The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells.

Studies in many systems have led to the model that the human beta-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the beta-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous beta-globin locus in an erythroid background, transcription of all beta-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the beta-globin locus are not affected by the deletion. In contrast, deletion of 5'HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on beta-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an "open" conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the beta-like globin genes.

Effects of butyrate and glucocorticoids on gamma- to beta-globin gene switching in somatic cell hybrids.

Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that alpha-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the gamma- to beta-globin gene (gamma-to-beta) switch in the HFE x MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on gamma-globin expression is due to inhibition of gamma gene silencing rather than to induction of gamma gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the gamma-to-beta switch in HFE x MEL hybrids was examined. Dexamethasone strikingly accelerated the gamma-to-beta switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the gamma-to-beta switch of the HFE x MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.

Coexpression of gamma and beta globin mRNA in cells containing a single human beta globin locus: results from studies using single-cell reverse transcription polymerase chain reaction.

We developed a method detecting globin gene expression in single cells using reverse transcription polymerase chain reaction. epsilon and gamma globin cDNAs are coamplified by an epsilon gamma primer set whereas gamma and beta globin cDNAs are coamplified by a gamma beta primer set and the individual globin cDNAs are distinguished by restriction enzyme digestion. Analysis of RNA preparations from human fetal liver, neonatal red blood cells (RBCs), or adult RBCs showed the expected mRNA species for each stage of human development. Analysis of single cells from a human erythroleukemia line coexpressing gamma and beta globin chains showed heterogeneity in gamma and beta mRNA contents. The method was subsequently used to test whether only one or more than one globin genes are expressed in cells that contain a single human beta globin locus. We found that about 50% of single cells from MEL x fetal erythroid cell hybrids containing a single human beta globin locus coexpressed gamma and beta globin mRNA. This finding is best explained by assuming that both gamma and beta genes are simultaneously transcribed from the same beta globin locus implying that the LCR can simultaneously interact with more than one globin gene promoter.

Use of yeast artificial chromosomes (YACs) for studying control of gene expression: correct regulation of the genes of a human beta-globin locus YAC following transfer to mouse erythroleukemia cell lines.

We demonstrate that transfer of a yeast artificial chromosome (YAC) containing 230 kb of the human beta-globin locus into mouse erythroleukemia cells by fusion results in correct developmental regulation of the human beta-like globin genes. Additionally, we show that early after hybrid formation, human embryonic epsilon- and fetal gamma-globin genes are coexpressed with the adult beta gene but that after 10-20 weeks in culture, globin gene expression switches to predominantly adult. Thus, in contrast to shorter gene constructs, the globin genes of the beta-globin locus YAC are regulated like the chromosomal globin genes. These results indicate that transfer of YACs into established cell lines can be used for the analysis of the developmental control of multigenic and developmentally regulated human loci.

Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.

Survival curves, reproductive life span and age-related pathology of Mus caroli.

Although Mus caroli is being used in a number of laboratories as an experimental animal, basic information concerning its life span, reproductive ability, and age-related pathologies has been unavailable. Here we present this basic information, and discuss the similarities to and differences from the laboratory mouse, Mus musculus domesticus [strains A/StTrWo and (A/StTrWo x C57BL/6NNia)F1] and, from published data, wild-type Mus musculus.

Murine erythroleukemia cell line GM979 contains factors that can activate silent chromosomal human gamma-globin genes.

We introduced a normal chromosome 11 into GM979 murine erythroleukemia cells by fusing them with Epstein-Barr virus-transformed lymphocytes from a normal individual. In contrast to previous data obtained with other murine erythroleukemia cells, we detected activation of human chromosomal gamma-globin genes in GM979 cells. GM979, unlike previously used murine erythroleukemia cell lines, expresses murine embryonic globin in addition to adult globin. While all the hybrids expressed gamma- and beta-globin, they displayed a wide range of gamma-globin expression in relation to that of beta-globin. No correlation, however, was found in quantitative expression between murine embryonic globin and human gamma-globin in these hybrids, suggesting that the two globins are regulated independently, at least in this cell line. These data indicate that gamma-globin genes from normal, nonerythroid chromosomes are not irreversibly silenced, and they can be activated by a positive trans factor(s) present in GM979 cells.

Murine "housekeeping" enzyme (genetic locus: Idh-1) is regulated in an allele-specific manner.

The murine "housekeeping" enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C.1.1.1.42) (genetic locus: Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1 alpha allele, but did not vary as a function of sex. Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-homodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1 alpha allele. While cytosolic NADP-IDH is a "housekeeping" enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.

Effects of thyroid hormones on cardiac hypertrophy and beta-adrenergic receptors during aging.

Administration of thyroxine daily at a dosage of 6.4 mu g/g body weight stimulates cardiac hypertrophy in both 9--13-month-old (mature, n = 34) and 22--24-month-old (senescent, n = 45) Wistar rats. The increase due to thyroxine as well as the time course of the effect do not change with age, although ventricular wet weight to tibial length ratios are higher in senescent animals at 0, 3, and 7 days of treatment (senescent controls = 0.331; senescent hypertrophied = 0.395; mature controls = 0.295; mature hypertrophied = 0.336). Hypertrophy is maximal after 3 days. beta-Adrenergic receptor levels as measured by stereospecific binding of dihydroalprenolol in mature rats are increased about two-fold after 7 days of thyroxine treatment (mature controls = 35 +/- 3 fmol/mg protein, mature treated = 65 +/- 6 fmol/mg protein). Although both stimulated and control values do not differ significantly between mature and senescent groups (senescent controls = 45 +/- 4 fmol/mg protein, senescent treated = 56 +/- 9 fmol/mg protein), the effect of thyroxine on senescent hearts is not statistically significant. In addition, 3-day levels do not increase over 0-day controls at either age. No significant differences in either beta-adrenergic receptor concentrations or affinities between age groups are observed at 0, 3, and 7 days of treatment. However, senescent membrane preparations contain 33% more sialic acid (a rough plasma membrane marker) per unit of protein than mature preparations. Thus, the beta-adrenergic receptor density per unit of sialic acid may be reduced very slightly in the senescent hearts.

Contractile and biochemical correlates of beta-adrenergic stimulation of the aged heart.

The age dependence of contractile and associated biochemical parameters of basal- and catecholamine-stimulated myocardial contractile performance was investigated using isolated perfused septa from adult and senescent rats. Base-line maximum rate of force development (dF/dt), beta-receptor number and affinity, cAMP levels, and cAMP-dependent protein kinase activity were not different in the two age groups. During maximal isoproterenol stimulation, the increase in dF/dt was 40% less in the senescent hearts, and the cAMP levels and cAMP activation of protein kinase increased two fold but to the same extent in both age groups. The maximum contractile response to dibutyryl cAMP (DBcAMP) in the senescent was half that observed in the adult hearts. However, adult and senescent septa responded equally to an increase in perfusate [Ca2+] to 1.0 mM, which enhanced contractility to the same extent as that obtained with isoproterenol and DBcAMP in adult septa. These data taken together suggest that the factors that limit the contractile response to catecholamines in the senescent heart act subsequent to protein kinase activation but proximal to the Ca2+-troponin interaction.