Regulation of ILT3 gene expression by processing of precursor transcripts in human endothelial cells.

Immunoglobulin-like transcript (ILT)-3 is a transmembrane receptor, which belongs to the immunoglobulin superfamily. In previous studies, we showed that allospecific CD8+CD28- T suppressor cells (Ts) induce the expression of ILT3 in human endothelial cells (EC) rendering them tolerogenic. Using a polymerase chain reaction (PCR)-based approach, we now demonstrate by cell fractionation and sequencing studies that ILT3 precursor RNA is expressed and retained in nuclei of resting EC. Ts interaction with EC or exposure of EC to interleukin-10 (IL-10) and interferon alpha (IFN-alpha) triggers processing of ILT3 pre-mRNA. Western blot analysis showed that the expression of the mature ILT3 transcript is accompanied by production of ILT3 protein.

An apparent interlocus gene conversion-like event at a putative tumor suppressor gene locus on human chromosome 6q27 in a Burkitt's lymphoma cell line.

A region of minimal deletion in B-cell non-Hodgkin's lymphoma (B-NHL) has recently been defined between D6S186 and D6S227 spanning 5-9 Mb at 6q26-q27, predicting the presence of at least one tumor suppressor gene (TSG) at this locus. During the construction of a deletion map in the B-NHL tumor panel, we report the identification of a Burkitt's lymphoma cell line, BL74, having an apparent homozygous deletion at the D6S347 locus, internal to the critical region. Since this case may facilitate the localization of the target TSG, a detailed structural molecular characterization and search for candidate genes were undertaken at this locus. While BL74 underwent a loss of heterozygosity at 6q26-q27, D6S347 was also likely subjected to a somatic interlocus gene conversion-like event between two homologous but distinct loci, resulting in the homozygous replacement of a 1860- to 2067-bp segment of one locus with the corresponding segment copied from the other locus. Two genes at this locus were identified, but their lack of expression in B-cell lineages tentatively excludes them as candidate TSGs. Another still unidentified gene at this locus may be disrupted by the gene conversion-like event, which would represent a novel mechanism of TSG inactivation.

Cloning and mapping of human chromosome 6q26-q27 deleted in B-cell non-Hodgkin lymphoma and multiple tumor types.

Frequent deletions of the distal region on the long arm of chromosome 6 have been reported in multiple human tumors including B-cell non-Hodgkin lymphoma (B-NHL), suggesting the presence of one or more tumor suppressor genes (TSGs) at this locus. Previously, we identified a region of minimal molecular deletion at 6q25-q27 (RMD-1) in B-NHL cases. To facilitate positional cloning efforts to identify the RMD-1 TSG(s), a yeast artificial chromosome (YAC) contig consisting of 110 clones was constructed across 6q26-q27 by sequence-tagged site/probe content mapping. The contig integrates 79 ordered markers including restriction fragment length polymorphisms, minisatellites, microsatellites, YAC-insert termini, expressed sequence tags, and known genes. It spans 34 cM and has a minimal tiling path of approximately 12 clones, covering an estimated 9-14 Mb with nearly every marker on the map showing at least double linkage to its adjacent markers. Dual-color fluorescence in situ hybridization of selected marker pairs on normal pachytene chromosome 6 further confirmed the YAC-based mappings. Utilizing a loss of constitutional heterozygosity assay in the B-NHL tumor panel, 24 additional 6q26-q27 polymorphic markers (21 mapping to the contig) further defined RMD-1 between markers D6S186 proximally and D6S227 distally. The minimal tiling path of the B-NHL RMD-1 consists of approximately 8 YAC clones, providing a size estimate of 5-9 Mb. This interval contains, in their entirety, several smaller candidate TSG critical regions previously delimited in other tumor systems. The AF-6 gene, mapping within RMD-1, revealed no mutations in a small subset of B-NHL. The deletion and physical maps presented herein provide a framework for the identification of the gene(s) involved in B-NHL as well as other malignancies and diseases mapped to this region and provide the initial reagents for large-scale genomic sequencing.

Analysis of a 69-kb contiguous genomic sequence at a putative tumor suppressor gene locus on human chromosome 6q27.

Multiple neoplasias including B-cell non-Hodgkin's lymphoma, breast carcinoma, and ovarian carcinoma, have been associated with frequent deletions of the distal region on the long arm of human chromosome 6, suggesting the presence of one or more tumor suppressor gene(s) at this locus. Loss of heterozygosity analysis of breast and ovarian tumors has further restricted the minimal region of loss within 6q27. To further characterize this genomic region for gene content including putative tumor suppressor genes as well as other elements that may contribute to tumorigenesis, a 68940-bp contiguous sequence, encompassing markers D6S193 and D6S297, was generated by random shotgun sequencing of a cosmid, P1, and PAC contig. In addition, exon trapping was performed utilizing a subset of these clones. Sixteen trapped exons, ranging in size from 44 to 399 bp, span this approximately 69-kb region. Many other putative exons have been identified computationally. Further analysis has identified 13 potential promoters and 13 putative polyadenylation sites in the region. Northern analysis identified a transcript mapping within this interval that is expressed in ovarian, breast, and lymphoid-derived tumor cell lines. Consideration of these data, together with the demonstration of several regions of high CpG content, suggests the possibility of several genes at this locus.

Post-transcriptional control regulates transforming growth factor alpha in the human carcinoma KB cell line.

Expression of epidermal growth factor receptor (EGF-R) antisense RNA results in a drastic reduction of EGF-R levels in the human carcinoma KB cell line and induces a reversion of their transformed phenotype (Moroni, M. C., Willingham, M. C., and Beguinot, L. (1992) J. Biol. Chem. 267, 2714-2722). We used parental and EGF-R antisense KB clones as a genetic system to study, in the same cell line, the role of transforming growth factor alpha (TGF-alpha) in the establishment and maintenance of the transformed phenotype. KB cells produce TGF-alpha mRNA, and their conditioned medium is able to sustain growth of antisense cells, mimicking the effect of exogenous EGF or TGF-alpha. In antisense cells there is a marked reduction of TGF-alpha mRNA steady-state levels. In addition, the decrease in TGF-alpha parallels the levels of residual EGF-R in the various antisense clones, indicating a direct correlation between receptors and growth factor levels. The addition of exogenous TGF-alpha (10 ng/ml) to antisense clones induces TGF-alpha levels. The half-life of TGF-alpha mRNA is 40-60 min in antisense cells and more than 8 h in parental KB cells, as determined by actinomycin D decay curves. This result indicates a predominant regulation of TGF-alpha mRNA at the post-transcriptional level. Nuclear run-on experiments show that there is only a marginal effect at the transcriptional level. We conclude that the autocrine loop responsible for the transformed phenotype of the human carcinoma KB cell line is dependent on both elevated levels of EGF-R and the presence of TGF-alpha. In addition, TGF-alpha is able to induce its own mRNA via a signal due to activation of the EGF-R acting predominantly at the post-transcriptional level.

Antisense epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells.

Human rhabdomyosarcoma cells express membrane epidermal growth factor receptor (ECF-R), which could confer responsiveness to EGF and transforming growth factor-alpha (TGF-alpha) of autocrine or paracrine origin. To study the role played by this growth factor circuit in the proliferation and differentiation of myogenic neoplastic cells, human rhabdomyosarcoma EGF-R-expressing cells (RD/18 clone) have been transfected with a plasmid containing a fragment of the EGF-R cDNA in the antisense orientation. In vitro growth and differentiative ability were studied on six antisense-transfected clones (AS) in comparison to parental RD/18 cells and to cells transfected with the plasmid containing only the neomycin resistance gene (NEO). A reduced EGF-R membrane expression was found in AS clones by decreased immunofluorescence with an anti-EGF-R monoclonal antibody. All AS transfectants had a greatly impaired proliferative ability, even when cultured in fetal bovine serum-containing medium. Proliferation of AS clones was completely blocked in medium supplemented with 2% horse serum. The differentiation ability of AS clones was heterogeneous, ranging from clones with a percentage of myosin-positive cells higher than controls to clones with a negligible myosin expression. Therefore, the growth impairment determined by the loop interruption is not sufficient to switch on the differentiation program. The role played by EGF-R in the proliferation of human rhabdomyosarcoma cells suggests that this receptor could constitute a target for a therapeutic approach.

Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974-containing internalization motif.

The carboxyl-terminal regulatory domain of the epidermal growth factor (EGF) receptor is essential for its endocytosis and interaction with the clathrin-associated protein complex AP-2. To identify AP-2 binding motif in the receptor, several single and multiple-point mutations within the region between residues 966 and 977 of the human EGF receptor were made, and the mutant receptors were expressed in NIH3T3 cells. Mutation of tyrosine 974 alone or together with surrounding residues and the deletion of residues 973-975 essentially eliminated AP-2 co-immunoprecipitation with the EGF receptor. Furthermore, a synthetic peptide corresponding to receptor residues 964-978 blocked AP-2 association with the wild-type EGF receptor. These data suggest that AP-2 has only one high-affinity binding site in the EGF receptor composed of Tyr974-containing motif. Receptor mutants that did not bind AP-2 displayed a lower rate of internalization, down-regulation, and turnover compared to wild-type receptors when expressed at high levels. However, similar receptor mutants expressed at low levels were internalized and down-regulated as efficiently as wild-type receptors. Internalization of the mutant receptors lacking the high-affinity binding site for AP-2 was inhibited by K+-depletion of the cells, indicating that their endocytosis required intact coated pits. We suggest that whereas one mechanism of EGF receptor recruitment into coated pits involves high-affinity binding of AP-2 to Tyr974-containing motif, another pathway may be mediated by weak receptor/AP-2 interactions or by proteins other than AP-2.

Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-beta 1 gene.

Transcription of the human gene encoding transforming growth factor beta 1 (TGF-beta 1), which is a key regulator of cell growth and differentiation, is inducible by phorbol esters. DNA sequences resembling phorbol ester response elements (TREs) are present upstream and downstream of this gene. TREs are recognized by proteins from the AP-1 family of transcription factors. We examined a 16 basepair (bp) sequence downstream of the TGF-beta 1 gene that contains three putative TREs. This sequence had been shown to stimulate reporter gene expression from a downstream location in response to phorbol ester treatment. Electrophoretic mobility shift assays suggest that minor proteins from the related AP-1 and CREB families of transcription factors bind to the overlapping TREs within the 16 bp element. A site beginning at the end of this 16 bp element matches the consensus sequence of a DNA-binding protein called MDBP and was shown to bind to this protein. When the intact MDBP site was present in a reporter gene construct in addition to the TREs, the phorbol ester-induced stimulation of reporter gene expression was no longer observed. This suggests that MDBP can counteract the stimulation of transcription by AP-1/CREB-like proteins binding to this downstream enhancer element.

Analysis of transforming growth factor beta 1 messenger RNA degradation by the transcript-selective, 12-O-tetradecanoylphorbol-13-acetate-regulated ribonuclease system from U937 promonocytes.

Transforming growth factor (TGF) beta 1 mRNA is selectively stabilized during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of U937 promonocytes. In previous studies (R. Wager and R. Assoian, Mol. Cell. Biol., 10: 5983-5990, 1990), we showed that this phenotype results from the action of a RNase system that (a) recognizes the transcript selectively and (b) is inhibited upon exposure of cells to TPA. The studies reported here were designed to localize domains of TGF-beta 1 mRNA required for recognition by this TPA-regulated, transcript-selective RNase system. By examining the degradation of several truncated TGF-beta 1 in vitro transcripts with U937 cell extracts, we show that the coding domain is sufficient to allow selective degradation of the mRNA and that this process is enhanced by either the 5' or 3' untranslated regions. The 5' and 3' untranslated regions of TGF-beta 1 mRNA are also required for TPA-mediated inhibition of the transcript-selective RNase system. In contrast, an analysis of the half-lives of the 2.1- and 1.8-kilobase TGF-beta 1 mRNAs showed that the first 270 bases, unique to the larger TGF-beta 1 mRNA, minimally affect degradation of the transcript. Finally, a survey of several transcripts showed that gamma-actin mRNA levels are also controlled by the TPA-regulated RNase system. The regulated decay of TGF-beta 1 mRNA may reflect the behavior of a class of transcripts subject to similar posttranscriptional controls on overall gene expression.

A GC-rich domain with bifunctional effects on mRNA and protein levels: implications for control of transforming growth factor beta 1 expression.

Chimeric plasmids containing selected reporter coding domains and portions of the transforming growth factor beta 1 (TGF-beta 1) 3' untranslated region (UTR) were prepared and used to identify potential mechanisms involved in regulating the biosynthesis of TGF-beta 1. Transient transfections with core and chimeric constructs containing the chloramphenicol acetyltransferase (CAT) reporter showed that steady-state CAT mRNA levels were decreased two- to threefold in response to the TGF-beta 1 3' UTR. Interestingly, CAT activity was somewhat increased in the same transfectants. Thus, production of CAT protein per unit of mRNA was stimulated by the TGF-beta 1 3' UTR (approximately fourfold in three cell lines of distinct lineage). The translation-stimulatory effect of the TGF-beta 1 3' UTR suggested by these studies in vivo was confirmed in vitro by cell-free translation of core and chimeric transcripts containing the growth hormone coding domain. These studies showed that production of growth hormone was stimulated threefold by the TGF-beta 1 3' UTR. A deletion analysis in vivo indicated that the GC-rich domain in the TGF-beta 1 3' UTR was responsible for both the decrease in mRNA levels and stimulation of CAT activity-mRNA. We conclude that this GC-rich domain can have a bifunctional effect on overall protein expression. Moreover, the notable absence of this GC-rich domain in TGF-beta 2, TGF-beta 3, TGF-beta 4, and TGF-beta 5 indicates that expression of distinct TGF-beta family members can be differentially controlled in cells.

Cell lineage specificity of expression of the murine transforming growth factor beta 3 and transforming growth factor beta 1 genes.

We have cloned a murine transforming growth factor (TGF) beta 3 complementary (cDNA) from normal tissue by low stringency screening of a testicular cDNA library with a TGF beta 1 probe. The coding domain of this TGF beta 3 cDNA agrees completely with the sequence reported for the TGF beta 3 cDNA isolated from the AKR-2B cell line, but the testicular clone uses a distinct and unusual polyadenylation signal resulting in an altered 3' untranslated domain. Northern blot hybridization analysis of gonadal tissues showed that both TGF beta 3 and TGF beta 1 mRNAs are detectable in the mouse testis and ovary. A detailed analysis of TGF beta 3 and TGF beta 1 gene expression in normal and germ cell-deficient male mice showed that the somatic cell compartment of the mouse testis expresses the usual-sized transcripts for both genes. However, a smaller (1.8-kilobase) TGF beta 1 mRNA is expressed selectively in male germ cells, and expression of this transcript was constitutive throughout the spermatogenic stages examined. This result demonstrates a new pattern of TGF beta 1 gene expression, consistent with cell lineage-specific transcriptional regulation during spermatogenesis.

Type beta 1 transforming growth factor gene expression. A corrected mRNA structure reveals a downstream phorbol ester responsive element in human cells.

A combined approach of cDNA cloning and direct oligonucleotide mapping of TGF-beta 1 mRNA from several human cell lines has revealed that the major human TGF-beta 1 transcript is 381 bases shorter than originally reported, and that the reduced mRNA size is due to polyadenylation from an ATTAAA signal at position 2136 rather than use of the expected AATAAA signal at position 2517. Moreover, there is no evidence for a significant amount of structural heterogeneity, as a result of alternative polyadenylation, in the human TGF-beta 1 transcripts. Considering that the 381-base domain is not part of the major human TGF-beta 1 mRNA, we analyzed this sequence for potential transcriptional regulatory elements. We have identified a 16-base pair domain which contains three putative phorbol ester responsive elements (TREs) based on homology to the TRE consensus sequence. We also show that this 16-base pair fragment confers phorbol ester responsiveness to the chloramphenicol acetyltransferase gene after transient transfection of the heterologous construct in NIH-3T3 cells. The identification of a TRE immediately downstream of the last TGF-beta 1 exon suggests that a 3' enhancer may play an important role in human TGF-beta 1 gene transcription, and suggests a basis for growth factor-mediated regulation of TGF-beta 1 expression by activation of protein kinase C.

Three new class I HLA alleles: structure of mRNAs and alternative mechanisms of processing.

Sixteen HLA class I clones have been isolated from a SV40-transformed human fibroblast line (GM637) cDNA library. The clones, characterized by hybridization to ABC locus-specific probes and sequence analysis, correspond to transcripts from four different class I genes: A2, A10, Cw4, and Cw6 (or Cw7), as implied by cell typing. Only the A2 sequence was known. The nucleotide and deduced amino acid sequence of the new alleles are reported here, and their structural features are discussed. Two independent cDNAs of A2 specificity display an unusual polyadenylation site located 100 bp upstream from the canonical one. Moreover, two cDNAs pertaining to the same C allele display two alternative mechanisms of splicing, which cause either presence or absence in mature transcripts of the transmembrane exon 5 sequence. Transcripts missing this region are predicted to synthesize a nonmembrane-bound, secreted antigen. A soluble protein, specifically reacting with class I-specific HLA antibodies, is found in the supernatant of the GM637 cells. The significance of HLA class I transcripts generated by differential processing is discussed.

Posttranscriptional control of human homeobox gene expression in induced NTERA-2 embryonal carcinoma cells.

We have studied the expression of four human homeobox genes representative of four different clusters (i.e., HOX-1, HOX-2, HOX-3 and HOX-5) in the embryonal carcinoma (EC) cell line NT2/D1. Following treatment with retinoic acid (RA), these cells differentiate into several cell types, including neurons, and steadily accumulate polyadenylated transcripts derived from the genes in a period ranging from 18 hr to 14 days of RA treatment. The sizes of major transcripts in differentiated EC cells coincide with those previously detected by the same probes in human embryos. Nuclear run-on transcriptional analysis showed no difference in the transcription rate of the four homeobox genes in differentiated vs. undifferentiated EC cells. Inhibition of protein synthesis by 5-18 hr of treatment of undifferentiated cells with cycloeximide causes accumulation of some homeobox transcripts at levels comparable to those observed after 18 hr of RA induction, although it does not cause superinduction in fully differentiated cells. These data suggest that the activation of homeobox gene expression in RA-induced EC cells is controlled, at least in part, by posttranscriptional mechanisms.

Molecular analysis of the heterogeneity region of the human ribosomal spacer.

The human ribosomal non-transcribed spacers are 30 X 10(3) base-pairs (or 30 kb) in length with a limited length heterogeneity localized in a specific region downstream from the 3' end of the transcribed region. Total DNA digested with EcoRI and BamHI and hybridized with a probe containing the 3' end of the 28 S ribosomal RNA coding region shows four major bands of 3.9 kb, 4.6 kb, 5.4 kb and 6.2 kb. The 5.4 kb band is the most abundant in every individual, followed by the 4.6 kb band. The longest and the shortest size classes are less well-represented and may even be absent. Every individual shows his own pattern of relative abundance of non-transcribed spacer length classes that can be followed through generations. We decided to investigate the molecular structure of the heterogeneity region, in order to cast light onto the mechanisms underlying the origin and maintenance of this length heterogeneity. Pertinent spacer regions of eight ribosomal clones from two human genomic libraries were subcloned and analyzed by restriction mapping and nucleotide sequencing. In the minimal length class, there is a sequence of 700 base-pairs that appears to be tandemly duplicated once, twice or three times in the other length classes. This repeated DNA module contains a region consisting of repetitions of simple pyrimidine groups like C-T, C-T-T-T or C-C-C-T. DNA module repeats may differ by the length of this pyrimidine-rich region. However, these length variations are not continuous, as revealed by Southern transfer analysis of several individuals and different cloned gene units: instead, the repeated modules fall into two discrete length classes of about 700 base-pairs and 800 base-pairs. An imperfect duplication of a short sequence of 86/89 base-pairs is present at the boundary between the heterogeneity region and the upstream flanking region, representing a very ancient duplication event.

Isolation of a series of HLA class I clones from a human chromosome 6 genomic library.

Human metaphase chromosomes were fractionated by a fluorescent-activated cell sorter (FACS II) and the chromosome 6 fraction was sorted. A genomic library was constructed in lambda gtWES cloning arms using a partial EcoRI digest of the chromosomal DNA. We estimate that at least 60% of our library represents chromosome 6 material, and as 1.2 x 10(5) recombinants were obtained, this indicates that the majority of clonable chromosome 6 sequences are represented. The library was screened with a mouse H-2 class I clone and 18 HLA class I recombinants were isolated from 4 x 10(4) plaques.

Purification and characterization of myosin from calf brain.

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.