Biomedical subjects

Sherman M Weissman

Publications and source records attributed to Sherman M Weissman.

At least 19 recordsLinked to original sources

PubMed2006

A limited number of genes are involved in the differentiation of germinal center B cells.

Mature B cells, upon activation, progressively differentiate through centroblasts into centrocytes and finally to plasmacytes that express large amounts of selected immunoglobulins. A significant part of this maturation is thought to involve induction of the unfolded protein response (UPR). We have compared gene expression in normal germinal center centroblasts, centrocytes, lymphoblastoid cells undergoing induced UPR, and the CCL155 plasmacytoma cell line. In the centroblast to centrocyte transition there is a change in the expression of a relatively small number of genes. These include a limited subset of the genes upregulated by a fully activated UPR as well as a small number of other transcription factors, some disulphide isomerases, and other genes. This is consistent with a model in which this transition is mediated by changes in the levels of expression of transcription factor B-lymphocyte-induced maturation protein 1 (Blimp1) (PRDM1), BACH2, X-box binding protein 1 (XBP1), interferon regulatory factor 4 (IRF4), and possibly vitamin D receptor (VDR) expression, together with post-transcriptional changes and a limited induction of aspects of the UPR.

Animals↗

PubMed2006

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Adult Stem Cells↗

PubMed2006

Identification of coding single nucleotide polymorphisms and mutations by combination of genome tiling arrays and enrichment/depletion of mismatch cDNAs.

Genome tiling array technology combined with a method for both enrichment and depletion of mismatch-containing cDNA fragments offers a useful approach for detecting coding single nucleotide polymorphisms (cSNPs) and mutations in pooled cDNA samples. Enriched mismatch and perfect match cDNA samples from human primary melanoma cells and normal melanocytes were obtained by selection using mismatch repair thymine DNA glycosylase-bound beads. These cDNA samples were then labeled and hybridized to Encyclopedia of DNA Elements genome tiling arrays. The results revealed that the hybridization intensity values of potential cDNA variation regions of the enriched mismatch samples increased, whereas the hybridization intensity values of corresponding regions of the enriched perfect match samples decreased. Six potential mutations were confirmed by polymerase chain reaction product sequencing, including two novel heterozygous mutations in melanoma cells. We suggest that this strategy should increase the efficiency of both cSNP and mutation detection throughout the entire human genome and decrease the cost and complexity of genomewide analysis of cDNA variations.

Base Pair Mismatch↗

PubMed2006

High-resolution mapping of DNA copy alterations in human chromosome 22 using high-density tiling oligonucleotide arrays.

Deletions and amplifications of the human genomic sequence (copy number polymorphisms) are the cause of numerous diseases and a potential cause of phenotypic variation in the normal population. Comparative genomic hybridization (CGH) has been developed as a useful tool for detecting alterations in DNA copy number that involve blocks of DNA several kilobases or larger in size. We have developed high-resolution CGH (HR-CGH) to detect accurately and with relatively little bias the presence and extent of chromosomal aberrations in human DNA. Maskless array synthesis was used to construct arrays containing 385,000 oligonucleotides with isothermal probes of 45-85 bp in length; arrays tiling the beta-globin locus and chromosome 22q were prepared. Arrays with a 9-bp tiling path were used to map a 622-bp heterozygous deletion in the beta-globin locus. Arrays with an 85-bp tiling path were used to analyze DNA from patients with copy number changes in the pericentromeric region of chromosome 22q. Heterozygous deletions and duplications as well as partial triploidies and partial tetraploidies of portions of chromosome 22q were mapped with high resolution (typically up to 200 bp) in each patient, and the precise breakpoints of two deletions were confirmed by DNA sequencing. Additional peaks potentially corresponding to known and novel additional CNPs were also observed. Our results demonstrate that HR-CGH allows the detection of copy number changes in the human genome at an unprecedented level of resolution.

Base Sequence↗

PubMed2006

Multi-protein complexes at the beta-globin locus.

Biochemical analysis of the beta-globin gene function has led to the identification of several multi-protein complexes at the locus control region (LCR), insulator and promoters. This review briefly summarizes these multi-protein complexes and discusses their contribution towards the regulation of the beta-globin gene expression.

Gene Expression Regulation↗

PubMed2006

Synthetic messenger RNA as a tool for gene therapy.

Transfection of human cells with DNA in biomedical applications carries the risk of insertional mutagenesis. Transfection with mRNA avoids this problem; however, in vitro production of mRNA, based on preliminary DNA template cloning in special vectors, is a laborious and time-consuming procedure. We report an efficient vectorfree method of mRNA production from polymerase chain reaction-generated DNA templates. For all cell types tested mRNA was transfected more readily than DNA, and its expression was highly uniform in cell populations. Even cell types relatively resistant to transfection with DNA could express transfected mRNA well. The level of mRNA expression could be controlled over a wide range by changing the amount of input RNA. Cells could be efficiently and simultaneously loaded with several different transcripts. To test a potential clinical application of this method, we transfected human T lymphocytes with mRNA encoding a chimeric immune receptor directed against CD19, a surface antigen widely expressed in leukemia and lymphoma. The transfected mRNA conferred powerful cytotoxicity to T cells against CD19+ targets from the same donor. These results demonstrate that this method can be applied to generate autologous T lymphocytes directed toward malignant cells.

Antigens, CD19↗

PubMed2006

FAT10/diubiquitin-like protein-deficient mice exhibit minimal phenotypic differences.

The FAT10 gene encodes a diubiquitin-like protein containing two tandem head-to-tail ubiquitin-like domains. There is a high degree of similarity between murine and human FAT10 sequences at both the mRNA and protein levels. In various cell lines, FAT10 expression was shown to be induced by gamma interferon or by tumor necrosis factor alpha. In addition, FAT10 expression was found to be up-regulated in some Epstein-Barr virus-infected B-cell lines, in activated dendritic cells, and in several epithelial tumors. However, forced expression of FAT10 in cultured cells was also found to produce apoptotic cell death. Overall, these findings suggest that FAT10 may modulate cellular growth or cellular viability. Here we describe the steps to generate, by genetic targeting, a FAT10 gene knockout mouse model. The FAT10 knockout homozygous mice are viable and fertile. No gross lesions or obvious histological differences were found in these mutated mice. Examination of lymphocyte populations from spleen, thymus, and bone marrow did not reveal any abnormalities. However, flow cytometry analysis demonstrated that the lymphocytes of FAT10 knockout mice were, on average, more prone to spontaneous apoptotic death. Physiologically, these mice demonstrated a high level of sensitivity toward endotoxin challenge. These findings indicate that FAT10 may function as a survival factor.

Amino Acid Sequence↗

PubMed2005

Two types of precursor cells in a multipotential hematopoietic cell line.

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.

Animals↗

PubMed2005

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the beta-globin locus control region.

Locus control regions (LCRs) are regulatory DNA sequences that are situated many kilobases away from their cognate promoters. LCRs protect transgenes from position effect variegation and heterochromatinization and also promote copy-number dependence of the levels of transgene expression. In this work, we describe the biochemical purification of a previously undescribed LCR-associated remodeling complex (LARC) that consists of heterogeneous nuclear ribonucleoprotein C1/C2, nucleosome remodeling SWI/SNF, and nucleosome remodeling and deacetylating (NuRD)/MeCP1 as a single homogeneous complex. LARC binds to the hypersensitive 2 (HS2)-Maf recognition element (MARE) DNA in a sequence-specific manner and remodels nucleosomes. Heterogeneous nuclear ribonucleoprotein C1/C2, previously known as a general RNA binding protein, provides a sequence-specific DNA recognition element for LARC, and the LARC DNA-recognition sequence is essential for the enhancement of transcription by HS2. Independently of the initiation of transcription, LARC becomes associated with beta-like globin promoters.

Amino Acid Sequence↗

PubMed2005

Werner protein protects nonproliferating cells from oxidative DNA damage.

Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of gammaH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.

Adenosine Triphosphatases↗

PubMed2004

Lineage specificity of gene expression patterns.

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.

Cell Lineage↗

PubMed2004

A panorama of lineage-specific transcription in hematopoiesis.

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.

Animals↗

PubMed2003

Gene expression in mature neutrophils: early responses to inflammatory stimuli.

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.

Chromatin Assembly and Disassembly↗

PubMed2003

Gene expression in human neutrophils during activation and priming by bacterial lipopolysaccharide.

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/v89.page.html).

Cluster Analysis↗

PubMed2003

Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit.

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3' end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from "stem cell genes" being highly expressed at 0 h and turned off at 48 h, while "cell division" genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

Animals↗

PubMed2002

Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line.

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.

Animals↗

PubMed2002

Nucleolar localization of hTERT protein is associated with telomerase function.

Telomerase is a ribonucleoprotein (RNP) complex that prevents telomeric erosion in eukaryotic cells. Although there are also other associated proteins in this complex, the catalytic activity of this complex is composed of two components. One is a reverse transcriptase subunit, TERT (telomerase reverse transcriptase); another is an RNA template subunit, TR (telomerase RNA). However, where these two parts are assembled in mammalian cells is unclear. In the present study, we investigated the intracellular distribution of human TERT (hTERT) protein and observed that hTERT protein in individual cells could concentrate in or be excluded from the nucleolus. Further we have identified a nucleolar targeting signal in the hTERT protein. Point mutations that disrupted this signal region interrupted telomerase RNP complex formation, decreased telomerase activity, and caused telomere shortening in cells transfected with mutated hTERT. Our results indicate that the amino acid sequence of the extreme N-terminus (1-15) of hTERT, which targets nucleolar localization of the protein, is required for full telomerase function.

Amino Acid Sequence↗

PubMed2002

An approach for global scanning of single nucleotide variations.

Efficient global scanning of single nucleotide variations in DNA sequences between related, complex DNA samples remains a challenge. In the present article we present an approach to this problem. We have used immobilized thymidine DNA glycosylases to capture and enrich DNA fragments containing internal mismatched base pairs and separate these fragments as a pool from perfectly base-paired fragments as another pool. Enrichments of up to several hundredfold were obtained with one cycle of treatment, and all of the four groups of single nucleotide mismatches were fully covered by combining use of two thymine DNA glycosylases generated here. We have used a heterohybrid-orientating strategy for selective amplification of duplexes with one strand derived from each of two input DNA samples, which can also be used for selective amplification of duplexes with both strands derived from one of two input samples when desired. By combining these methods, the single nucleotide variations either between two DNA pools or within one DNA pool can be obtained in one process. This approach has been applied to the total cDNA from a human cell line and has several potential applications in mapping genetic variations, particularly global scanning of cDNA single nucleotide variations or polymorphisms, and finally high-throughput mapping of complex genetic traits.

Base Pair Mismatch↗