Evolution and heterogeneity of non-hereditary colorectal cancer revealed by single-cell exome sequencing.

Recently single-cell whole-exome sequencing (scWES) has deeply expanded and sharpened our knowledge of cancer evolution and subclonality. Herein, with scWES and matched bulk whole-exome sequencing (bulk WES) on two colorectal cancer (CRC) patients with normal or adenomatous polyps, we found that both the adenoma and cancer were of monoclonal origin, and both shared partial mutations in the same signaling pathways, but each showed a specific spectrum of heterogeneous somatic mutations. In addition, the adenoma and cancer further developed intratumor heterogeneity with the accumulation of nonrandom somatic mutations specifically in GPCR, PI3K-Akt and FGFR signaling pathways. We identified novel driver mutations that developed during adenoma and cancer evolution, particularly in OR1B1 (GPCR signaling pathway) for adenoma evolution, and LAMA1 (PI3K-Akt signaling pathway) and ADCY3 (FGFR signaling pathway) for CRC evolution. In summary, we demonstrated that both colorectal adenoma and CRC are monoclonal in origin, and the CRCs further diversified into different subclones with heterogeneous mutation profiles accumulating in GPCR, PI3K-Akt and FGFR signaling pathways. ScWES provides evidence for the importance of mutations in certain pathways that would not be as apparent from bulk sequencing of tumors, and can potentially establish whether specific mutations are mutually exclusive or occur sequentially in the same subclone of cells.

Differential display of protein tyrosine kinase cDNAs from human fetal and adult brains.

Here we describe a differential display method for surveying the expression of most protein tyrosine kinases and applying it to cDNAs from human fetal and adult brains. The method involves two selective steps for processing the mRNA. At each step, degenerate oligonucleotide primers derived from highly conserved regions of the catalytic domain of the kinases are used. In the display with BstYI and BsiHKI digests of the cDNA, 65% and 59% of a total of 72 and 63 bands, respectively, represented fragments from a total of 27 different tyrosine kinases. The expression levels of the kinases in the display were comparable with those measured by RT-PCR. This method offers a relatively specific way to display differentially expressed gene families in any tissue and cell type.

Functional analysis of B144/LST1: a gene in the tumor necrosis factor cluster that induces formation of long filopodia in eukaryotic cells.

B144/LST1 is a gene encoded in the human major histocompatibility complex that produces multiple forms of alternatively spliced mRNA and encodes peptides fewer than 100 amino acids in length. B144/LST1 is strongly expressed in dendritic cells. Transfection of B144/LST1 into a variety of cells induces morphologic changes including the production of long, thin filopodia differing from those seen on transfection of a dominant active CDC42 gene. The structures are dynamically rearranging and sometimes connect one cell with another. The full effect of B144/LST1 protein on cell morphology requires the retention of at least one of the four cysteines of the peptide plus the presence of a hydrophobic segment in the protein, but requires only one of the two coding regions present in the terminal 3' exons.

Human MHC class III and IV genes and disease associations.

The major histocompatibility complex (MHC) was initially defined as the genetic locus encoding the Class I and Class II highly polymorphic cell surface antigens that are now known to present antigen to matched sets of T cell receptors. Genes for several diverse complement components, specifically Bf, C2, and C4 were found between the Class I and II genes, in a region later dubbed Class III. More recently, several genes have been described that are encoded in the telomeric end of the Class III region and that appear to be involved in both global and specific inflammatory responses. Due to this commonality of function this gene-rich region was dubbed Class IV, and includes the TNF family, AIF1, and HSP70. The genes of the Class III and Class IV regions are sufficiently divergent in sequence and structure so that clustering is not explicable in terms of gene duplication or divergence. We present some of the newer pertinent information and puzzling features of the genes embraced in the Class IV region and discuss possible roles in specific autoimmune diseases linked to this region.

The homeodomain gene Pitx2 is expressed in primitive hematopoietic stem/progenitor cells but not in their differentiated progeny.

OBJECTIVE: Hematopoietic stem cells (HSCs) represent a rare and incompletely characterized fraction of marrow cells that are capable of both self-renewal and differentiation into all of the mature cells in the peripheral blood. We undertook to identify genes expressed preferentially by HSCs as an initial step toward better understanding the molecular mechanisms that underlie HSC behavior. METHODS: We modified the representational difference analysis technique to isolate gene fragments present in amplified cDNA prepared from highly purified murine hematopoietic stem/progenitor cells (Lin(-)/Hoechst(low)/rhodamine(low)) and absent (or much less abundant) in amplified cDNA prepared from lineage-committed marrow cells. We went on to use one potentially important gene fragment that we isolated in this way, to screen a cDNA library prepared from these cells and to characterize the pattern of expression of the gene in hematopoietic and other cells. RESULTS: We isolated a fragment of the homeobox transcription factor Pitx2 from amplified cDNA prepared from murine hematopoietic stem/progenitor cells. From a cDNA library prepared from these cells, a full-length cDNA was isolated that corresponds to one of the three known isoforms of Pitx2 (Pitx2c). Pitx2c is expressed in murine embryonic stem (ES) cells and in hematopoietic stem/progenitor cells but not in more differentiated hematopoietic cells or in a large panel of established murine hematopoietic cell lines. Pitx2c expression was not detected after 48 hours of in vitro cytokine stimulation of hematopoietic stem/progenitor cells. CONCLUSIONS: Pitx2c is expressed in hematopoietic stem/progenitor cells but not in their differentiated progeny. The pattern of expression of Pitx2c in primitive hematopoietic stem/progenitor cells suggests that it may play a role in hematopoietic stem-cell biology.

BXMAS1 identifies a cluster of homologous genes differentially expressed in B cells.

Characterization of genes activated by anti-IgM crosslinking of BL2 cells identified one gene, designated BXMAS1, that is predicted to be a novel cell surface receptor. The time course of activation indicates maximal transcriptional induction after 24 h. The predicted protein contains 977 aa residues, with a cytoplasmic domain containing 2 ITIM motifs. The ectodomain of the protein contains 6 repeats of characteristic 93 aa sequences which we have designated BXMAS1 domains. These domains correspond to 6 out of 8 Ig-like domains in BXMAS1. A search of the human genome revealed 5 additional closely linked homologous genes many of which contain BXMAS1 domains as well. Analysis of expression in cell lines and tissues suggests a general restriction of expression of these genes to B cells. These genes may be involved in B cell development and differentiation in peripheral lymphoid organs and may be useful markers of B cell stages.

Genomic and proteomic analysis of the myeloid differentiation program.

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)

RNA expression patterns change dramatically in human neutrophils exposed to bacteria.

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.

Werner protein recruits DNA polymerase delta to the nucleolus.

Werner syndrome is a Mendelian disorder of man that produces a number of manifestations resembling human aging. This disorder is caused by inactivation of the wrn gene, a member of the RecQ family of DNA helicases. The helicase and exonuclease activities of the Werner protein (WRN) suggest that it functions in DNA transactions, but the physiological function of WRN remains elusive. We present several lines of evidence that WRN interacts specifically with the p50 subunit of polymerase delta, the major DNA polymerase required for chromosomal DNA replication. P50, identified by yeast two-hybrid screening, interacts physically with the C terminus of WRN. Native WRN protein coimmunoprecipitates with p50 in a cellular fraction enriched in nucleolar proteins, and this immunocomplex also includes p125, the catalytic subunit of polymerase delta. In subcellular localization studies of cells transfected with WRN, p50 and p125 redistribute to the nucleolus and colocalize with WRN. These results suggest that one of the functions of WRN protein is to directly modify DNA replication via its interaction with p50 and abet dynamic relocalization of the DNA polymerase delta complexes within the nucleus.

Characterization of the promoter of human leukocyte-specific transcript 1. A small gene with a complex pattern of alternative transcripts.

The gene for the human leukocyte-specific transcript 1 (LST1) encodes a small protein that modulates immune responses and cellular morphogenesis. The LST1 transcripts are expressed at high levels in dendritic cells. Because of the complex splicing pattern, use of alternative 5'-untranslated exons, and a biologically interesting pattern of expression of LST1 mRNA, we studied the human LST1 gene promoter and regulatory elements. We identified an additional upstream 5'-untranslated exon in U937 monocytic cells. Transient transfection studies demonstrated that the combination of regions from -1363 to -621 with -112 to -54, relative to the translation start codon, produced the highest level of transcripts from among the various constructs tested, but the pattern of transcripts produced was only a subset of those produced from the endogenous gene. DNase I footprinting analysis and electrophoretic mobility shift assays showed that oligonucleotide probes corresponding to three regions, -1171 to -1142 (BI), -1136 to -1111 (BII), and -783 to -751 (BIV), bound proteins in U937 nuclear extracts. Competition and supershift electrophoretic mobility shift assay did not identify any known transcription factors responsible for BII probe binding. These studies suggest that a novel DNA-binding site and interaction of multiple regulatory elements may be involved in mediating the expression of the various forms of LST1 mRNA.

Expression and cellular localization of the protein encoded by the 1C7 gene: a recently described component of the MHC.

The major histocompatibility complex (MHC) is located on human Chromosome 6 and includes clusters of class I, class II, and class III genes. Centromeric to the class I region is a cluster of genes designated as MHC class IV encoding genes involved in immunity and inflammation, including the 1C7 gene. The human 1C7 gene has several alternatively spliced forms and potentially codes for proteins with at least three unique carboxy termini. 1C7 mRNA in human (h1C7) is present in spleen, tonsil, B and NK cell lines, and with a different splicing pattern in liver. The 1C7 RNA and protein are present at highest levels in the germinal center of the lymphoid follicles in tonsil. The protein is expressed in NKL cells, tonsil, and unexpectedly in brain. In contrast, the mouse 1C7 gene is transcribed in liver but is predicted to be a pseudogene. However, the 1C7 homologue expressed in rat is predicted to have long stretches of amino acids essentially identical to the human protein.

WRN or telomerase constructs reverse 4-nitroquinoline 1-oxide sensitivity in transformed Werner syndrome fibroblasts.

WRN encodes a RecQ helicase, which is mutated in Werner syndrome. Werner syndrome is a genetic condition of young adults characterized by premature aging, limited replicative capacity of cells in vitro, and increased cancer risk. Telomerase is a reverse transcriptase that extends the G-rich strand of telomeric DNA. Primary cells in vitro typically lack telomerase activity and undergo senescence, whereas telomerase is reactivated in many, but not all, tumors. The roles of the two genes are not known to be related. Here we report the development of an effective colony-forming assay in which a SV40-transformed Werner fibroblast cell line is 6-18-fold more sensitive to 4-nitroquinoline 1-oxide than SV40-transformed normal cell lines. The sensitivity can be partially reversed by transfecting a normal WRN gene but not a mutated WRN gene into the cells. Curiously, the sensitivity can be reversed equally well by transfecting a telomerase gene (TERT) into the cells. These data indicate the possibility of an interdependent function of these two genes.

Global analysis of neutrophil gene expression.

A widespread, but incorrect, view of the neutrophil portrays it as a short-lived, terminally differentiated cell that has a highly condensed nucleus and hence is unable to induce gene expression. However, these cells express mRNA encoding phagocytic receptors, modulate RNA synthesis in response to lectin stimulation or glucocorticoid treatment, and upregulate genes involved in phagocytic function, such as respiratory burst activity and cytokine secretion. Most studies of neutrophil gene expression have examined cytokine stimulation and have focused on a few specific genes of known interest, rather than the global genetic repertoire of the cell. In part stimulated by the availability of gene and expressed sequence tag databases, several approaches have been developed to assess the levels of all mRNA species found in single RNA preparations. We have analyzed the regulation of gene expression in neutrophils using a gel-based method that displays 3' end fragments of cDNA generated by restriction enzymes. Our data indicate that neutrophils are capable of extensive, rapid, and complex changes in gene expression, involving at least several percent of all mRNAs present in the cell. The number and magnitude of mRNA responses are comparable to those measured on activation of normal T cells. The data also indicate that activated neutrophils are a source of newly synthesized, physiologically significant, intercellular signaling molecules.

A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2.

Recently a number of nonclass I genes were discovered in the human MHC class I region. One of these, FAT10, encodes a protein consisting of two domains with homology to ubiquitin. FAT10 mRNA is expressed constitutively in some lymphoblastoid lines and dendritic cells and in certain other cells after gamma-interferon induction. FAT10 protein expression is controlled at several levels including transcription, translation, and protein stability. Yeast two-hybrid screening of a human lymphocyte library and immunoprecipitation studies revealed that FAT10 noncovalently associated with MAD2, a protein implicated in a cell-cycle checkpoint for spindle assembly during anaphase. Thus, FAT10 may modulate cell growth during B cell or dendritic cell development and activation.

Comparison of methods for identifying transcription units and transcription map of the Werner syndrome gene region.

To isolate a human disease gene by positional cloning, a critical step is the identification of candidate genes from a targeted genomic region. We used cDNA selection, exon trapping, and genomic sequencing to identify 12 transcription units from a 1.4-Mb genomic region containing the Werner syndrome gene (WRN). This included sequencing of 650 kb in the region of the WRN gene, to date, the most DNA sequenced as part of a positional cloning effort. The result of this combined method was significant overlap among the transcription units identified by each method; yet, no one method identified all of the transcription units. We present here a comparison of the effectiveness and efficiency of these methods and present a transcription map of the Werner syndrome gene region.

An expression map from human chromosome 14q24.3.

We have constructed an expression map of chromosome 14q24.3 between markers D14S42 and D14S63. cDNA selection with YACs from 14q24.3 was used to generate expressed sequence tags (ESTs). The localization of ESTs was confirmed on a YAC contig. PCR products of ESTs were used as probes to screen cDNA libraries leading to the isolation of transcripts for known and unknown genes. In total, the expression map contains 7 known genes previously mapped to 14q24.3, 6 cDNA transcripts, and 15 anonymous ESTs. The addition of 21 unique transcribed loci from an approximately 5- to 7-Mb region of chromosome 14q24.3 will facilitate future efforts to identify human disease genes from this region.

FGFR1 is fused with a novel zinc-finger gene, ZNF198, in the t(8;13) leukaemia/lymphoma syndrome.

Various histological subtypes of leukaemia and lymphoma are associated with diagnostic chromosome translocations, and substantial strides have been made in determining the specific oncogenes targetted by those translocations. We report the cloning of a novel fusion oncogene associated with a unique leukaemia/lymphoma syndrome. Patients afflicted with this syndrome present with lymphoblastic lymphoma and a myeloproliferative disorder, often accompanied by pronounced peripheral eosinophilia and/or prominent eosinophilic infiltrates in the affected bone marrow, which generally progress to full-blown acute myelogenous leukaemia within a year of diagnosis. A specific chromosome translocation, t(8;13)(p11;q11-12), is found in both lymphoma and myeloid leukaemia cells from these patients, supporting bi-lineage differentiation from a transformed stem cell. We find that the 8p11 translocation breakpoints, in each of four patients, interrupt intron 8 of the fibroblast growth factor receptor 1 gene (FGFR1). These translocations are associated with aberrant transcripts in which four predicted zinc-finger domains, contributed by a novel and widely expressed chromosome-13 gene (ZNF198), are fused to the FGFR1 tyrosine-kinase domain. Transient expression studies show that the ZNF198-FGFR1 fusion transcript directs the synthesis of an approximately 87-kD polypeptide, localizing predominantly to the cytoplasm. Our studies demonstrate an FGFR1 oncogenic role and suggest a tumorigenic mechanism in which ZNF198-FGFR1 activation results from ZNF198 zinc-finger-mediated homodimerization.