Margaret C. Cam

Loss of signal transducer and activator of transcription 5 leads to hepatosteatosis and impaired liver regeneration

Growth hormone controls many facets of a cells biology through the transcription factors Stat5a and Stat5b (Stat5). However, whole body deletion of these genes from the mouse does not provide portentous information on cell‐specific cytokine signaling. To explore liver‐specific functions of Stat5, the entire Stat5 locus was deleted in hepatocytes using Cre‐mediated recombination. Notably, Stat5‐mutant mice developed fatty livers and displayed impaired proliferation of hepatocytes upon partial hepatectomy (PHx). Loss of Stat5 led to molecular consequences beyond the reduced expression of Stat5 target genes, such as those encoding suppressor of cytokine signaling 2 (SOCS2), Cish, and insulin‐like growth factor 1 (IGF‐1). In particular, circulating growth hormone levels were increased and correlated with insulin resistance and increased insulin levels. Aberrant growth hormone (GH)‐induced activation of the transcription factors Stat1 and Stat3 was observed in mutant livers. To test whether some of the defects observed in liver‐specific Stat5 deficient mice were due to aberrant Stat1 expression and activation, we generated Stat1−/− mice with a hepatocyte‐specific deletion of Stat5. Concomitant loss of both Stat5 and Stat1 restored cell proliferation upon PHx but did not reverse fatty liver development. Thus the molecular underpinnings of some defects observed in the absence of Stat5 are the consequence of a deregulated activation of other signal transducers and activators of transcription (STAT) family members. Conclusion: Aberrant cytokine‐Stat5 signaling in hepatocytes alters their physiology through increased activity of Stat1 and Stat3. Such cross‐talk between different pathways could add to the complexity of syndromes observed in disease. (HEPATOLOGY 2007.)

Transcript-based redefinition of grouped oligonucleotide probe sets using AceView: High-resolution annotation for microarrays

BackgroundExtracting biological information from high-density Affymetrix arrays is a multi-step process that begins with the accurate annotation of microarray probes. Shortfalls in the original Affymetrix probe annotation have been described; however, few studies have provided rigorous solutions for routine data analysis.ResultsUsing AceView, a comprehensive human transcript database, we have reannotated the probes by matching them to RNA transcripts instead of genes. Based on this transcript-level annotation, a new probe set definition was created in which every probe in a probe set maps to a common set of AceView gene transcripts. In addition, using artificial data sets we identified that a minimal probe set size of 4 is necessary for reliable statistical summarization. We further demonstrate that applying the new probe set definition can detect specific transcript variants contributing to differential expression and it also improves cross-platform concordance.ConclusionWe conclude that our transcript-level reannotation and redefinition of probe sets complement the original Affymetrix design. Redefinitions introduce probe sets whose sizes may not support reliable statistical summarization; therefore, we advocate using our transcript-level mapping redefinition in a secondary analysis step rather than as a replacement. Knowing which specific transcripts are differentially expressed is important to properly design probe/primer pairs for validation purposes. For convenience, we have created custom chip-description-files (CDFs) and annotation files for our new probe set definitions that are compatible with Bioconductor, Affymetrix Expression Console or third party software.

Long CGG‐repeat tracts are toxic to human cells: Implications for carriers of Fragile X premutation alleles

People with 59–200 CGG · CCG‐repeats in the 5′ UTR of one of their FMR1 genes are at risk for Fragile X tremor and ataxia syndrome. Females are also at risk for premature ovarian failure. These symptoms are thought to be due to the presence of the repeats at the DNA and/or RNA level. We show here that long transcribed but untranslated CGG‐repeat tracts are toxic to human cells and alter the expression of a wide variety of different genes including caspase‐8, CYFIP, Neurotensin and UBE3A.

Age-specific signatures of glioblastoma at the genomic, genetic, and epigenetic levels.

Age is a powerful predictor of survival in glioblastoma multiforme (GBM) yet the biological basis for the difference in clinical outcome is mostly unknown. Discovering genes and pathways that would explain age-specific survival difference could generate opportunities for novel therapeutics for GBM. Here we have integrated gene expression, exon expression, microRNA expression, copy number alteration, SNP, whole exome sequence, and DNA methylation data sets of a cohort of GBM patients in The Cancer Genome Atlas (TCGA) project to discover age-specific signatures at the transcriptional, genetic, and epigenetic levels and validated our findings on the REMBRANDT data set. We found major age-specific signatures at all levels including age-specific hypermethylation in polycomb group protein target genes and the upregulation of angiogenesis-related genes in older GBMs. These age-specific differences in GBM, which are independent of molecular subtypes, may in part explain the preferential effects of anti-angiogenic agents in older GBM and pave the way to a better understanding of the unique biology and clinical behavior of older versus younger GBMs.

HIV-protease inhibitors induce expression of suppressor of cytokine signaling-1 in insulin-sensitive tissues and promote insulin resistance and type 2 diabetes mellitus

Insulin resistance, hyperglycemia, and type 2 diabetes are among the sequelae of metabolic syndromes that occur in 60-80% of human immunodeficiency virus (HIV)-positive patients treated with HIV-protease inhibitors (PIs). Studies to elucidate the molecular mechanism(s) contributing to these changes, however, have mainly focused on acute, in vitro actions of PIs. Here, we examined the chronic (7 wk) in vivo effects of the PI indinavir (IDV) in male Zucker diabetic fatty (fa/fa) (ZDF) rats. IDV exposure accelerated the diabetic state and dramatically exacerbated hyperglycemia and oral glucose intolerance in the ZDF rats, compared with vehicle-treated ZDF rats. Oligonucleotide gene array analyses revealed upregulation of suppressor of cytokine signaling-1 (SOCS-1) expression in insulin-sensitive tissues of IDV rats. SOCS-1 is a known inducer of insulin resistance and diabetes, and immunoblotting analyses revealed increases in SOCS-1 protein expression in adipose, skeletal muscle, and liver tissues of IDV-administered ZDF rats. This was associated with increases in the upstream regulator TNF-alpha and downstream effector sterol regulatory element-binding protein-1 and a decrease in IRS-2. IDV and other PIs currently in clinical use induced the SOCS-1 signaling cascade also in L6 myotubes and 3T3-L1 adipocytes exposed acutely to PIs under normal culturing conditions and in tissues from Zucker wild-type lean control rats administered PIs for 3 wk, suggesting an effect of these drugs even in the absence of background hyperglycemia/hyperlipidemia. Our findings therefore indicate that induction of the SOCS-1 signaling cascade by PIs could be an important contributing factor in the development of metabolic dysregulation associated with long-term exposures to HIV-PIs.

Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation.

Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4+ T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation.

G-CIMP Status Prediction of Glioblastoma Samples Using mRNA Expression Data

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.

Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well‐established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10−5 M) or spermine (10−5 M) compared to a control culture containing 10−8 M spermidine. 247 genes were upregulated > two‐fold and 11 genes were upregulated >10‐fold after spermidine addition. Functional categorization of the genes showed induction of transport‐related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two‐fold, and six genes were downregulated > eight‐fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Copyright

Coordinated Gene Expression of Neuroinflammatory and Cell Signaling Markers in Dorsolateral Prefrontal Cortex during Human Brain Development and Aging

Background Age changes in expression of inflammatory, synaptic, and neurotrophic genes are not well characterized during human brain development and senescence. Knowing these changes may elucidate structural, metabolic, and functional brain processes over the lifespan, as well vulnerability to neurodevelopmental or neurodegenerative diseases. Hypothesis Expression levels of inflammatory, synaptic, and neurotrophic genes in the human brain are coordinated over the lifespan and underlie changes in phenotypic networks or cascades. Methods We used a large-scale microarray dataset from human prefrontal cortex, BrainCloud, to quantify age changes over the lifespan, divided into Development (0 to 21 years, 87 brains) and Aging (22 to 78 years, 144 brains) intervals, in transcription levels of 39 genes. Results Gene expression levels followed different trajectories over the lifespan. Many changes were intercorrelated within three similar groups or clusters of genes during both Development and Aging, despite different roles of the gene products in the two intervals. During Development, changes were related to reported neuronal loss, dendritic growth and pruning, and microglial events; TLR4, IL1R1, NFKB1, MOBP, PLA2G4A, and PTGS2 expression increased in the first years of life, while expression of synaptic genes GAP43 and DBN1 decreased, before reaching plateaus. During Aging, expression was upregulated for potentially pro-inflammatory genes such as NFKB1, TRAF6, TLR4, IL1R1, TSPO, and GFAP, but downregulated for neurotrophic and synaptic integrity genes such as BDNF, NGF, PDGFA, SYN, and DBN1. Conclusions Coordinated changes in gene transcription cascades underlie changes in synaptic, neurotrophic, and inflammatory phenotypic networks during brain Development and Aging. Early postnatal expression changes relate to neuronal, glial, and myelin growth and synaptic pruning events, while late Aging is associated with pro-inflammatory and synaptic loss changes. Thus, comparable transcriptional regulatory networks that operate throughout the lifespan underlie different phenotypic processes during Aging compared to Development.

Micro-Environment Causes Reversible Changes in DNA Methylation and mRNA Expression Profiles in Patient-Derived Glioma Stem Cells

In vitro and in vivo models are widely used in cancer research. Characterizing the similarities and differences between a patients tumor and corresponding in vitro and in vivo models is important for understanding the potential clinical relevance of experimental data generated with these models. Towards this aim, we analyzed the genomic aberrations, DNA methylation and transcriptome profiles of five parental tumors and their matched in vitro isolated glioma stem cell (GSC) lines and xenografts generated from these same GSCs using high-resolution platforms. We observed that the methylation and transcriptome profiles of in vitro GSCs were significantly different from their corresponding xenografts, which were actually more similar to their original parental tumors. This points to the potentially critical role of the brain microenvironment in influencing methylation and transcriptional patterns of GSCs. Consistent with this possibility, ex vivo cultured GSCs isolated from xenografts showed a tendency to return to their initial in vitro states even after a short time in culture, supporting a rapid dynamic adaptation to the in vitro microenvironment. These results show that methylation and transcriptome profiles are highly dependent on the microenvironment and growth in orthotopic sites partially reverse the changes caused by in vitro culturing.