Nuclease sensitivity of the human growth hormone-chorionic somatomammotropin locus in pituitary and placenta suggest different mechanisms for tissue-specific regulation.

The five human growth hormone (GH) and chorionic somatomammotropin (CS) genes are located at a single locus on chromosome 17. These genes share extensive nucleotide sequence similarity (approximately 94%) even in their flanking DNA, yet GH-N is expressed efficiently in the pituitary under the control of the pituitary-specific factor GHF-1/Pit-1 and the remaining CS-A, CS-B, CS-L and GH-V genes are transcriptionally active in the placenta. Despite this specificity in vivo, a truncated CS-A promoter can bind GHF-1/Pit-1 and allow CS-A promoter activity in pituitary cells in vitro. With a view to assessing whether the placental genes of the GH/CS locus possess a different chromatin structure in the pituitary and are, thus, less transcriptionally active than the GH-N gene, we have compared the DNAase I sensitivity of GH/CS in isolated pituitary and placenta cell nuclei. Our data indicate that these genes are equally sensitive in isolated human pituitary nuclei. By contrast, the CS-A, CS-B and CS-L genes were significantly (P < 0.05) more sensitive than the GH-N gene in isolated human placenta nuclei. Although just not significant, the GH-V gene was slightly more sensitive than the GH-N gene. This pattern was also seen with nuclei from human choriocarcinoma BeWo and JEG-3 cells, which express low and extremely low levels of CS RNA, respectively, but was distinct from the pattern observed in the non placental human cervical carcinoma HeLa cell line. These data indicate that the inactivity of the CS genes in the pituitary does not correlate with a 'closed' chromatin structure. However, they are consistent with a role for a more 'open' chromatin conformation in placenta-specific expression, but not necessarily high levels of transcriptional activity.

Pituitary-specific repression of placental members of the human growth hormone gene family. A possible mechanism for locus regulation.

Five members of the human growth hormone (GH) gene family are located at a single locus on chromosome 17. Growth hormone is expressed in the pituitary under the control of the tissue-specific factor Pit 1/GHF-1, and chorionic somatomammotropin (CS) -A, -B, and -L, as well as placental GH variant, are expressed specifically in the placental syncytiotrophoblast. Despite this specificity in vivo, the CS-A promoter can bind Pit 1/GHF-1 and allow CS-A promoter activity in pituitary tumor cells after gene transfer. We have identified and characterized PSF sequences associated with only the placental members in the GH/CS locus which repress placental promoter activity > 90% in transfected pituitary cells. These sequences do not significantly affect promoter function in placental cells after gene transfer. Repressor activity correlates with binding of protein at two sites (PSF-A and PSF-B) with pituitary, but not placental, nuclear extracts. Competition studies suggest an interaction between PSF and Pit 1/GHF-1 proteins. These results indicate that PSF protein can repress CS-A promoter activity in a tissue-specific manner in vitro and provide a possible mechanism by which expression of placental members of the GH family are inhibited in the pituitary in vivo.

Differential expression of human placental growth hormone variant and chorionic somatomammotropin genes in choriocarcinoma cells treated with methotrexate.

Chorionic somatomammotropin (hCS) genes (hCS-A and hCS-B) and the placental growth hormone variant (hGH-V) gene are expressed in the syncytiotrophoblast in vivo, and at low levels in cytotrophoblast-like choriocarcinoma (BeWo) cells. Treatment of choriocarcinoma cells with methotrexate (MTX) will induce a cell type intermediate between a cytotrophoblast and syncytiotrophoblast. After treatment with MTX, hCS/hGH-V mRNA levels were decreased in BeWo cells, and only hGH-V and minor hCS-A related transcripts of 1.6, 2.1 and 4.2 kilobases, termed hCS-A2, hCS-A3 and hCS-A4, respectively, were detected. By contrast, chorionic gonadotropin RNA levels were increased. This pattern of hCS/hGH-V expression resembles that observed when BeWo cells are grown in thyroid hormone (T3)-depleted serum, where hGH-V/hCS RNA increases in response to T3. This increase is blunted by MTX treatment, but is not due to a decrease in number or affinity of T3 receptors. These data indicate that the hGH-V and hCS genes can be differentially regulated by MTX, and are consistent with MTX interfering with T3 responsiveness of these genes. Also, if BeWo cells treated with MTX do represent a transitional state, these data raise the possibility that hGH-V and hCS possess a different temporal pattern of expression in the developing trophoblast.

Differential binding of rat pituitary-specific nuclear factors to the 5'-flanking region of pituitary and placental members of the human growth hormone gene family.

Placental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5'-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (-140/-107) and proximal site (-97/-66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (-140/-116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific expression and thyroid hormone regulation of the endogenous placental growth hormone variant and chorionic somatomammotropin genes in a human choriocarcinoma cell line.

Human (h) placenta-derived choriocarcinoma cell lines (BeWo, JAR, and JEG-3) were examined for expression of pituitary GH (hGH-N) as well as placental GH variant (hGH-V) and chorionic somatomammotropin (hCS, encoded by the hCS-A or hCS-B gene). RNA was isolated and assessed using hGH-N complementary DNA since hGH and hCS genes share more than 90% sequences similarity. The relative expression is BeWo greater than JAR greater than JEG-3. In BeWo cells expression of placental hCS-A, hCS-B, and hGH-V genes, but not pituitary hGH-N, is observed using polyadenylated RNA and oligonucleotide probes specific for the different family members. The absence of hGH-N expression in BeWo cells is not due to deletion or gross rearrangement of the gene. No difference was seen between the hGH/hCS genes in genomic DNA from these cells and the DNA from placenta and pituitary when analyzed by restriction digestion and blotting. Treatment of BeWo cells with 10 nM T3 results in a 6-fold increase in messenger RNA from placental members of the hGH gene family. Levels of hCS-A, hCS-B, and hGH-V transcripts are all elevated. Cellular and secreted proteins from BeWo cells were analyzed by Western blotting, and a band of about 22 kilodaltons was detected using a polyclonal antibody which cross-reacts with hGH-V and hCS. The level of 22 kilodalton band in samples of cellular as well as released protein was increased by T3 treatment. BeWo cells provide a model system for studying hGH-V and hCS regulation as well as tissue-specific expression.

Differential expression of human placental growth-hormone variant and chorionic somatomammotropin in culture.

Regulation of human placental growth-hormone variant (hGH-V) in the presence of its own promoter has been studied. At term, 10-20% of placental mRNA is specific for chorionic somatomammotropin (hCS-A and -B) compared with 0.05% hGH-V, yet these genes show more than 90% sequence similarity at the nucleotide level. By using stable gene transfer of intact hGH-V and hCS-A genes into rat pituitary (GC) cells, synthesis and release of hGH-V and hCS are detected. This suggests that hGH-V as well as hCS is secreted during pregnancy. The hCS-A mRNA level is higher than that observed from the hGH-V gene in stably transfected GC cells. Also, a hybrid gene containing hGH-V 5'-flanking DNA was less active than a hybrid hCS-A gene containing equivalent sequences after transient transfection of these cells. This correlates with the binding of a known transcription factor to a proximal region (-97/-66) of the hCS-A promoter, and not the equivalent hGH-V gene sequences. These results indicate that differential expression of hGH-V and hCS in GC cells is related, in part, to the strength of their respective promoters, and suggest a similar mechanism may exist in the placenta.

Human growth hormone gene expression in rat but not human non-pituitary cells after stable gene transfer.

Tissue-specific expression of the rat growth hormone (rGH) gene requires binding of a pituitary-specific factor. Binding of this factor has been used to explain tissue-specific expression of the human growth hormone (hGH-N) gene in transfected rat pituitary (GC) tumour cells. Neither rat fibroblast (R2) nor human cervical carcinoma (HeLa) cells contain the rat pituitary-specific factor. Thus, no expression of hGH-N or rGH would be expected in these cells. R2 cell lines containing stably integrated hGH-N or rGH genes were generated. Expression of hGH-N but not rGH was detected. By contrast, stably transfected HeLa cells did not express the endogenous or transfected hGH-N genes. However, an hGH-N transcript was detected when hGH-N gene expression was directed by a viral promoter. This suggests that the block in expression occurs at the level of transcription and not mRNA stability. Hybrid genes containing 496 base pairs (bp) of hGH-N or 234 bp of rGH 5'-flanking DNA, including promoter sequences, fused to the bacterial gene coding for chloramphenicol acetyltransferase were used to stably transfect R2 cells. The hybrid hGH-N gene was more active than a promoterless construction in these cells. By contrast, the hybrid rGH gene was not. These data suggest that the hGH-N gene can be activated by rat transcription factors other than those found in pituitary cells.

The human placental growth hormone variant is mitogenic for rat lymphoma Nb2 cells.

Although there is evidence that human (h) placental GH variant (hGH-V) possesses a growth-promoting function, lactogenic activity by the hormone has not been demonstrated. Rat anterior pituitary tumor (GC) cells stably transfected with the hGH-V gene (GC [hGH-V] cells) synthesize and secrete hGH-V. This hormone shares considerable structural similarity with pituitary growth hormone (hGH-N) and chorionic somatomammotropin (hCS) at the nucleotide (greater than 90%) and amino acid (greater than 80%) levels. As expected, both hGH-N and hCS antibodies detect hGH-V by immunoblotting. However, hGH-V, but not hGH-N or hCS, cross-reacts with human or rat pituitary prolactin (PRL) antibodies. These data indicate that structural features shared by hGH-V and pituitary PRL are not present in hGH-N or hCS. Comparison of amino acid sequences implicates two regions that may account for a common epitope between hGH-V and hPRL, and structural difference from hGH-N and hCS. The possible lactogenic activity by hGH-V was assessed in a rat lymphoma Nb2 cell bioassay. Conditioned medium from GC[hGH-V] cells permitted growth of lactogen-dependent Nb2 lymphoma cells in culture. This activity was blocked by antibodies raised to rat PRL but not hPRL or hGH-N. Comparison of the hGH-V amino acid sequence with those from 14 other lactogenic hormones, including hPRL, hCS and hGH-N, reveals 6 conserved amino acids. These data indicate a lactogenic as well as growth-promoting function for the secreted hGH-V protein in vivo.

Human chorionic somatomammotropin and growth hormone gene expression in rat pituitary tumour cells is dependent on proximal promoter sequences.

Human placental chorionic somatomammotropin (hCS-A or hCS-B) and pituitary growth hormone (hGH-N) are related by structure and function. The hCS-A gene is expressed in rat pituitary tumour (GC) cells after gene transfer. Deletion of hCS-A 5'-flanking DNA reveals repressor activity upstream of nucleotide -132, and a region essential for expression in GC cells between nucleotides -94 and -61. The sequences in this region differ from the equivalent hGH-N gene DNA by one nucleotide, and include the binding site (-92 to -65) for a pituitary-specific factor (GHF-1), required for hGH-N expression in GC cells. Exchange of hGH-N with hCS-A gene DNA in this region maintains expression in GC cells. By contrast, modification of these sequences blocks expression. These data indicate that proximal promoter sequences, equivalent to those bound by GHF-1 on the hGH-N gene, are required for hCS-A expression in GC cells.

Structure of polyubiquitinated histone H2A.

We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.

Ubiquitinated histone H2B is preferentially located in transcriptionally active chromatin.

Using an anti-ubiquitin antibody in Western blotting experiments, we detected polyubiquitinated species of histones H2A, H2A.Z, and H2B in histone preparations of bovine thymus, chicken erythrocyte, and Tetrahymena macro- and micronuclei. Histone H2A had the greatest level of polyubiquitinated species, with tetra- to hexaubiquitinated forms of this histone being observed. The fraction of bovine thymus and chicken erythrocyte chromatin enriched in transcriptionally active gene sequences was enriched in mono- and polyubiquitinated species of histones H2A, H2B, and H2A.Z, especially in the ubiquitinated forms of histone H2B. Histones H2A and H2B were ubiquitinated in the transcriptionally active Tetrahymena macronucleus, with monoubiquitinated (u) H2B being the predominant ubiquitinated histone species. Ubiquitinated forms of histones H2A and H2B were found in transcriptionally inert micronuclei, but at lower levels than seen in macronuclear histones. Also, the level of micronuclear uH2A was greater than that of uH2B which may be from macronuclei that contaminate the preparation. These results indicate that the mono- and polyubiquitinated species of histone H2B are preferentially located in transcriptionally active chromatin regions. Ubiquitinated histone H2A is located in both expressed and repressed chromatin domains, but expressed chromatin is enriched in mono- and polyubiquitinated forms of this histone. These observations are consistent with the hypothesis that ubiquitinated histones have a role maintaining the structure of transcriptionally active chromatin.

The protamine gene chromatin in developing trout testis exists in an altered state.

Micrococcal nuclease was used to probe the nucleosomal organization of the rainbow trout germ-line-specific protamine multi-gene family in testis and erythrocytes. In erythrocyte chromatin, the repressed protamine genes show a distinct nucleosomal repeat pattern. However, in early-stage testis chromatin, where the protamine genes are expressed, they lack a distinct nucleosomal repeat pattern, indicating that the disrupted chromatin structure is related to their transcriptional activity. Micrococcal nuclease-digested testis and erythrocyte chromatin was separated into soluble and insoluble fractions. Transcriptionally active/competent genes of testis that had been labeled by nuclear nick-translation were enriched in the low-salt eluted, micrococcal nuclease-sensitive chromatin fraction. This fraction was not enriched in protamine DNA sequences. In testis, but not erythrocytes, protamine DNA sequences were slightly enriched in chromatin that fractionated with insoluble nuclear material, suggesting that transcriptionally active protamine gene chromatin has an insoluble character. Since the different protamine genes may not be simultaneously expressed, our results show the distribution of both transcriptionally active and inactive protamine genes. However, our observations indicate that the active germ-line-specific protamine gene chromatin shares several, but not all, of the features associated with other active tissue-specific genes.

Reduced levels of histones H1o and H1b, and unaltered content of methylated DNA in rainbow trout hepatocellular carcinoma chromatin.

The levels of histone subtypes and DNA methylation of aflatoxin-induced rainbow trout hepatocellular carcinoma and adult liver nuclei were compared. The hepatocellular carcinoma nuclei were enriched in the ubiquitinated species of histone H2A and depleted in histones H1o and H1b. The 5-methylcytosine content and methylation patterns of the vitellogenin genes and the transcriptionally inactive TPG-3 protamine gene were not altered in the trout hepatocellular carcinoma DNA. Thus, undermethylation of DNA is not a general feature of chemically induced tumors in vivo.

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions.

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis.

The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.

Brief clinical report: ring chromosome 17 in a mentally retarded young man - clinical, cytogenetic, and biochemical investigations.

A young, mentally retarded man with seizures was discovered to have a ring chromosome 17. He had no major anomalies. The phenotype associated with r(17) probably is variable. The patient's deletion and genotype allowed us to reduce further the chromosome location of the acid alpha-glucosidase gene.

Extension of human acid alpha-glucosidase polymorphism by isoelectric focusing in polyacrylamide gel.

1. A method to analyse acid alpha-glucosidase (GAA) activity in human tissues by flatbed polyacrylamide gel isoelectric focusing has been devised. 2. With this method the GAA polymorphism has been extended from three to six commonly occurring phenotypes. 3. After isoelectric focusing the GAA2 isozyme migrated cathodally with respect to the GAA1 isozyme, whereas with the previous starch-gel electrophoresis method their relative positions were reversed. 4. The six common phenotypes are generated by three alleles, GAA*1, GAA*2 and GAA*4 with frequencies of 0.91, 0.03 and 0.06, respectively.