Yuval Kluger, Ph.D.

PNAS Plus: Genome-wide remodeling of the epigenetic landscape during myogenic differentiation
Asp, Patrik; Blum, Roy; Vethantham, Vasupradha; Parisi, Fabio; Micsinai, Mariann; Cheng, Jemmie; Bowman, Christopher; Kluger, Yuval; Dynlacht, Brian David
2011 May 31;108(22):E149-E158, Proceedings of the National Academy of Sciences of the United States of America
We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a 'fail-safe' mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells
— id: 133464, year: 2011, vol: 108, page: E149, stat: Journal Article,

RUNX Transcription Factor-Mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation
Collins, Amelie; Hewitt, Susannah L; Chaumeil, Julie; Sellars, Maclean; Micsinai, Mariann; Allinne, Jeanne; Parisi, Fabio; Nora, Elphege P; Bolland, Dan J; Corcoran, Anne E; Kluger, Yuval; Bosselut, Remy; Ellmeier, Wilfried; Chong, Mark M W; Littman, Dan R; Skok, Jane A
2011 Mar 25;34(3):303-314, Immunity
T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells
— id: 129323, year: 2011, vol: 34, page: 303, stat: Journal Article,

Plasma markers for identifying patients with metastatic melanoma
Kluger, Harriet M; Hoyt, Kathleen; Bacchiocchi, Antonella; Mayer, Tina; Kirsch, Jonathan; Kluger, Yuval; Sznol, Mario; Ariyan, Stephan; Molinaro, Annette; Halaban, Ruth
2011 Apr 15;17(8):2417-2425, Clinical cancer research
PURPOSE: With the rising incidence of melanoma, more patients are undergoing surveillance for disease recurrence. Our purpose was to study levels of proteins that might be secreted in the blood of patients with metastatic melanoma that can be used for monitoring these individuals. Methods: Genome-wide gene expression data were used to identify abundantly expressed genes in melanoma cells that encode for proteins likely to be present in the blood of cancer patients, based on high expression levels in tumors. ELISA assays were employed to measure proteins in plasma of 216 individuals; 108 metastatic melanoma patients and 108 age- and gender-matched patients with resected stage I/II disease split into equal-sized training and test cohorts. RESULTS: Levels of seven markers, CEACAM (carcinoembryonic antigen-related cell adhesion molecule), ICAM-1 (intercellular adhesion molecule 1), osteopontin, MIA (melanoma inhibitory activity), GDF-15 (growth differentiation factor 15), TIMP-1 (tissue inhibitor of metalloproteinase 1), and S100B, were higher in patients with unresected stage IV disease than in patients with resected stage I/II disease. About 81% of the stage I/II patients in the training set had no marker elevation, whereas 69% of the stage IV patients had elevation of at least one marker (P < 0.0001). Receiver operating characteristic curves for the markers in combination in these two patient populations had an area under curve (AUC) of 0.79 in the training set and 0.8 in the test set. A CART (Classification and Regression Trees) model developed in the training set further improved the AUC in the test set to 0.898. CONCLUSIONS: Plasma markers, particularly when assessed in combination, can be used to monitor patients for disease recurrence and can compliment currently used lactate dehydrogenase and imaging studies; prospective validation is warranted
— id: 141320, year: 2011, vol: 17, page: 2417, stat: Journal Article,

Detecting copy number status and uncovering subclonal markers in heterogeneous tumor biopsies
Parisi, Fabio; Ariyan, Stephan; Narayan, Deepak; Bacchiocchi, Antonella; Hoyt, Kathleen; Cheng, Elaine; Xu, Fang; Li, Peining; Halaban, Ruth; Kluger, Yuval
2011 ;12:230-230, BMC genomics
BACKGROUND: Genomic aberrations can be used to determine cancer diagnosis and prognosis. Clinically relevant novel aberrations can be discovered using high-throughput assays such as Single Nucleotide Polymorphism (SNP) arrays and next-generation sequencing, which typically provide aggregate signals of many cells at once. However, heterogeneity of tumor subclones dramatically complicates the task of detecting aberrations. RESULTS: The aggregate signal of a population of subclones can be described as a linear system of equations. We employed a measure of allelic imbalance and total amount of DNA to characterize each locus by the copy number status (gain, loss or neither) of the strongest subclonal component. We designed simulated data to compare our measure to existing approaches and we analyzed SNP-arrays from 30 melanoma samples and transcriptome sequencing (RNA-Seq) from one melanoma sample.We showed that any system describing aggregate subclonal signals is underdetermined, leading to non-unique solutions for the exact copy number profile of subclones. For this reason, our illustrative measure was more robust than existing Hidden Markov Model (HMM) based tools in inferring the aberration status, as indicated by tests on simulated data. This higher robustness contributed in identifying numerous aberrations in several loci of melanoma samples. We validated the heterogeneity and aberration status within single biopsies by fluorescent in situ hybridization of four affected and transcriptionally up-regulated genes E2F8, ETV4, EZH2 and FAM84B in 11 melanoma cell lines. Heterogeneity was further demonstrated in the analysis of allelic imbalance changes along single exons from melanoma RNA-Seq. CONCLUSIONS: These studies demonstrate how subclonal heterogeneity, prevalent in tumor samples, is reflected in aggregate signals measured by high-throughput techniques. Our proposed approach yields high robustness in detecting copy number alterations using high-throughput technologies and has the potential to identify specific subclonal markers from next-generation sequencing data
— id: 141319, year: 2011, vol: 12, page: 230, stat: Journal Article,

VDA, a Method of Choosing a Better Algorithm with Fewer Validations
Strino, Francesco; Parisi, Fabio; Kluger, Yuval
2011 ;6(10):e26074-e26074, PLoS ONE
The multitude of bioinformatics algorithms designed for performing a particular computational task presents end-users with the problem of selecting the most appropriate computational tool for analyzing their biological data. The choice of the best available method is often based on expensive experimental validation of the results. We propose an approach to design validation sets for method comparison and performance assessment that are effective in terms of cost and discrimination power.Validation Discriminant Analysis (VDA) is a method for designing a minimal validation dataset to allow reliable comparisons between the performances of different algorithms. Implementation of our VDA approach achieves this reduction by selecting predictions that maximize the minimum Hamming distance between algorithmic predictions in the validation set. We show that VDA can be used to correctly rank algorithms according to their performances. These results are further supported by simulations and by realistic algorithmic comparisons in silico.VDA is a novel, cost-efficient method for minimizing the number of validation experiments necessary for reliable performance estimation and fair comparison between algorithms.Our VDA software is available at http://sourceforge.net/projects/klugerlab/files/VDA/
— id: 140682, year: 2011, vol: 6, page: e26074, stat: Journal Article,

PLX4032, a selective BRAF(V600E) kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAF melanoma cells
Halaban, Ruth; Zhang, Wengeng; Bacchiocchi, Antonella; Cheng, Elaine; Parisi, Fabio; Ariyan, Stephan; Krauthammer, Michael; McCusker, James P; Kluger, Yuval; Sznol, Mario
2010 Apr;23(2):190-200, Pigment cell & melanoma research
BRAF(V600E/K) is a frequent mutationally active tumor-specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAF(V600E/K) and BRAF(WT) showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAF(V600E/K), it activated the pathway in the resistant BRAF(WT) cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAF(WT) melanoma cells and induced changes in the expression of a wide-spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor-dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAF(WT) primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses
— id: 141321, year: 2010, vol: 23, page: 190, stat: Journal Article,

Benefits of biomarker selection and clinico-pathological covariate inclusion in breast cancer prognostic models
Parisi F; Gonzalez AM; Nadler Y; Camp RL; Rimm DL; Kluger HM; Kluger Y
2010 Sep 1;12(5):R66-R66, Breast cancer research
ABSTRACT: INTRODUCTION: Multi-marker molecular assays have impacted management of early stage breast cancer, facilitating adjuvant chemotherapy decisions. We generated prognostic models that incorporate protein-based molecular markers and clinico-pathological variables to improve survival prediction. METHODS: We used a quantitative immunofluorescence method to study protein expression of 14 markers included in the Oncotype DX assay on a 638 breast cancer patient cohort with 15-year follow-up. We performed cross-validation analyses to assess performance of multivariate Cox models consisting of these markers and standard clinico-pathological covariates, using an average time-dependent Area Under the Receiver Operating Characteristic curves and compared it to nested Cox models obtained by robust backward selection procedures. RESULTS: A prognostic index derived from of a multivariate Cox regression model incorporating molecular and clinico-pathological covariates (nodal status, tumor size, nuclear grade, and age) is superior to models based on molecular studies alone or clinico-pathological covariates alone. Performance of this composite model can be further improved using feature selection techniques to prune variables. When stratifying patients by Nottingham Prognostic Index (NPI), the most prognostic markers in high and low NPI groups differed. Similarly, for the node-negative, hormone receptor-positive sub-population, we derived a compact model with three clinico-pathological variables and two protein markers that was superior to the full model. CONCLUSIONS: Prognostic models that include both molecular and clinico-pathological covariates can be more accurate than models based on either set of features alone. Furthermore, feature selection can decrease the number of molecular variables needed to predict outcome, potentially resulting in less expensive assays
— id: 140043, year: 2010, vol: 12, page: R66, stat: Journal Article,

The Mammalian sin3 proteins are required for muscle development and sarcomere specification
van Oevelen, Chris; Bowman, Christopher; Pellegrino, Jessica; Asp, Patrik; Cheng, Jemmie; Parisi, Fabio; Micsinai, Mariann; Kluger, Yuval; Chu, Alphonse; Blais, Alexandre; David, Gregory; Dynlacht, Brian D
2010 Dec;30(24):5686-5697, Molecular & cellular biology
The highly related mammalian Sin3A and Sin3B proteins provide a versatile platform for chromatin-modifying activities. Sin3-containing complexes play a role in gene repression through deacetylation of nucleosomes. Here, we explore a role for Sin3 in myogenesis by examining the phenotypes resulting from acute somatic deletion of both isoforms in vivo and from primary myotubes in vitro. Myotubes ablated for Sin3A alone, but not Sin3B, displayed gross defects in sarcomere structure that were considerably enhanced upon simultaneous ablation of both isoforms. Massively parallel sequencing of Sin3A- and Sin3B-bound genomic loci revealed a subset of target genes directly involved in sarcomere function that are positively regulated by Sin3A and Sin3B proteins. Both proteins were coordinately recruited to a substantial number of genes. Interestingly, depletion of Sin3B led to compensatory increases in Sin3A recruitment at certain target loci, but Sin3B was never found to compensate for Sin3A loss. Thus, our analyses describe a novel transcriptional role for Sin3A and Sin3B proteins associated with maintenance of differentiated muscle cells
— id: 114827, year: 2010, vol: 30, page: 5686, stat: Journal Article,

Phosphatidylinositol-3-kinase as a therapeutic target in melanoma
Aziz, Saadia A; Davies, Michael; Pick, Elah; Zito, Christopher; Jilaveanu, Lucia; Camp, Robert L; Rimm, David L; Kluger, Yuval; Kluger, Harriet M
2009 May 1;15(9):3029-3036, Clinical cancer research
PURPOSE: Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and survival, and are targets of drugs in clinical development. We assessed expression of PI3K in melanomas and nevi, and studied associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002. EXPERIMENTAL DESIGN: Using Automated Quantitative Analysis, we quantified expression of p85 and p110alpha subunits in 540 nevi and 523 melanomas. We determined the IC(50) for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002. RESULTS: p85 and p110alpha tend to be coexpressed (P < 0.0001); expression was higher in melanomas than nevi (P < 0.0001) for both subunits, and higher in metastatic than primary melanomas for p85 (P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable. We found no association by t tests between baseline p85, p110alpha, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser(235) and Ser(240) were lower in the more resistant cell lines (P = 0.01 and P = 0.004, respectively). CONCLUSIONS: Expression of p85 and p110alpha subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials
— id: 141322, year: 2009, vol: 15, page: 3029, stat: Journal Article,

Integrative analysis of epigenetic modulation in melanoma cell response to decitabine: clinical implications
Halaban, Ruth; Krauthammer, Michael; Pelizzola, Mattia; Cheng, Elaine; Kovacs, Daniela; Sznol, Mario; Ariyan, Stephan; Narayan, Deepak; Bacchiocchi, Antonella; Molinaro, Annette; Kluger, Yuval; Deng, Min; Tran, Nam; Zhang, Wengeng; Picardo, Mauro; Enghild, Jan J
2009 ;4(2):e4563-e4563, PLoS ONE
Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21(Cip1) in a p53-independent manner, identified the TGFbeta pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated beta-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy
— id: 95727, year: 2009, vol: 4, page: e4563, stat: Journal Article,

Expression patterns and prognostic value of Bag-1 and Bcl-2 in breast cancer
Nadler, Yasmine; Camp, Robert L; Giltnane, Jennifer M; Moeder, Christopher; Rimm, David L; Kluger, Harriet M; Kluger, Yuval
2008 ;10(2):R35-R35, Breast cancer research
INTRODUCTION: Bcl-2 antanogene-1 (Bag-1) binds the anti-apoptotic mediator Bcl-2, and enhances its activity. Bcl-2 and Bag-1 are associated with chemotherapy resistance in cancer cells. Drugs that target Bcl-2 are currently in clinical development. The purpose of the present study was to examine expression patterns of Bag-1 in a large cohort of breast tumors and to assess the association with Bcl-2, estrogen receptor, progesterone receptor and Her2/neu, and other clinical/pathological variables. METHODS: Tissue microarrays containing primary specimens from 638 patients with 10-year follow-up were employed, and the expression of Bag-1, Bcl-2, estrogen receptor, progesterone receptor and Her2/neu was assessed using our automated quantitative analysis method. We used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and we measured biomarker expression within the mask using Cy5 conjugated antibodies. RESULTS: High Bcl-2 expression was associated with improved survival in the entire cohort and in the node-positive subset (P = 0.008 and P = 0.002, respectively). High Bag-1 expression was associated with improved survival in the node-positive subset (P = 0.006). On multivariable analysis, neither Bcl-2 nor Bag-1 retained their independence as prognostic markers. Strong associations were found between Bag-1, Bcl-2, estrogen receptor and progesterone receptor. CONCLUSION: Bag-1 and Bcl-2 expression in breast tumors is associated with improved outcome and steroid receptor positivity. Evaluation of Bcl-2 and Bag-1 expression in breast cancer may identify a subset of patients with a favorable prognosis, who might not benefit from chemotherapy or who might benefit from Bcl-2 targeting agents in addition to antihormonal therapy
— id: 80344, year: 2008, vol: 10, page: R35, stat: Journal Article,

Expression of Aurora A (but not Aurora B) is predictive of survival in breast cancer
Nadler, Yasmine; Camp, Robert L; Schwartz, Candice; Rimm, David L; Kluger, Harriet M; Kluger, Yuval
2008 Jul 15;14(14):4455-4462, Clinical cancer research
PURPOSE: The cell cycle mediators Aurora A and B are targets of drugs currently in clinical development. As with other targeted therapies in breast cancer, response to therapy might be associated with target expression in tumors. We therefore assessed expression of Aurora A and B in breast tumors and studied associations with clinical/pathologic variables. EXPERIMENTAL DESIGN: Tissue microarrays containing primary specimens from 638 patients with 15-year follow-up were employed to assess expression of Aurora A and B using our automated quantitative analysis method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured Aurora A and B expression within the mask using Cy5-conjugated antibodies. RESULTS: Aurora A and B expression was variable in primary breast tumors. High Aurora A expression was strongly associated with decreased survival (P = 0.0005). On multivariable analysis, it remained an independent prognostic marker. High Aurora A expression was associated with high nuclear grade and high HER-2/neu and progesterone receptor expression. Aurora B expression was not associated with survival. CONCLUSIONS: Aurora A expression defines a population of patients with decreased survival, whereas Aurora B expression does not, suggesting that Aurora A might be the preferred drug target in breast cancer. Aurora A expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of Aurora A as well as the predictive role of Aurora A expression in patients treated with Aurora A inhibitors
— id: 95728, year: 2008, vol: 14, page: 4455, stat: Journal Article,

A role for mammalian Sin3 in permanent gene silencing
van Oevelen, Chris; Wang, Jinhua; Asp, Patrik; Yan, Qin; Kaelin, William G Jr; Kluger, Yuval; Dynlacht, Brian David
2008 Nov 7;32(3):359-370, Molecular cell
The multisubunit Sin3 corepressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. Here, we have investigated genome-wide binding of mouse Sin3 and RBP2 as well as histone modifications and nucleosome positioning as a function of myogenic differentiation. Remarkably, we find that Sin3 complexes spread immediately downstream of the transcription start site on repressed and transcribed genes during differentiation. We show that RBP2 is part of a Sin3 complex and that on a subset of E2F4 target genes, the coordinated activity of Sin3 and RBP2 leads to deacetylation, demethylation, and repositioning of nucleosomes. Our work provides evidence for coordinated binding of Sin3, chromatin modifications, and chromatin remodeling within discrete regulatory regions, suggesting a model in which spreading of Sin3 binding is ultimately linked to permanent gene silencing on a subset of E2F4 target genes
— id: 90059, year: 2008, vol: 32, page: 359, stat: Journal Article,

XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks
Acosta-Alvear, Diego; Zhou, Yiming; Blais, Alexandre; Tsikitis, Mary; Lents, Nathan H; Arias, Carolina; Lennon, Christen J; Kluger, Yuval; Dynlacht, Brian David
2007 Jul 6;27(1):53-66, Molecular cell
Using genome-wide approaches, we have elucidated the regulatory circuitry governed by the XBP1 transcription factor, a key effector of the mammalian unfolded protein response (UPR), in skeletal muscle and secretory cells. We identified a core group of genes involved in constitutive maintenance of ER function in all cell types and tissue- and condition-specific targets. In addition, we identified a cadre of unexpected targets that link XBP1 to neurodegenerative and myodegenerative diseases, as well as to DNA damage and repair pathways. Remarkably, we found that XBP1 regulates functionally distinct targets through different sequence motifs. Further, we identified Mist1, a critical regulator of differentiation, as an important target of XBP1, providing an explanation for developmental defects associated with XBP1 loss of function. Our results provide a detailed picture of the regulatory roadmap governed by XBP1 in distinct cell types as well as insight into unexplored functions of XBP1
— id: 73301, year: 2007, vol: 27, page: 53, stat: Journal Article,

Genome wide expression profiles associated with 5-Aza-2'-deoxy-cytidine-induced apoptosis in melanoma cells
Molinaro, A; Krauthammer, M; Kluger, Y; Cheng, E; Deng, M; Mor, G; Brailey, L; Sznol, M; Kluger, H; Ariyan, S; McNiff, J; Narayan, D; Shivakumar, P; Pelizzola, M; Kovacs, D; Picardo, M; Halaban, R
2007 APR ;127(2):S28-S28, Journal of investigative dermatology
— id: 71617, year: 2007, vol: 127, page: S28, stat: Journal Article,

High HSP90 expression is associated with decreased survival in breast cancer
Pick, Elah; Kluger, Yuval; Giltnane, Jennifer M; Moeder, Christopher; Camp, Robert L; Rimm, David L; Kluger, Harriet M
2007 Apr 1;67(7):2932-2937, Cancer research
The heat shock protein HSP90 chaperones proteins implicated in breast cancer progression, including Her2/neu. HSP90-targeting agents are in clinical trials for breast cancer. HSP90 expression is high in breast cancer cell lines, yet no large studies have been conducted on expression in human tumors and the association with clinical/pathologic variables. Tissue microarrays containing 10 cell lines and primary specimens from 655 patients with 10-year follow-up were assessed using our automated quantitative analysis (AQUA) method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured HSP90 expression within the mask using Cy5-conjugated antibodies. We similarly assessed estrogen receptor, progesterone receptor, and Her2/neu expression. HSP90 expression was more variable in human tumors than in cell lines (P < 0.0001). High HSP90 expression was associated with decreased survival (P = 0.0024). On multivariable analysis, high HSP90 expression remained an independent prognostic marker. High HSP90 expression was associated with high Her2/neu and estrogen receptor, large tumors, high nuclear grade, and lymph node involvement. Although HSP90 levels were high in all our cell lines, expression in tumors was more variable. High HSP90 expression in primary breast cancer defines a population of patients with decreased survival. Evaluation of HSP90 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.
— id: 72894, year: 2007, vol: 67, page: 2932, stat: Journal Article,

Inter- and intra-combinatorial regulation by transcription factors and microRNAs
Yiming Zhou; Ferguson, J.; Chang, J.T.; Kluger, Y.
Proceedings of the 2007 International Conference on Bioinformatics & Computational Biology. BIOCOMP 2007 [S.l.] : CSREA press, 2007,
MicroRNAs (miRNAs) are a class of small RNAs whose functions remain unclear. Here, we studied' the nature of coordination among transcriptional and miRNA regulatory layers by assessment of regulatory 'interactions' among the sets of predicted targets of miRNAs and of transcription factors (TFs). We found that regulation by TF pairs and miRNA pairs is more abundant than the regulation by TF-miRNA pairs, perhaps due to the higher probability of evolutionary duplication events of shorter DNA sequences. Application of significance measures for evaluating the degree of interaction between regulators shows that strongly interacting regulatory pairs tend to include regulators with large numbers of genes. Therefore, we introduced a Bayesian score that combines the significance and strength of an interaction. We found that strongly interacting TF-miRNA pairs are more likely to form feed forward loops. This may be attributed to processes that prevent waste of resources or accelerate mRNA degradation
— id: 5078, year: 2007, vol: , page: 598, stat: Chapter,

Inter- and intra-combinatorial regulation by transcription factors and microRNAs
Zhou, Yiming; Ferguson, John; Chang, Joseph T; Kluger, Yuval
2007 ;8:396-396, BMC genomics
BACKGROUND: MicroRNAs (miRNAs) are a novel class of non-coding small RNAs. In mammalian cells, miRNAs repress the translation of messenger RNAs (mRNAs) or degrade mRNAs. miRNAs play important roles in development and differentiation, and they are also implicated in aging, and oncogenesis. Predictions of targets of miRNAs suggest that they may regulate more than one-third of all genes. The overall functions of mammalian miRNAs remain unclear. Combinatorial regulation by transcription factors alone or miRNAs alone offers a wide range of regulatory programs. However, joining transcriptional and post-transcriptional regulatory mechanisms enables higher complexity regulatory programs that in turn could give cells evolutionary advantages. Investigating coordinated regulation of genes by miRNAs and transcription factors (TFs) from a statistical standpoint is a first step that may elucidate some of their roles in various biological processes. RESULTS: Here, we studied the nature and scope of coordination among regulators from the transcriptional and miRNA regulatory layers in the human genome. Our findings are based on genome wide statistical assessment of regulatory associations ('interactions') among the sets of predicted targets of miRNAs and sets of putative targets of transcription factors. We found that combinatorial regulation by transcription factor pairs and miRNA pairs is much more abundant than the combinatorial regulation by TF-miRNA pairs. In addition, many of the strongly interacting TF-miRNA pairs involve a subset of master TF regulators that co-regulate genes in coordination with almost any miRNA. Application of standard measures for evaluating the degree of interaction between pairs of regulators show that strongly interacting TF-miRNA, TF-TF or miRNA-miRNA pairs tend to include TFs or miRNAs that regulate very large numbers of genes. To correct for this potential bias we introduced an additional Bayesian measure that incorporates not only how significant an interaction is but also how strong it is. Putative pairs of regulators selected by this procedure are more likely to have biological coordination. Importantly, we found that the probability of a TF-miRNA pair forming feed forward loops with its common target genes (where the miRNA simultaneously suppresses the TF and many of its targets) is increased for strongly interacting TF-miRNA pairs. CONCLUSION: Genes are more likely to be co-regulated by pairs of TFs or pairs of miRNAs than by pairs of TF-miRNA, perhaps due to higher probability of evolutionary duplication events of shorter DNA sequences. Nevertheless, many gene sets are reciprocally regulated by strongly interacting pairs of TF-miRNA, which suggests an effective mechanism to suppress functionally related proteins. Moreover, the particular type of feed forward loop (with two opposing modes where the TF activates its target genes or the miRNA simultaneously suppresses this TF and the TF-miRNA joint target genes) is more prevalent among strongly interacting TF-miRNA pairs. This may be attributed to a process that prevents waste of cellular resources or a mechanism to accelerate mRNA degradation
— id: 76761, year: 2007, vol: 8, page: 396, stat: Journal Article,

Unraveling condition specific gene transcriptional regulatory networks in Saccharomyces cerevisiae
Kim, Hyunsoo; Hu, William; Kluger, Yuval
2006 ;7:165-165, BMC bioinformatics
BACKGROUND: Gene expression and transcription factor (TF) binding data have been used to reveal gene transcriptional regulatory networks. Existing knowledge of gene regulation can be presented using gene connectivity networks. However, these composite connectivity networks do not specify the range of biological conditions of the activity of each link in the network. RESULTS: We present a novel method that utilizes the expression and binding patterns of the neighboring nodes of each link in existing experimentally-based, literature-derived gene transcriptional regulatory networks and extend them in silico using TF-gene binding motifs and a compendium of large expression data from Saccharomyces cerevisiae. Using this method, we predict several hundreds of new transcriptional regulatory TF-gene links, along with experimental conditions in which known and predicted links become active. This approach unravels new links in the yeast gene transcriptional regulatory network by utilizing the known transcriptional regulatory interactions, and is particularly useful for breaking down the composite transcriptional regulatory network to condition specific networks. CONCLUSION: Our methods can facilitate future binding experiments, as they can considerably help focus on the TFs that must be surveyed to understand gene regulation.(Supplemental material and the latest version of the MATLAB implementation of the United Signature Algorithm is available online at 1 or [see Additional files 1, 2, 3, 4, 5, 6, 7, 8, 9, 10])
— id: 66458, year: 2006, vol: 7, page: 165, stat: Journal Article,

Association between pathways in regulatory networks
Kluger, Yuval; Kluger, Harriet; Tuck, David
2006 ;1:2036-2040, Conference Proceedings (IEEE Engineering in Medicine & Biology Society)
During cell progression from one state to another, such as transformation from benign to malignant conditions, cells undergo changes in gene regulation. To reveal state-dependent circuitries in human regulatory networks, we employed drafts of normal and malignant cell networks. Using these condition specific networks, gene profiles and annotated pathways we studied: a) the capacity to separate samples or cell states based on the collective expression of all the genes in each pathway rather than individual genes, b) the degree of regulatory network connectivity within and between pathways. Distinct cell types reveal notable differences in transcriptional activity in numerous pathways. On the other hand, in datasets from breast cancer patients with variable outcome the capacity of single pathway expression signatures to predict disease outcome is very limited, though this can be somewhat improved by combining multiple pathways. Remarkable connectivity between pathways on the transcriptional regulatory level revealed a non-modular network structure. Overall, network blueprints enable us to quantify the degree of interaction between condition specific co-regulated pathways. This can contribute to understanding deregulated processes associated with cancer
— id: 95729, year: 2006, vol: 1, page: 2036, stat: Journal Article,

Melanoma biomarker discovery through serum antibody profiting on protein microarrays
Mattoon, D; Love, B; Kluger, Y; Michaud, G; Schweitzer, B; Predki, P; Ritter, G; Halaban, R
2006 AUG ;119(4):S28-S28, Clinical immunology
— id: 67555, year: 2006, vol: 119, page: S28, stat: Journal Article,

Characterizing disease states from topological properties of transcriptional regulatory networks
Tuck, David P; Kluger, Harriet M; Kluger, Yuval
2006 ;7:236-236, BMC bioinformatics
BACKGROUND: High throughput gene expression experiments yield large amounts of data that can augment our understanding of disease processes, in addition to classifying samples. Here we present new paradigms of data Separation based on construction of transcriptional regulatory networks for normal and abnormal cells using sequence predictions, literature based data and gene expression studies. We analyzed expression datasets from a number of diseased and normal cells, including different types of acute leukemia, and breast cancer with variable clinical outcome. RESULTS: We constructed sample-specific regulatory networks to identify links between transcription factors (TFs) and regulated genes that differentiate between healthy and diseased states. This approach carries the advantage of identifying key transcription factor-gene pairs with differential activity between healthy and diseased states rather than merely using gene expression profiles, thus alluding to processes that may be involved in gene deregulation. We then generalized this approach by studying simultaneous changes in functionality of multiple regulatory links pointing to a regulated gene or emanating from one TF (or changes in gene centrality defined by its in-degree or out-degree measures, respectively). We found that samples can often be separated based on these measures of gene centrality more robustly than using individual links.We examined distributions of distances (the number of links needed to traverse the path between each pair of genes) in the transcriptional networks for gene subsets whose collective expression profiles could best separate each dataset into predefined groups. We found that genes that optimally classify samples are concentrated in neighborhoods in the gene regulatory networks. This suggests that genes that are deregulated in diseased states exhibit a remarkable degree of connectivity. CONCLUSION: Transcription factor-regulated gene links and centrality of genes on transcriptional networks can be used to differentiate between cell types. Transcriptional network blueprints can be used as a basis for further research into gene deregulation in diseased states
— id: 69043, year: 2006, vol: 7, page: 236, stat: Journal Article,

An initial blueprint for myogenic differentiation
Blais, Alexandre; Tsikitis, Mary; Acosta-Alvear, Diego; Sharan, Roded; Kluger, Yuval; Dynlacht, Brian David
2005 Mar 1;19(5):553-569, Genes & development
We have combined genome-wide transcription factor binding and expression profiling to assemble a regulatory network controlling the myogenic differentiation program in mammalian cells. We identified a cadre of overlapping and distinct targets of the key myogenic regulatory factors (MRFs)--MyoD and myogenin--and Myocyte Enhancer Factor 2 (MEF2). We discovered that MRFs and MEF2 regulate a remarkably extensive array of transcription factor genes that propagate and amplify the signals initiated by MRFs. We found that MRFs play an unexpectedly wide-ranging role in directing the assembly and usage of the neuromuscular junction. Interestingly, these factors also prepare myoblasts to respond to diverse types of stress. Computational analyses identified novel combinations of factors that, depending on the differentiation state, might collaborate with MRFs. Our studies suggest unanticipated biological insights into muscle development and highlight new directions for further studies of genes involved in muscle repair and responses to stress and damage
— id: 51096, year: 2005, vol: 19, page: 553, stat: Journal Article,

Using a xenograft model of human breast cancer metastasis to find genes associated with clinically aggressive disease
Kluger, Harriet M; Chelouche Lev, Dina; Kluger, Yuval; McCarthy, Mary M; Kiriakova, Galina; Camp, Robert L; Rimm, David L; Price, Janet E
2005 Jul 1;65(13):5578-5587, Cancer research
Metastasis is the primary cause of death from breast cancer. A xenograft model was used to identify genes potentially involved with metastasis, comparing expression in the poorly metastatic GI101A human breast cancer cell line and a highly metastatic variant, GILM2. cDNA microarray analyses of these isogenic variants were done using 16K Operon 70-mer oligonucleotide microarray slides. Differentially expressed genes were identified by ANOVA, and differences of > or =2.5-fold were found for 106 genes. Changes in protein or RNA expression were confirmed for 10 of 12 genes. Three markers, heat shock protein 70 (HSP-70), chemokine (C-X-C motif) ligand 1 (CXCL-1), and secreted leukocyte protease inhibitor (SLPI), were studied further with breast cancer tissue microarrays using a novel method of automated quantitative analysis. This uses cytokeratin to define pixels as breast cancer (tumor mask) within the tissue array spot and then measures intensity of marker expression using a cyanine 5-conjugated antibody within the mask. Scores were correlated with clinicopathologic variables. High HSP-70 expression and high nuclear CXCL-1 expression in primary tumors were both associated with decreased survival (P = 0.05 and 0.027, respectively). Expression of each marker was strongly associated with lymph node involvement (P = 0.0002, 0.008, 0.0012, and 0.012 for HSP-70, nuclear CXCL-1, cytoplasmic CXCL-1, and SLPI, respectively). Identification of genes associated with metastasis in experimental models may have clinical implications for the management of breast cancer, because some of these are associated with lymph node metastasis and survival and might be useful as prognostic markers or molecular targets for novel therapies.
— id: 72896, year: 2005, vol: 65, page: 5578, stat: Journal Article,

Two types of precursor cells in a multipotential hematopoietic cell line
Ye, Zhi-jia; Kluger, Yuval; Lian, Zheng; Weissman, Sherman M
2005 Dec 20;102(51):18461-18466, Proceedings of the National Academy of Sciences of the United States of America
The biochemistry of early stages of hematopoietic differentiation is difficult to study because only relatively small numbers of precursor cells are available. The murine EML cell line is a multipotential cell line that can be used to model some of these steps. We found that the lineage- EML precursor cells can be separated into two populations based on cell surface markers including CD34. Both populations contain similar levels of stem cell factor (SCF) receptor (c-Kit) but only the CD34+ population shows a growth response when treated with SCF. Conversely, the CD34- population will grow in the presence of the cytokine IL-3. The human beta-globin locus control region hypersensitive site 2 plays different roles on beta-globin transcription in the CD34+ and CD34- populations. The two populations are present in about equal amounts in culture, and the CD34+ population rapidly regenerates the mixed population when grown in the presence of SCF. We suggest that this system may mimic a normal developmental transition in hematopoiesis.
— id: 72895, year: 2005, vol: 102, page: 18461, stat: Journal Article,

A common set of gene regulatory networks links metabolism and growth inhibition
Cam, Hugh; Balciunaite, Egle; Blais, Alexandre; Spektor, Alexander; Scarpulla, Richard C; Young, Richard; Kluger, Yuval; Dynlacht, Brian David
2004 Nov 5;16(3):399-411, Molecular cell
Using genome-wide analysis of transcription factor occupancy, we investigated the mechanisms underlying three mammalian growth arrest pathways that require the pRB tumor suppressor family. We found that p130 and E2F4 cooperatively repress a common set of genes under each growth arrest condition and showed that growth arrest is achieved through repression of a core set of genes involved not only in cell cycle control but also mitochondrial biogenesis and metabolism. Motif-finding algorithms predicted the existence of nuclear respiratory factor-1 (NRF1) binding sites in E2F target promoters, and genome-wide factor binding analysis confirmed our predictions. We showed that NRF1, a factor known to regulate expression of genes involved in mitochondrial function, is a coregulator of a large number of E2F target genes. Our studies provide insights into E2F regulatory circuitry, suggest how factor occupancy can predict the expression signature of a given target gene, and reveal pathways deregulated in human tumors
— id: 47810, year: 2004, vol: 16, page: 399, stat: Journal Article,

cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model
Kluger, Harriet M; Kluger, Yuval; Gilmore-Hebert, Maureen; DiVito, Kyle; Chang, Joseph T; Rodov, Sofya; Mironenko, Olga; Kacinski, Barry M; Perkins, Archibald S; Sapi, Eva
2004 Mar;84(3):320-331, Laboratory investigation
The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets
— id: 42813, year: 2004, vol: 84, page: 320, stat: Journal Article,

A panorama of lineage-specific transcription in hematopoiesis
Kluger, Yuval; Lian, Zheng; Zhang, Xueqing; Newburger, Peter E; Weissman, Sherman M
2004 Dec;26(12):1276-1287, Bioessays
The hematopoietic system consists of more than ten differentiated cell types, all of which are derived from a single type of hematopoietic stem cell. The accessibility and interest of this system have made it a model for understanding normal and abnormal differentiation of mammalian cells. Newer techniques have generated a mass of data that requires integrative approaches for analysis and interpretation. The traditional view of the differentiation program holds that a small number of regulators are involved in each stage of cell specification. However, this may not be the case. Recent analyses have shown that almost all substantial subsets of genes, including the set of broadly expressed transcription factors, are expressed in patterns that are unique for each lineage. Further, much of this difference between lineages can be captured in two-dimensional graphs. Understanding the biologic significance, mechanisms and constraints underlying these differences is a challenge for experimentalists and computational biologists alike
— id: 48239, year: 2004, vol: 26, page: 1276, stat: Journal Article,

Lineage specificity of gene expression patterns
Kluger, Yuval; Tuck, David P; Chang, Joseph T; Nakayama, Yasuhiro; Poddar, Ranjana; Kohya, Naohiko; Lian, Zheng; Ben Nasr, Abdelhakim; Halaban, H Ruth; Krause, Diane S; Zhang, Xueqing; Newburger, Peter E; Weissman, Sherman M
2004 Apr 27;101(17):6508-6513, Proceedings of the National Academy of Sciences of the United States of America
The hematopoietic system offers many advantages as a model for understanding general aspects of lineage choice and specification. Using oligonucleotide microarrays, we compared gene expression patterns of multiple purified hematopoietic cell populations, including neutrophils, monocytes, macrophages, resting, centrocytic, and centroblastic B lymphocytes, dendritic cells, and hematopoietic stem cells. Some of these cells were studied under both resting and stimulated conditions. We studied the collective behavior of subsets of genes derived from the Biocarta database of functional pathways, hand-tuned groupings of genes into broad functional categories based on the Gene Ontology database, and the metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes database. Principal component analysis revealed strikingly pervasive differences in relative levels of gene expression among cell lineages that involve most of the subsets examined. These results indicate that many processes in these cells behave differently in different lineages. Much of the variation among lineages was captured by the first few principal components. Principal components biplots were found to provide a convenient visual display of the contributions of the various genes within the subsets in lineage discrimination. Moreover, by applying tree-constructing methodologies borrowed from phylogenetics to the expression data from differentiated cells and stem cells, we reconstructed a tree of relationships that resembled the established hematopoietic program of lineage development. Thus, the mRNA expression data implicitly contained information about developmental relationships among cell types
— id: 42812, year: 2004, vol: 101, page: 6508, stat: Journal Article,

Gene expression in mature neutrophils: early responses to inflammatory stimuli
Zhang, Xueqing; Kluger, Yuval; Nakayama, Yasuhiro; Poddar, Ranjana; Whitney, Constance; DeTora, Adam; Weissman, Sherman M; Newburger, Peter E
2004 Feb;75(2):358-372, Journal of leukocyte biology
Neutrophils provide an essential defense against bacterial and fungal infection and play a major role in tissue damage during inflammation. Using oligonucleotide microarrays, we have examined the time course of changes in gene expression induced by stimulation with live, opsonized Escherichia coli, soluble lipopolysaccharide, and the chemoattractant formyl-methionyl-leucyl-phenylalanine. The results indicate that activated neutrophils generate a broad and vigorous set of alterations in gene expression. The responses included changes in the levels of transcripts encoding 148 transcription factors and chromatin-remodeling genes and 95 regulators of protein synthesis or stability. Clustering analysis showed distinct temporal patterns with many rapid changes in gene expression within the first hour of exposure. In addition to the temporal clustering of genes, we also observed rather different profiles associated with each stimulus, suggesting that even a nonvirulent organism such as E. coli is able to play a dynamic role in shaping the inflammatory response. Principal component analysis of transcription factor genes demonstrated clear separation of the neutrophil-response clusters from those of resting and stimulated human monocytes. The present study indicates that combinatorial transcriptional regulation including alterations of chromatin structure may play a role in the rapid changes in gene expression that occur in these terminally differentiated cells
— id: 42814, year: 2004, vol: 75, page: 358, stat: Journal Article,

A Bayesian networks approach for predicting protein-protein interactions from genomic data
Jansen, Ronald; Yu, Haiyuan; Greenbaum, Dov; Kluger, Yuval; Krogan, Nevan J; Chung, Sambath; Emili, Andrew; Snyder, Michael; Greenblatt, Jack F; Gerstein, Mark
2003 Oct 17;302(5644):449-453, Science
We have developed an approach using Bayesian networks to predict protein-protein interactions genome-wide in yeast. Our method naturally weights and combines into reliable predictions genomic features only weakly associated with interaction (e.g., messenger RNAcoexpression, coessentiality, and colocalization). In addition to de novo predictions, it can integrate often noisy, experimental interaction data sets. We observe that at given levels of sensitivity, our predictions are more accurate than the existing high-throughput experimental data sets. We validate our predictions with TAP (tandem affinity purification) tagging experiments. Our analysis, which gives a comprehensive view of yeast interactions, is available at genecensus.org/intint.
— id: 72898, year: 2003, vol: 302, page: 449, stat: Journal Article,

Spectral biclustering of microarray data: coclustering genes and conditions
Kluger, Yuval; Basri, Ronen; Chang, Joseph T; Gerstein, Mark
2003 Apr;13(4):703-716, Genome research
Global analyses of RNA expression levels are useful for classifying genes and overall phenotypes. Often these classification problems are linked, and one wants to find "marker genes" that are differentially expressed in particular sets of "conditions." We have developed a method that simultaneously clusters genes and conditions, finding distinctive "checkerboard" patterns in matrices of gene expression data, if they exist. In a cancer context, these checkerboards correspond to genes that are markedly up- or downregulated in patients with particular types of tumors. Our method, spectral biclustering, is based on the observation that checkerboard structures in matrices of expression data can be found in eigenvectors corresponding to characteristic expression patterns across genes or conditions. In addition, these eigenvectors can be readily identified by commonly used linear algebra approaches, in particular the singular value decomposition (SVD), coupled with closely integrated normalization steps. We present a number of variants of the approach, depending on whether the normalization over genes and conditions is done independently or in a coupled fashion. We then apply spectral biclustering to a selection of publicly available cancer expression data sets, and examine the degree to which the approach is able to identify checkerboard structures. Furthermore, we compare the performance of our biclustering methods against a number of reasonable benchmarks (e.g., direct application of SVD or normalized cuts to raw data).
— id: 72900, year: 2003, vol: 13, page: 703, stat: Journal Article,

Relationship between gene co-expression and probe localization on microarray slides
Kluger, Yuval; Yu, Haiyuan; Qian, Jiang; Gerstein, Mark
2003 Dec 10;4(1):49-49, BMC genomics
BACKGROUND: Microarray technology allows simultaneous measurement of thousands of genes in a single experiment. This is a potentially useful tool for evaluating co-expression of genes and extraction of useful functional and chromosomal structural information about genes. RESULTS: In this work we studied the association between the co-expression of genes, their location on the chromosome and their location on the microarray slides by analyzing a number of eukaryotic expression datasets, derived from the S. cerevisiae, C. elegans, and D. melanogaster. We find that in several different yeast microarray experiments the distribution of the number of gene pairs with correlated expression profiles as a function of chromosomal spacing is peaked at short separations and has two superimposed periodicities. The longer periodicity has a spacing of 22 genes (approximately 42 Kb), and the shorter periodicity is 2 genes (approximately 4 Kb). CONCLUSION: The relative positioning of DNA probes on microarray slides and source plates introduces subtle but significant correlations between pairs of genes. Careful consideration of this spatial artifact is important for analysis of microarray expression data. It is particularly relevant to recent microarray analyses that suggest that co-expressed genes cluster along chromosomes or are spaced by multiples of a fixed number of genes along the chromosome.
— id: 72897, year: 2003, vol: 4, page: 49, stat: Journal Article,

Identification and correction of spurious spatial correlations in microarray data
Qian, Jiang; Kluger, Yuval; Yu, Haiyuan; Gerstein, Mark
2003 Jul;35(1):42-4, 46, 48, Biotechniques
— id: 72899, year: 2003, vol: 35, page: 42, stat: Journal Article,

Gene expression in human neutrophils during activation and priming by bacterial lipopolysaccharide
Tsukahara, Yasuhiro; Lian, Zheng; Zhang, Xueqing; Whitney, Constance; Kluger, Yuval; Tuck, David; Yamaga, Shigeru; Nakayama, Yasuhiro; Weissman, Sherman M; Newburger, Peter E
2003 Jul 1;89(4):848-861, Journal of cellular biochemistry
Circulating neutrophils play a key role both in the systemic inflammatory response and in complications of bacterial infection such as septic shock and septic multiple organ dysfunction syndrome. We have analyzed gene expression patterns in human neutrophils stimulated by E. coli lipopolysaccharide (LPS), with or without prior exposure to LPS, using differential display and oligonucleotide chip techniques. We identified 307 genes that were activated or repressed after treatment with LPS at 10 ng/ml and 385 genes after LPS at 100 ng/ml, compared with untreated neutrophils. The two sets included many transcription factors, cytokines, chemokines, interleukins, and surface antigens, as well as members of the toll-like receptor, Rel/NF-kappaB, and immune mediator gene families. Time course analysis showed that the early and late neutrophil responses to LPS share some common mechanisms, but many changes in gene expression are transient or late to develop. Neutrophils also showed a priming response to LPS, in which 97 genes significantly changed expression on re-exposure to lower dose LPS and were analyzed by unsupervised hierarchical clustering. These findings indicate that the neutrophil is a transcriptionally active cell responsive to environmental stimuli and capable of a complex series of both early and late changes in gene expression. Supplementary material for this article can be found on the Journal of Cellular Biochemistry website (http://jws-edci.interscience.wiley.com:8998/jpages/0730-2312/suppmat/89/v 89.page.html)
— id: 42815, year: 2003, vol: 89, page: 848, stat: Journal Article,

Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line
Lian, Zheng; Kluger, Yuval; Greenbaum, Dov S; Tuck, David; Gerstein, Mark; Berliner, Nancy; Weissman, Sherman M; Newburger, Peter E
2002 Nov 1;100(9):3209-3220, Blood
We have used an approach using 2-dimensional gel electrophoresis with mass spectrometry analysis combined with oligonucleotide chip hybridization for a comprehensive and quantitative study of the temporal patterns of protein and mRNA expression during myeloid development in the MPRO murine cell line. This global analysis detected 123 known proteins and 29 'new' proteins out of 220 protein spots identified by tandem mass spectroscopy, including proteins in 12 functional categories such as transcription factors and cytokines. Bioinformatic analysis of these proteins revealed clusters with functional importance to myeloid differentiation. Previous analyses have found that for a substantial number of genes the absolute amount of protein in the cell is not strongly correlated to the amount of mRNA. These conclusions were based on simultaneous measurement of mRNA and protein at just a single time point. Here, however, we are able to investigate the relationship between mRNA and protein in terms of simultaneous changes in their levels over multiple time points. This is the first time such a relationship has been studied, and we find that it gives a much stronger correlation, consistent with the hypothesis that a substantial proportion of protein change is a consequence of changed mRNA levels, rather than posttranscriptional effects. Cycloheximide inhibition also showed that most of the proteins detected by gel electrophoresis were relatively stable. Specific investigation of transcription factor mRNA representation showed considerable similarity to those of mature human neutrophils and highlighted several transcription factors and other functional nuclear proteins whose mRNA levels change prominently during MPRO differentiation but which have not been investigated previously in the context of myeloid development. Data are available online at http://bioinfo.mbb.yale.edu/expression/myelopoiesis
— id: 42816, year: 2002, vol: 100, page: 3209, stat: Journal Article,

Genomic and proteomic analysis of the myeloid differentiation program
Lian Z; Wang L; Yamaga S; Bonds W; Beazer-Barclay Y; Kluger Y; Gerstein M; Newburger PE; Berliner N; Weissman SM
2001 Aug 1;98(3):513-524, Blood
Although the mature neutrophil is one of the better characterized mammalian cell types, the mechanisms of myeloid differentiation are incompletely understood at the molecular level. A mouse promyelocytic cell line (MPRO), derived from murine bone marrow cells and arrested developmentally by a dominant-negative retinoic acid receptor, morphologically differentiates to mature neutrophils in the presence of 10 microM retinoic acid. An extensive catalog was prepared of the gene expression changes that occur during morphologic maturation. To do this, 3'-end differential display, oligonucleotide chip array hybridization, and 2-dimensional protein electrophoresis were used. A large number of genes whose mRNA levels are modulated during differentiation of MPRO cells were identified. The results suggest the involvement of several transcription regulatory factors not previously implicated in this process, but they also emphasize the importance of events other than the production of new transcription factors. Furthermore, gene expression patterns were compared at the level of mRNA and protein, and the correlation between 2 parameters was studied. (Blood. 2001;98:513-524)
— id: 42817, year: 2001, vol: 98, page: 513, stat: Journal Article,

RNA expression patterns change dramatically in human neutrophils exposed to bacteria
Subrahmanyam YV; Yamaga S; Prashar Y; Lee HH; Hoe NP; Kluger Y; Gerstein M; Goguen JD; Newburger PE; Weissman SM
2001 Apr 15;97(8):2457-2468, Blood
A comprehensive study of changes in messenger RNA (mRNA) levels in human neutrophils following exposure to bacteria is described. Within 2 hours there are dramatic changes in the levels of several hundred mRNAs including those for a variety of cytokines, receptors, apoptosis-regulating products, and membrane trafficking regulators. In addition, there are a large number of up-regulated mRNAs that appear to represent a common core of activation response genes that have been identified as early-response products to a variety of stimuli in a number of other cell types. The activation response of neutrophils to nonpathogenic bacteria is greatly altered by exposure to Yersinia pestis, which may be a major factor contributing to the virulence and rapid progression of plague. Several gene clusters were created based on the patterns of gene induction caused by different bacteria. These clusters were consistent with those found by a principal components analysis. A number of the changes could be interpreted in terms of neutrophil physiology and the known functions of the genes. These findings indicate that active regulation of gene expression plays a major role in the neutrophil contribution to the cellular inflammatory response. Interruption of these changes by pathogens, such as Y pestis, could be responsible, at least in part, for the failure to contain infections by highly virulent organisms
— id: 42818, year: 2001, vol: 97, page: 2457, stat: Journal Article,