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We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human beta-globin locus as a model. Full transcriptional activity of the human beta-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous beta-globin locus are dissociable and dependent on distinct cis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed beta-globin-promoter and gene. On the basis of these results, we suggest a multistep model for gene activation; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetylation is required for high-level transcription of the beta-globin genes.

We demonstrated previously that chicken embryo fibroblasts accumulate approximately 100 copies of embryonic globin RNA after transformation by Rous sarcoma virus. Here we demonstrate that the globin gene in chicken embryo fibroblasts is activated by infection with two other oncogenic retroviruses, avian erythroblastosis virus and strain MC-29 of avian myeloblastosis virus, which contain transforming genes unrelated in nucleotide sequence content to each other or to the Rous sarcoma virus src gene. In addition, we have measured the genetic complexity of transformation by using established techniques for determining the number of different RNA sequences in specific populations of cells. Our results indicate that transformation of chicken embryo fibroblasts by Rous sarcoma virus results in the accumulation of RNA from approximately 1000 average-sized new transcription units.

The timing of DNA replication of the human beta-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span greater than 120 kilobases across the human beta-globin gene locus, we show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-gamma-globin gene region and approximately 20 kilobases 5' to the epsilon-globin gene and 20 kilobases 3' to the beta-globin gene, replicate later and throughout S phase. A similar area is also present in the alpha-globin gene region in K562 cells. We suggest that these regions may represent sites of termination of replication forks.

Serum amyloid A (SAA) proteins are secreted by mammalian liver in response to inflammatory stimuli. Both transcriptional and posttranscriptional mechanisms have been shown to regulate the 2,000-fold increase in SAA mRNA after injection of endotoxin into mice. We report here the characterization of a cell line derived from mouse liver (BNL) in which the expression of SAA3 mRNA is regulated. In this model, SAA3 mRNA accumulated in response to conditioned medium from the mouse macrophage P388D1 cell line with kinetics similar to that seen in mouse liver (C. A. Lowell, R. S. Stearman, and J. F. Morrow, J. Biol. Chem. 261:8453-8461, 1986). In in vitro nuclear transcription assays, the SAA3 gene was transcribed equally in induced and uninduced cells. In addition, in steady-state RNA studies treatment with conditioned medium allowed the cells to rapidly accumulate SAA3 mRNA without an apparent change in half-life. These observations suggest that conditioned medium contains a factor(s) that acts directly on hepatocytes to regulate SAA3 mRNA processing.

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.

A new promoter of the human c-myc gene called P0, with multiple RNA start sites, was mapped over 500 bases upstream of the two previously identified promoters, P1 and P2. Sequencing full-length cDNA clones of P0 RNAs revealed two open reading frames upstream of that for the P64c-myc protein. P0 RNA is located on polyribosomes and released by puromycin, indicating that it functions as an mRNA. In vitro translation of RNA synthesized from the cloned cDNAs predicts that P0 transcripts are translated into a novel 12.5-kilodalton protein corresponding to the first open reading frame. The regulation of P0 RNA was studied in the B-cell lymphoma cell line Manca, in which only the translocated c-myc allele lacking exon 1 was thought to be active. However, we found that P0 transcription and the DNase I-hypersensitive site associated with this promoter persist on the untranslocated allele, even though P1/P2 transcription as measured by a nuclear runoff assay was repressed. These results suggest that allelic exclusion of c-myc expression in this B-cell lymphoma is caused by a repression of transcription which is specific to the P1/P2 promoters. We previously reported a block to elongation of transcription near the 3' end of exon 1 in the wild-type c-myc gene, which results in an excess of exon 1 over exon 2 transcription (5a). In contrast, we found that in the Daudi B-cell lymphoma, which retains exon 1 in the active allele, equimolar transcription of exons 1 and 2 occurs. This result suggests a model for the activation of c-myc in B-cell lymphomas.

Several aspects of the structure of the chicken thymidine kinase gene (tk) have been resolved as a result of genetic experiments and nucleotide sequencing. Deletion mapping established the locations of two functional boundaries in a region thought to correspond to the 5' terminus of the gene. One such boundary coincides with a transcriptional promoter, and the other coincides with the translation start codon of the chicken tk polypeptide. Similar deletion mapping assays identified a functional boundary at the 3' terminus of the gene. DNA sequence analysis confirms the prediction that this 3' region encodes the carboxyl terminus of the tk polypeptide. A recombinant cDNA clone complementary to genomic tk sequences was isolated. A comparison between genomic and cDNA sequences reveals the locations of six intervening sequences and allows prediction of the complete amino acid sequence of the chicken tk polypeptide.

Avian leukosis virus (ALV) induces bursal lymphomas in chickens, after proviral integration next to the cellular myc proto-oncogene, and subsequent c-myc hyperexpression. Our previous work suggested that labile or short-lived cellular proteins interact with the viral long terminal repeat (LTR) enhancer, and binding of these proteins appeared to be essential for high rates of LTR-enhanced transcription (A. Ruddell, M. Linial, W. Schubach, and M. Groudine, J. Virol. 62:2728-2735, 1988). This lability is specific for B-lymphoid cell types, since T cells and fibroblasts show stable high rates of LTR-enhanced transcription and stable LTR-binding activity. Moreover, the lability of these proteins may be important in determining susceptibility to bursal lymphoma. In this study, we separated and characterized the labile and stable LTR-binding proteins and examined their lability and expression in different cell types. Gel shift and DNase I footprinting analyses indicated that at least five proteins interact with the 140-base-pair LTR enhancer region. These proteins were distinct by several criteria, including lability or stability after inhibition of protein synthesis, resistance to heat denaturation, chromatographic behavior, and expression in different cell types. Two binding proteins were present in many cell types and were specifically labile in B cells. A third binding protein showed hematopoietic-cell-type-specific expression and was also labile in B cells. These findings indicate that there is tissue-specific modulation of the lability and expression of ALV LTR-binding proteins, which may be important for regulation of LTR transcription enhancement and ALV bursal lymphomagenesis.

We have isolated and characterized a novel member (CAT3) of the cationic amino acid transporter (CAT) family. In oocyte injection assays, CAT3 cRNA exhibited a saturable, sodium ion-independent transport activity with high affinity for L-arginine and L-lysine (Km = 40-60 and 115-165 microM, respectively). Transport of L-arginine was effectively competed only by cationic amino acids in L-form: arginine, lysine, ornithine, and 2,4-diamino-n-butyric acid but not by 2,3-diaminopropionic acid. The presence of L-arginine in the incubation medium stimulated the efflux rate of L-arginine, indicating that CAT3 is subject to trans-stimulation. All these results are consistent with the idea that CAT3, along with CAT1 and CAT2, constitutes the transport activity originally assigned to system y+. Like CAT2, but unlike CAT1, the expression of CAT3 is regulated in a highly tissue-specific manner; when various adult tissues were examined, significant levels of CAT3 transcript were detectable only in brain. In situ hybridization on brain sections revealed that CAT3 transcripts were localized predominantly along the midbrain-thalamus-hypothalamus axis, whereas neither CAT1 nor CAT2 transcripts demonstrated a similar localization. In contrast to its highly localized expression during the primitive streak stage and in the adult stage, CAT3 expression was detected more widely in 13.5 day post-coitum mouse embryos.

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

The chicken c-myc gene, as defined by its homology to the v-myc gene of MC29 virus, is comprised of two exons. Using the techniques of runoff transcription, primer extension, and S1 nuclease protection, we demonstrate that there is a third c-myc exon of approximately equal to 345 base pairs (bp) located 0.7 kbp upstream of the 5' end of the v-myc homology. This first exon is transcribed and present in myc mRNA in normal chicken cells. We also examined RNA from five cell lines derived from avian leukosis virus-induced bursal lymphomas. In all these lines, the level of transcription of the 2.2- to 2.5-kbp myc mRNA is increased 30- to 60-fold over normal cells. The myc mRNA in four of these lines also contains increased levels of the first noncoding exon, and evidence is presented that the long terminal repeat (LTR) in the vicinity of c-myc is functioning as an enhancer of c-myc transcription rather than as a promoter in several of these cell lines. In two cell lines in which the viral LTR has integrated between the first and second exons in the proper orientation for downstream promotion of myc, the LTR does not exhibit promoter function. The pattern of c-myc transcription observed by others in a vast majority of avian leukosis virus-induced neoplasms is not observed in any of the five cell lines examined.

Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.

The DNase I-hypersensitive sites in the human embryonic beta-globin gene region have been mapped in erythroid-enriched fractions of disaggregated fetal livers, in adult nucleated red blood cells, and in fetal brain tissue. Our analysis of a region extending 11 kilobases (kb) 5' of the epsilon-globin gene reveals many minor nuclease-hypersensitive sites and one major site located 6.1 kb upstream of the epsilon-globin gene. All of these hypersensitive sites are erythroid-specific, and the major site is stable throughout erythroid development. As assayed by nuclear runoff transcription, little or no epsilon-globin gene expression is detectable in fetal or adult erythroid cells. Thus, the presence of the major hypersensitive site 5' of the epsilon-globin gene in both fetal and adult erythroid cells demonstrates that this site is not specifically correlated with transcription of the gene or with a particular stage of development. Rather, this site may reflect an early event in erythroid differentiation. In addition, DNase I has been used to probe the overall sensitivity of epsilon-globin chromatin in fetal erythroid cells. Our findings indicate that the epsilon-globin gene as well as the other genes in the beta-globin cluster reside within the chromatin domain that is more DNase I-sensitive than "bulk" chromatin.

Fetal hemoglobin production can be reactivated in vivo in adult persons with various hemoglobinopathies and other hemopoietic disorders, and in cultures of adult erythroid progenitors. We show that the activation of Hb F in adult cells is transcriptional in nature and is accompanied by the appearance of DNase I-hypersensitive sites and undermethylation of Hpa II sites 5' to the gamma-globin genes. Production of Hb F in culture is strongly modulated by the environment, and the repression of Hb F synthesis by specific culture conditions has been reported. By nuclear runoff, chromatin, and methylation analyses, we show that this inhibition of Hb F production in vitro is at the level of transcription with the concomitant loss of characteristic gamma hypersensitive sites and methylation of gamma Hpa II sites. These data indicate, first, that the organization of globin chromatin of adult cells that produce fetal hemoglobin resembles that of fetal erythroid cells and, second, that this organization switches from a fetal to an adult pattern in response to changes in the environment of the erythroid cells.

The translocation of the c-abl oncogene from chromosome 9 to the bcr gene on chromosome 22 in cases of Philadelphia chromosome-positive chronic myelogenous leukemia (CML) generates an aberrant bcr-abl fusion transcript which may be intimately related to the pathogenesis of CML. Because factors controlling normal bcr expression might also be involved in the expression of this aberrant bcr-abl transcript, we studied the patterns of expression of the normal bcr gene in different cell types. We found that the normal bcr gene was expressed in many different types of human cells. Moreover, the bcr gene was evolutionarily conserved, and homologous bcr genomic sequences and RNA transcripts were readily detected in chick tissue. The highest level of bcr expression in chick tissue was in brain tissue, the lowest level was in liver tissue, and a truncated bcr mRNA was noted in chick testes. Normal bcr transcripts, in addition to the aberrant bcr-abl hybrid transcripts, have been found in all Philadelphia chromosome-positive CML cells studied to date. Within a given CML sample, the relative amounts of normal bcr RNA and aberrant bcr-abl RNA were similar. In addition, the normal bcr and the aberrant bcr-abl hybrid transcripts demonstrated similarly prolonged half-lives compared with that of the normal abl-related transcripts in CML cells. These findings suggest that in CML cells, similar cellular mechanisms control the steady-state levels of both the normal bcr and the bcr-abl fusion RNAs.

Human teratocarcinoma cells were used to examine structural features associated with expression of the major immediate-early (IE) gene of human cytomegalovirus. By immunofluorescence, comparison of RNA levels, and in vitro transcription of nuclei, we showed that the major IE gene is inactive in undifferentiated but active in differentiated cells. Therefore, the block in human cytomegalovirus replication in teratocarcinoma cells appears to be at the transcriptional level, in one of the initial genes transcribed. In addition, the in vitro transcription experiments demonstrated that in permissive infections the gene was transcriptionally inactive late in infection. A comparison of the structural features of the promoter region with the active and inactive IE genes showed the presence of constitutive and inducible DNase I-hypersensitive sites. The majority of the constitutive sites existed at -175, -275, -375, -425, and -525 relative to the cap site in an area which has been shown to be capable of simian virus 40 enhancer function. In contrast, the inducible DNase I sites were located outside this region at -650, -775, -875, and -975.