metric is novel in this respect and that it provides a measure of both inter and intra cluster cohesiveness. Best of all, computation of the proposed metric is easy and it provides a single quantitative value, which makes comparison of different algorithms easier. The maximum cluster cohesiveness and the maximum intra-cluster separation are indicated by the metric when its value is 0. We have demonstrated the metric by applying it to a data set with gene behavioral groupings such as biological process and molecular functions. The metric can be easily extended to other features of a gene such as DNA binding sites and protein-protein interactions of the gene product, special features of the intron-exon structure, promoter characteristics, etc. The metric can also be used in other domains that use two different parametric spaces; one for clustering and the other one for measuring the effectiveness

Desmocollin switching in colorectal cancer

The desmocollins are members of the desmosomal cadherin family of cell–cell adhesion molecules. They are essential constituents of desmosomes, intercellular junctions that play a critical role in the maintenance of tissue integrity in epithelia and cardiac muscle. In humans, three desmocollins (Dsc1, Dsc2 and Dsc3) have been described. The desmocollins exhibit tissue-specific patterns of expression; only Dsc2 is expressed in normal colonic epithelium. We have found switching between desmocollins in sporadic colorectal adenocarcinoma with a reduction in Dsc2 protein (in 8/16 samples analysed by immunohistochemistry) being accompanied by de novo expression of Dsc1 (16/16) and Dsc3 (7/16). Similar results were obtained by western blotting of a further 16 samples. No change was found in Dsc2 mRNA, but de novo expression of Dscs 1 and 3 was accompanied by increased message levels. Loss of Dsc2 (8/19) and de novo expression of Dsc1 (11/19) and Dsc3 (6/19) was also found in colorectal adenocarcinomas on a background of colitis. The data raise the possibility that switching of desmocollins could play an important role in the development of colorectal cancer

GPR50 Interacts with TIP60 to Modulate Glucocorticoid Receptor Signalling

GPR50 is an orphan G-protein coupled receptor most closely related to the melatonin receptors. The physiological function of GPR50 remains unclear, although our previous studies implicate the receptor in energy homeostasis. Here, we reveal a role for GPR50 as a signalling partner and modulator of the transcriptional co-activator TIP60. This interaction was identified in a yeast-two-hybrid screen, and confirmed by co-immunoprecipitation and co-localisation of TIP60 and GPR50 in HEK293 cells. Co-expression with TIP60 increased perinuclear localisation of full length GPR50, and resulted in nuclear translocation of the cytoplasmic tail of the receptor, suggesting a functional interaction of the two proteins. We further demonstrate that GPR50 can enhance TIP60-coactiavtion of glucocorticoid receptor (GR) signalling. In line with in vitro results, repression of pituitary Pomc expression, and induction of gluconeogenic genes in liver in response to the GR agonist, dexamethasone was attenuated in Gpr50−/− mice. These results identify a novel role for GPR50 in glucocorticoid receptor signalling through interaction with TIP60

Missense mutations in Desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro

<p>Abstract</p> <p>Background</p> <p>Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement.</p> <p>We screened 54 ARVC probands for mutations in desmocollin-2 (<it>DSC2</it>), the only desmocollin isoform expressed in cardiac tissue.</p> <p>Methods</p> <p>Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing.</p> <p>To evaluate the pathogenic potentials of the <it>DSC2 </it>mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells.</p> <p>Results</p> <p>We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions.</p> <p>In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm.</p> <p>Conclusion</p> <p>The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.</p

Epigenetic silencing of DSC3 is a common event in human breast cancer

INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure

Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

MYCN is a transcription factor that is expressed during the development of the neural crest and its dysregulation plays a major role in the pathogenesis of pediatric cancers such as neuroblastoma, medulloblastoma and rhabdomyosarcoma. MeCP2 is a CpG methyl binding protein which has been associated with a number of cancers and developmental disorders, particularly Rett syndrome.Using an integrative global genomics approach involving chromatin immunoprecipitation applied to microarrays, we have determined that MYCN and MeCP2 co-localize to gene promoter regions, as well as inter/intragenic sites, within the neuroblastoma genome (MYCN amplified Kelly cells) at high frequency (70.2% of MYCN sites were also positive for MeCP2). Intriguingly, the frequency of co-localization was significantly less at promoter regions exhibiting substantial hypermethylation (8.7%), as determined by methylated DNA immunoprecipitation (MeDIP) applied to the same microarrays. Co-immunoprecipitation of MYCN using an anti-MeCP2 antibody indicated that a MYCN/MeCP2 interaction occurs at protein level. mRNA expression profiling revealed that the median expression of genes with promoters bound by MYCN was significantly higher than for genes bound by MeCP2, and that genes bound by both proteins had intermediate expression. Pathway analysis was carried out for genes bound by MYCN, MeCP2 or MYCN/MeCP2, revealing higher order functions.Our results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency, and that the patterns of binding of these proteins can be associated with significant differences in transcriptional activity. Although it is not yet known if this interaction contributes to neuroblastoma disease pathogenesis, it is intriguing that the interaction occurs at the promoter regions of several genes important for the development of neuroblastoma, including ALK, AURKA and BDNF

The Methyl-CpG Binding Proteins Mecp2, Mbd2 and Kaiso Are Dispensable for Mouse Embryogenesis, but Play a Redundant Function in Neural Differentiation

The precise molecular changes that occur when a neural stem (NS) cell switches from a programme of self-renewal to commit towards a specific lineage are not currently well understood. However it is clear that control of gene expression plays an important role in this process. DNA methylation, a mark of transcriptionally silent chromatin, has similarly been shown to play important roles in neural cell fate commitment in vivo. While DNA methylation is known to play important roles in neural specification during embryonic development, no such role has been shown for any of the methyl-CpG binding proteins (Mecps) in mice.. No evidence for functional redundancy between these genes in embryonic development or in the derivation or maintenance of neural stem cells in culture was detectable. However evidence for a defect in neuronal commitment of triple knockout NS cells was found.Although DNA methylation is indispensable for mammalian embryonic development, we show that simultaneous deficiency of three methyl-CpG binding proteins genes is compatible with apparently normal mouse embryogenesis. Nevertheless, we provide genetic evidence for redundancy of function between methyl-CpG binding proteins in postnatal mice

Cell Cycle- and Cancer-Associated Gene Networks Activated by Dsg2: Evidence of Cystatin A Deregulation and a Potential Role in Cell-Cell Adhesion

This work was supported by grants from the National Institutes of Health (Mahoney, R01AR056067; Riobo, RO1 GM088256). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets

<p>Abstract</p> <p>Background</p> <p>MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. <it>MECP2 </it>mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand movements and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur <it>de novo </it>during spermatogenesis. Located at Xq28, <it>MECP2 </it>is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy.</p> <p>Methods</p> <p>To identify the pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out in cerebellum of <it>Mecp2 </it>mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document <it>in vivo </it>MeCP2 binding to promoter regions of candidate target genes.</p> <p>Results</p> <p>Of several hundred genes with altered expression levels in the mutants, twice as many were increased than decreased, and only 27 were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with the exon 3 deletion (<it>Mecp2</it>^tm1.1Jae) than in mice with the larger deletion (<it>Mecp2</it>^tm1.1Bird). Between the mutants, few genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: <it>Irak1</it>, interleukin-1 receptor-associated kinase 1; <it>Fxyd1</it>, phospholemman, associated with Na, K-ATPase;<it>Reln</it>, encoding an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and <it>Gtl2/Meg3</it>, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented <it>in vivo </it>MeCP2 binding to promoter regions of <it>Fxyd1, Reln</it>, and <it>Gtl2</it>.</p> <p>Conclusion</p> <p>Transcriptional profiling of cerebellum failed to detect significant global changes in <it>Mecp2</it>-mutant mice. Increased transcript levels of <it>Irak1, Fxyd1, Reln</it>, and <it>Gtl2 </it>may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on.</p

Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism

The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation