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1.
Nucleic Acids Res ; 42(14): 9424-35, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25056318

ABSTRACT

We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3' and/or 5' end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5' differences and in support of this we report that a 5' isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5' isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.


Subject(s)
MicroRNAs/metabolism , Animals , Argonaute Proteins/metabolism , Cell Line , Evolution, Molecular , Humans , Mice , MicroRNAs/chemistry , MicroRNAs/genetics , RNA Precursors/chemistry , RNA, Messenger/metabolism , Stem Cells/metabolism
2.
Hum Mol Genet ; 22(25): 5107-20, 2013 Dec 20.
Article in English | MEDLINE | ID: mdl-23912340

ABSTRACT

Smooth muscle cell (SMC) proliferation is a hallmark of vascular injury and disease. Global hypomethylation occurs during SMC proliferation in culture and in vivo during neointimal formation. Regardless of the programmed or stochastic nature of hypomethylation, identifying these changes is important in understanding vascular disease, as maintenance of a cells' epigenetic profile is essential for maintaining cellular phenotype. Global hypomethylation of proliferating aortic SMCs and concomitant decrease of DNMT1 expression were identified in culture during passage. An epigenome screen identified regions of the genome that were hypomethylated during proliferation and a region containing Collagen, type XV, alpha 1 (COL15A1) was selected by 'genomic convergence' for characterization. COL15A1 transcript and protein levels increased with passage-dependent decreases in DNA methylation and the transcript was sensitive to treatment with 5-Aza-2'-deoxycytidine, suggesting DNA methylation-mediated gene expression. Phenotypically, knockdown of COL15A1 increased SMC migration and decreased proliferation and Col15a1 expression was induced in an atherosclerotic lesion and localized to the atherosclerotic cap. A sequence variant in COL15A1 that is significantly associated with atherosclerosis (rs4142986, P = 0.017, OR = 1.434) was methylated and methylation of the risk allele correlated with decreased gene expression and increased atherosclerosis in human aorta. In summary, hypomethylation of COL15A1 occurs during SMC proliferation and the consequent increased gene expression may impact SMC phenotype and atherosclerosis formation. Hypomethylated genes, such as COL15A1, provide evidence for concomitant epigenetic regulation and genetic susceptibility, and define a class of causal targets that sit at the intersection of genetic and epigenetic predisposition in the etiology of complex disease.


Subject(s)
Atherosclerosis/genetics , Cellular Senescence/genetics , Collagen/genetics , Epigenesis, Genetic , Atherosclerosis/pathology , Cell Movement/genetics , Cell Proliferation , Cells, Cultured , DNA Methylation/genetics , Gene Expression Regulation , Humans , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Neointima/genetics
3.
BMC Med Genomics ; 4: 64, 2011 Aug 10.
Article in English | MEDLINE | ID: mdl-21827714

ABSTRACT

BACKGROUND: It is widely accepted that atherosclerosis and inflammation are intimately linked. Monocytes play a key role in both of these processes and we hypothesized that activation of inflammatory pathways in monocytes would lead to, among others, proatherogenic changes in the monocyte transcriptome. Such differentially expressed genes in circulating monocytes would be strong candidates for further investigation in disease association studies. METHODS: Endotoxin, lipopolysaccharide (LPS), or saline control was infused in healthy volunteers. Monocyte RNA was isolated, processed and hybridized to Hver 2.1.1 spotted cDNA microarrays. Differential expression of key genes was confirmed by RT-PCR and results were compared to in vitro data obtained by our group to identify candidate genes. RESULTS: All subjects who received LPS experienced the anticipated clinical response indicating successful stimulation. One hour after LPS infusion, 11 genes were identified as being differentially expressed; 1 down regulated and 10 up regulated. Four hours after LPS infusion, 28 genes were identified as being differentially expressed; 3 being down regulated and 25 up regulated. No genes were significantly differentially expressed following saline infusion. Comparison with results obtained in in vitro experiments lead to the identification of 6 strong candidate genes (BATF, BID, C3aR1, IL1RN, SEC61B and SLC43A3) CONCLUSION: In vivo endotoxin exposure of healthy individuals resulted in the identification of several candidate genes through which systemic inflammation links to atherosclerosis.


Subject(s)
Atherosclerosis/genetics , Inflammation/genetics , Lipopolysaccharides/administration & dosage , Atherosclerosis/metabolism , Gene Expression Regulation , Humans , Injections, Intravenous , Male , Monocytes/immunology , Monocytes/metabolism , Oligonucleotide Array Sequence Analysis , Young Adult
4.
Neuro Oncol ; 12(7): 664-78, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20164239

ABSTRACT

We studied the status of chromosomes 1 and 19 in 363 astrocytic and oligodendroglial tumors. Whereas the predominant pattern of copy number abnormality was a concurrent loss of the entire 1p and 19q regions (total 1p/19q loss) among oligodendroglial tumors and partial deletions of 1p and/or 19q in astrocytic tumors, a subset of apparently astrocytic tumors also had total 1p/19q loss. The presence of total 1p/19q loss was associated with longer survival of patients with all types of adult gliomas independent of age and diagnosis (P = .041). The most commonly deleted region on 19q in astrocytic tumors spans 885 kb in 19q13.33-q13.41, which is telomeric to the previously proposed region. Novel regions of homozygous deletion, including a part of DPYD (1p21.3) or the KLK cluster (19q13.33), were observed in anaplastic oligodendrogliomas. Amplifications encompassing AKT2 (19q13.2) or CCNE1 (19q12) were identified in some glioblastomas. Deletion mapping of the centromeric regions of 1p and 19q in the tumors that had total 1p/19q loss, indicating that the breakpoints lie centromeric to NOTCH2 within the pericentromeric regions of 1p and 19q. Thus, we show that the copy number abnormalities of 1p and 19q in human gliomas are complex and have distinct patterns that are prognostically predictive independent of age and pathological diagnosis. An accurate identification of total 1p/19q loss and discriminating this from other 1p/19q changes is, however, critical when the 1p/19q copy number status is used to stratify patients in clinical trials.


Subject(s)
Brain Neoplasms/diagnosis , Brain Neoplasms/genetics , Chromosome Deletion , Chromosomes, Human, Pair 19/genetics , Chromosomes, Human, Pair 1/genetics , Glioma/diagnosis , Glioma/genetics , Brain Neoplasms/classification , Chromosome Aberrations , Glioma/classification , Humans , Prognosis
5.
Nat Methods ; 7(2): 130-2, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20081834

ABSTRACT

We report an alternative approach to transcriptome sequencing for the Illumina Genome Analyzer, in which the reverse transcription reaction takes place on the flowcell. No amplification is performed during the library preparation, so PCR biases and duplicates are avoided, and because the template is poly(A)(+) RNA rather than cDNA, the resulting sequences are necessarily strand-specific. The method is compatible with paired- or single-end sequencing.


Subject(s)
Chromosome Mapping/methods , Fluorescence Resonance Energy Transfer/methods , Gene Expression Profiling/methods , Sequence Analysis, DNA/methods , Transcription Factors/genetics , Nucleic Acid Amplification Techniques
6.
J Invest Dermatol ; 130(6): 1726-36, 2010 Jun.
Article in English | MEDLINE | ID: mdl-19924136

ABSTRACT

T-pro are tumor-infiltrating TCRalphabeta(+)CD8(+) cells of reduced cytotoxic potential that promote experimental two-stage chemical cutaneous carcinogenesis. Toward understanding their mechanism of action, this study uses whole-genome expression analysis to compare T-pro with systemic CD8(+) T cells from multiple groups of tumor-bearing mice. T-pro show an overt T helper 17-like profile (high retinoic acid-related orphan receptor-(ROR)gammat, IL-17A, IL-17F; low T-bet and eomesodermin), regulatory potential (high FoxP3, IL-10, Tim-3), and transcripts encoding epithelial growth factors (amphiregulin, Gro-1, Gro-2). Tricolor flow cytometry subsequently confirmed the presence of TCRbeta(+) CD8(+) IL-17(+) T cells among tumor-infiltrating lymphocytes (TILs). Moreover, a time-course analysis of independent TIL isolates from papillomas versus carcinomas exposed a clear association of the "T-pro phenotype" with malignant progression. This molecular characterization of T-pro builds a foundation for elucidating the contributions of inflammation to cutaneous carcinogenesis, and may provide useful biomarkers for cancer immunotherapy in which the widely advocated use of tumor-specific CD8(+) cytolytic T cells should perhaps accommodate the cells' potential corruption toward the T-pro phenotype. The data are also likely germane to psoriasis, in which the epidermis may be infiltrated by CD8(+) IL-17-producing T cells.


Subject(s)
CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Gene Expression Profiling , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , 9,10-Dimethyl-1,2-benzanthracene/adverse effects , Amphiregulin , Animals , Cell Differentiation , Disease Models, Animal , EGF Family of Proteins , Forkhead Transcription Factors/metabolism , Glycoproteins/metabolism , Hepatitis A Virus Cellular Receptor 2 , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-10/metabolism , Interleukin-17/metabolism , Mice , Mice, Knockout , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Virus/metabolism , Skin Neoplasms/chemically induced
7.
Chromosome Res ; 17(8): 987-1000, 2009.
Article in English | MEDLINE | ID: mdl-19941159

ABSTRACT

Injection-site-associated sarcomas (ISAS), commonly arising at the site of routine vaccine administration, afflict as many as 22,000 domestic cats annually in the USA. These tumors are typically more aggressive and prone to recurrence than spontaneous sarcomas (non-ISAS), generally receiving a poorer long-term prognosis and warranting a more aggressive therapeutic approach. Although certain clinical and histological factors are highly suggestive of ISAS, timely diagnosis and optimal clinical management may be hindered by the absence of definitive markers that can distinguish between tumors with underlying injection-related etiology and their spontaneous counterpart. Specific nonrandom chromosome copy number aberrations (CNAs) have been associated with the clinical behavior of a vast spectrum of human tumors, providing an extensive resource of potential diagnostic and prognostic biomarkers. Although similar principles are now being applied with great success in other species, their relevance to feline molecular oncology has not yet been investigated in any detail. We report the construction of a genomic microarray platform for detection of recurrent CNAs in feline tumors through cytogenetic assignment of 210 large-insert DNA clones selected at intervals of approximately 15 Mb from the feline genome sequence assembly. Microarray-based profiling of 19 ISAS and 27 non-ISAS cases identified an extensive range of genomic imbalances that were highly recurrent throughout the combined panel of 46 sarcomas. Deletions of two specific regions were significantly associated with the non-ISAS phenotype. Further characterization of these regions may ultimately permit molecular distinction between ISAS and non-ISAS, as a tool for predicting tumor behavior and prognosis, as well as refining means for therapeutic intervention.


Subject(s)
DNA Copy Number Variations , Sarcoma/genetics , Animals , Cat Diseases/diagnosis , Cats , Cytogenetic Analysis , Gene Dosage , Gene Expression Profiling , Injections/adverse effects , Oligonucleotide Array Sequence Analysis , Sarcoma/diagnosis , Sarcoma/veterinary
8.
BMC Med ; 7: 62, 2009 Oct 22.
Article in English | MEDLINE | ID: mdl-19845972

ABSTRACT

BACKGROUND: Autism comprises a spectrum of behavioral and cognitive disturbances of childhood development and is known to be highly heritable. Although numerous approaches have been used to identify genes implicated in the development of autism, less than 10% of autism cases have been attributed to single gene disorders. METHODS: We describe the use of high-resolution genome-wide tilepath microarrays and comparative genomic hybridization to identify copy number variants within 119 probands from multiplex autism families. We next carried out DNA methylation analysis by bisulfite sequencing in a proband and his family, expanding this analysis to methylation analysis of peripheral blood and temporal cortex DNA of autism cases and matched controls from independent datasets. We also assessed oxytocin receptor (OXTR) gene expression within the temporal cortex tissue by quantitative real-time polymerase chain reaction (PCR). RESULTS: Our analysis revealed a genomic deletion containing the oxytocin receptor gene, OXTR (MIM accession no.: 167055), previously implicated in autism, was present in an autism proband and his mother who exhibits symptoms of obsessive-compulsive disorder. The proband's affected sibling did not harbor this deletion but instead may exhibit epigenetic misregulation of this gene through aberrant gene silencing by DNA methylation. Further DNA methylation analysis of the CpG island known to regulate OXTR expression identified several CpG dinucleotides that show independent statistically significant increases in the DNA methylation status in the peripheral blood cells and temporal cortex in independent datasets of individuals with autism as compared to control samples. Associated with the increase in methylation of these CpG dinucleotides is our finding that OXTR mRNA showed decreased expression in the temporal cortex tissue of autism cases matched for age and sex compared to controls. CONCLUSION: Together, these data provide further evidence for the role of OXTR and the oxytocin signaling pathway in the etiology of autism and, for the first time, implicate the epigenetic regulation of OXTR in the development of the disorder.See the related commentary by Gurrieri and Neri: http://www.biomedcentral.com/1741-7015/7/63.


Subject(s)
Autistic Disorder/genetics , DNA/genetics , DNA/metabolism , Receptors, Oxytocin/deficiency , Adolescent , Adult , Child , Child, Preschool , Comparative Genomic Hybridization , DNA Methylation , Female , Humans , Male , Microarray Analysis , Sequence Deletion , Young Adult
9.
Leuk Lymphoma ; 50(9): 1528-34, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19672776

ABSTRACT

Lymphoplasmacytic lymphoma (LPL) is not a sharply delineated lymphoma entity, either morphologically, phenotypically, or clinically. The diagnosis is often made by excluding other small cell lymphomas with plasmacytic differentiation, thus a genetic diagnostic marker would be of great benefit. Conventional cytogenetic techniques have previously demonstrated a deletion of 6q in a proportion of cases, varying from 7 to 55%. In this report, we apply array-based comparative genomic hybridization on 11 LPL samples. Genomic aberrations were detected in 9 of 11 cases, and included gains and losses. In general, the number of genetic aberrations was relatively low (two to three abnormalities per case). Recurrent aberrations detected were deletion of 6q (two cases), deletion of chromosome 17 (two cases), gain of 3q (two cases), and gain of chromosome 7 (two cases). This report not only confirms the reported loss of 6q in a proportion of cases but also highlights the genetic heterogeneity of LPL, in accordance with the known immunophenotypical, morphological, and clinical diversity of the disease.


Subject(s)
Chromosome Aberrations , Comparative Genomic Hybridization/methods , Waldenstrom Macroglobulinemia/genetics , Adult , Aged , Aged, 80 and over , Chromosome Mapping/methods , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 3 , Chromosomes, Human, Pair 6 , Chromosomes, Human, Pair 7 , Female , Genome-Wide Association Study , Humans , Male , Microarray Analysis/methods , Middle Aged , Waldenstrom Macroglobulinemia/mortality
10.
Epigenetics Chromatin ; 2(1): 7, 2009 Jun 08.
Article in English | MEDLINE | ID: mdl-19505295

ABSTRACT

BACKGROUND: DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. RESULTS: Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. CONCLUSION: The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

11.
Stem Cells ; 27(11): 2637-2645, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19544422

ABSTRACT

Histone H3 methylation at R17 and R26 recently emerged as a novel epigenetic mechanism regulating pluripotency in mouse embryos. Blastomeres of four-cell embryos with high H3 methylation at these sites show unrestricted potential, whereas those with lower levels cannot support development when aggregated in chimeras of like cells. Increasing histone H3 methylation, through expression of coactivator-associated-protein-arginine-methyltransferase 1 (CARM1) in embryos, elevates expression of key pluripotency genes and directs cells to the pluripotent inner cell mass. We demonstrate CARM1 is also required for the self-renewal and pluripotency of embryonic stem (ES) cells. In ES cells, CARM1 depletion downregulates pluripotency genes leading to their differentiation. CARM1 associates with Oct4/Pou5f1 and Sox2 promoters that display detectable levels of R17/26 histone H3 methylation. In CARM1 overexpressing ES cells, histone H3 arginine methylation is also at the Nanog promoter to which CARM1 now associates. Such cells express Nanog at elevated levels and delay their response to differentiation signals. Thus, like in four-cell embryo blastomeres, histone H3 arginine methylation by CARM1 in ES cells allows epigenetic modulation of pluripotency.


Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Animals , Arginine/metabolism , Blotting, Western , Cell Differentiation/genetics , Cell Line , Chromatin Immunoprecipitation , Histones/chemistry , Histones/metabolism , Homeodomain Proteins/genetics , Methylation , Mice , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/physiology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , SOXB1 Transcription Factors/genetics
12.
Chromosome Res ; 17(3): 365-377, 2009.
Article in English | MEDLINE | ID: mdl-19337847

ABSTRACT

Recurrent chromosomal aberrations in solid tumors can reveal the genetic pathways involved in the evolution of a malignancy and in some cases predict biological behavior. However, the role of individual genetic backgrounds in shaping karyotypes of sporadic tumors is unknown. The genetic structure of purebred dog breeds, coupled with their susceptibility to spontaneous cancers, provides a robust model with which to address this question. We tested the hypothesis that there is an association between breed and the distribution of genomic copy number imbalances in naturally occurring canine tumors through assessment of a cohort of Golden Retrievers and Rottweilers diagnosed with spontaneous appendicular osteosarcoma. Our findings reveal significant correlations between breed and tumor karyotypes that are independent of gender, age at diagnosis, and histological classification. These data indicate for the first time that individual genetic backgrounds, as defined by breed in dogs, influence tumor karyotypes in a cancer with extensive genomic instability.


Subject(s)
Chromosome Aberrations/veterinary , Dog Diseases/genetics , Genetic Predisposition to Disease/genetics , Osteosarcoma/veterinary , Animals , Comparative Genomic Hybridization , Dogs , Karyotyping/veterinary , Osteosarcoma/genetics , Species Specificity
13.
J Neurooncol ; 94(3): 333-49, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19333554

ABSTRACT

Numerous attributes render the domestic dog a highly pertinent model for cancer-associated gene discovery. We performed microarray-based comparative genomic hybridization analysis of 60 spontaneous canine intracranial tumors to examine the degree to which dog and human patients exhibit aberrations of ancestrally related chromosome regions, consistent with a shared pathogenesis. Canine gliomas and meningiomas both demonstrated chromosome copy number aberrations (CNAs) that share evolutionarily conserved synteny with those previously reported in their human counterpart. Interestingly, however, genomic imbalances orthologous to some of the hallmark aberrations of human intracranial tumors, including chromosome 22/NF2 deletions in meningiomas and chromosome 1p/19q deletions in oligodendrogliomas, were not major events in the dog. Furthermore, and perhaps most significantly, we identified highly recurrent CNAs in canine intracranial tumors for which the human orthologue has been reported previously at low frequency but which have not, thus far, been associated intimately with the pathogenesis of the tumor. The presence of orthologous CNAs in canine and human intracranial cancers is strongly suggestive of their biological significance in tumor development and/or progression. Moreover, the limited genetic heterogenity within purebred dog populations, coupled with the contrasting organization of the dog and human karyotypes, offers tremendous opportunities for refining evolutionarily conserved regions of tumor-associated genomic imbalance that may harbor novel candidate genes involved in their pathogenesis. A comparative approach to the study of canine and human intracranial tumors may therefore provide new insights into their genetic etiology, towards development of more sophisticated molecular subclassification and tailored therapies in both species.


Subject(s)
Brain Neoplasms/genetics , Chromosome Aberrations , Genome/genetics , Meningeal Neoplasms/genetics , Meningioma/genetics , Animals , Brain Neoplasms/pathology , Chromosome Mapping , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 22 , Cluster Analysis , Dogs , Female , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/pathology , Humans , In Situ Hybridization, Fluorescence , Male , Meningeal Neoplasms/pathology , Meningioma/pathology
14.
Genome Res ; 19(6): 994-1005, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19401398

ABSTRACT

The molecular events that contribute to, and result from, the in vivo binding of transcription factors to their cognate DNA sequence motifs in mammalian genomes are poorly understood. We demonstrate that variations within the DNA sequence motifs that bind the transcriptional repressor REST (NRSF) encode in vivo DNA binding affinity hierarchies that contribute to regulatory function during lineage-specific and developmental programs in fundamental ways. First, canonical sequence motifs for REST facilitate strong REST binding and control functional classes of REST targets that are common to all cell types, whilst atypical motifs participate in weak interactions and control those targets, which are cell- or tissue-specific. Second, variations in REST binding relate directly to variations in expression and chromatin configurations of REST's target genes. Third, REST clearance from its binding sites is also associated with variations in the RE1 motif. Finally, and most surprisingly, weak REST binding sites reside in DNA sequences that show the highest levels of constraint through evolution, thus facilitating their roles in maintaining tissue-specific functions. These relationships have never been reported in mammalian systems for any transcription factor.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Repressor Proteins/metabolism , Base Sequence , Binding Sites/genetics , Binding, Competitive , Cell Line , Chromatin/metabolism , Chromatin Immunoprecipitation , DNA/genetics , DNA-Binding Proteins/genetics , Gene Expression Profiling , HeLa Cells , Humans , Immunoblotting , K562 Cells , RNA, Small Interfering/genetics , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection
15.
Blood ; 113(19): e1-9, 2009 May 07.
Article in English | MEDLINE | ID: mdl-19228925

ABSTRACT

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.


Subject(s)
Bone Marrow Cells/physiology , Cell Differentiation/genetics , Gene Expression , Atlases as Topic , Cell Lineage , Cells, Cultured , Flow Cytometry , Gene Expression Profiling , Hematopoiesis , Humans , Oligonucleotide Array Sequence Analysis , Transcription Factors/metabolism
16.
Platelets ; 19(4): 258-67, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18569861

ABSTRACT

Platelet Glycoprotein VI (GPVI) is the activatory collagen signalling receptor that transmits an outside-in signal via the FcR gamma-chain. In Caucasians two GP6 haplotypes have been identified which encode GPVI isoforms that differ by five amino-acids. The minor haplotype is associated with a modest but statistically significant reduction in GPVI abundance and reduced downstream signalling events. As GPVI is also expressed on megakaryocytes, different GPVI isoforms may imprint on the platelet transcriptome. We investigated the association of GP6 haplotype with transcription by comparing the transcriptomes of platelets from individuals homozygous for the major ('a') and minor ('b') haplotypes to identify differentially expressed (DE) transcripts. Platelet RNA was isolated from apheresis concentrates from 16 'aa' donors and eight 'bb' donors. mRNA was amplified using a template-switching PCR based protocol and fluorescently labelled. Samples were randomly paired both within and between haplotypes and compared on a cDNA microarray. No consistently DE transcripts were identified within the 'aa' haplotype but 52 significantly DE transcripts were observed between haplotypes. Generally the fold differences were low (two to four-fold) but were confirmed by qRT-PCR for selected transcripts (TUBB1, P = 0.0004; VWF, P = 0.0126). The results of this study indicate that there are subtle differences between the platelet transcriptomes of individuals who differ by GP6 haplotype. The identification of DE genes may identify critical pathways and nodes not previously known to be involved in platelet development and function.


Subject(s)
Blood Platelets/chemistry , Gene Expression Profiling , Genetic Variation , Haplotypes , Platelet Membrane Glycoproteins/genetics , RNA, Messenger/analysis , Humans , Megakaryocytes
17.
J Hered ; 98(5): 474-84, 2007.
Article in English | MEDLINE | ID: mdl-17702974

ABSTRACT

The generation of a 7.5x dog genome assembly provides exciting new opportunities to interpret tumor-associated chromosome aberrations at the biological level. We present a genomic microarray for array comparative genomic hybridization (aCGH) analysis in the dog, comprising 275 bacterial artificial chromosome (BAC) clones spaced at intervals of approximately 10 Mb. Each clone has been positioned accurately within the genome assembly and assigned to a unique chromosome location by fluorescence in situ hybridization (FISH) analysis, both individually and as chromosome-specific BAC pools. The microarray also contains clones representing the dog orthologues of 31 genes implicated in human cancers. FISH analysis of the 10-Mb BAC clone set indicated excellent coverage of each dog chromosome by the genome assembly. The order of clones was consistent with the assembly, but the cytogenetic intervals between clones were variable. We demonstrate the application of the BAC array for aCGH analysis to identify both whole and partial chromosome imbalances using a canine histiocytic sarcoma case. Using BAC clones selected from the array as probes, multicolor FISH analysis was used to further characterize these imbalances, revealing numerous structural chromosome rearrangements. We outline the value of a combined aCGH/FISH approach, together with a well-annotated dog genome assembly, in canine and comparative cancer studies.


Subject(s)
Chromosome Mapping , Chromosomes, Artificial, Bacterial/genetics , Dogs/genetics , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Animals , Genome , In Situ Hybridization, Fluorescence , Sensitivity and Specificity
18.
Genome Res ; 17(6): 691-707, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17567990

ABSTRACT

We generated high-resolution maps of histone H3 lysine 9/14 acetylation (H3ac), histone H4 lysine 5/8/12/16 acetylation (H4ac), and histone H3 at lysine 4 mono-, di-, and trimethylation (H3K4me1, H3K4me2, H3K4me3, respectively) across the ENCODE regions. Studying each modification in five human cell lines including the ENCODE Consortium common cell lines GM06990 (lymphoblastoid) and HeLa-S3, as well as K562, HFL-1, and MOLT4, we identified clear patterns of histone modification profiles with respect to genomic features. H3K4me3, H3K4me2, and H3ac modifications are tightly associated with the transcriptional start sites (TSSs) of genes, while H3K4me1 and H4ac have more widespread distributions. TSSs reveal characteristic patterns of both types of modification present and the position relative to TSSs. These patterns differ between active and inactive genes and in particular the state of H3K4me3 and H3ac modifications is highly predictive of gene activity. Away from TSSs, modification sites are enriched in H3K4me1 and relatively depleted in H3K4me3 and H3ac. Comparison between cell lines identified differences in the histone modification profiles associated with transcriptional differences between the cell lines. These results provide an overview of the functional relationship among histone modifications and gene expression in human cells.


Subject(s)
Genome, Human/physiology , Histones/metabolism , Protein Processing, Post-Translational/physiology , Transcription, Genetic/physiology , HeLa Cells , Humans , K562 Cells
19.
Genome Res ; 17(6): 708-19, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17567991

ABSTRACT

Butyrate is a histone deacetylase inhibitor (HDACi) with anti-neoplastic properties, which theoretically reactivates epigenetically silenced genes by increasing global histone acetylation. However, recent studies indicate that a similar number or even more genes are down-regulated than up-regulated by this drug. We treated hepatocarcinoma HepG2 cells with butyrate and characterized the levels of acetylation at DNA-bound histones H3 and H4 by ChIP-chip along the ENCODE regions. In contrast to the global increases of histone acetylation, many genomic regions close to transcription start sites were deacetylated after butyrate exposure. In order to validate these findings, we found that both butyrate and trichostatin A treatment resulted in histone deacetylation at selected regions, while nucleosome loss or changes in histone H3 lysine 4 trimethylation (H3K4me3) did not occur in such locations. Furthermore, similar histone deacetylation events were observed when colon adenocarcinoma HT-29 cells were treated with butyrate. In addition, genes with deacetylated promoters were down-regulated by butyrate, and this was mediated at the transcriptional level by affecting RNA polymerase II (POLR2A) initiation/elongation. Finally, the global increase in acetylated histones was preferentially localized to the nuclear periphery, indicating that it might not be associated to euchromatin. Our results are significant for the evaluation of HDACi as anti-tumourogenic drugs, suggesting that previous models of action might need to be revised, and provides an explanation for the frequently observed repression of many genes during HDACi treatment.


Subject(s)
Adenocarcinoma/metabolism , Butyrates/pharmacology , Colonic Neoplasms/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Histones/metabolism , Neoplasm Proteins/metabolism , Protein Processing, Post-Translational/drug effects , Acetylation/drug effects , Cell Line, Tumor , Gene Expression Profiling , Histone Deacetylase Inhibitors , Histone Deacetylases/metabolism , Humans , Oligonucleotide Array Sequence Analysis
20.
Blood ; 109(8): 3260-9, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17192395

ABSTRACT

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.


Subject(s)
Cell Differentiation/physiology , Erythroblasts/metabolism , Gene Expression Regulation/physiology , Megakaryocytes/metabolism , Platelet Membrane Glycoproteins/biosynthesis , Erythroblasts/cytology , Gene Expression Profiling , Humans , Megakaryocytes/cytology , Oligonucleotide Array Sequence Analysis , Platelet Membrane Glycoproteins/genetics
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