Stephen A. Liebhaber

Pit-1 binding sites at the somatotrope-specific DNase I hypersensitive sites I, II of the human growth hormone locus control region are essential for in vivo hGH-N gene activation.

The human growth hormone gene cluster is composed of five closely related genes. The 5′-most gene in the cluster,hGH-N, is expressed exclusively in somatotropes and lactosomatotropes of the anterior pituitary. Although thehGH-N promoter contains functional binding sites for multiple transcription factors, including Sp1, Zn-15, and Pit-1, predictable and developmentally appropriate expression ofhGH-N transgenes in the mouse pituitary requires the presence of a previously characterized locus control region (LCR) composed of multiple chromatin DNase I hypersensitive sites (HS). LCR determinant(s) necessary for hGH-N transgene activation are largely conferred by two closely spaced HS (HS I,II) located 14.5 kilobase pairs upstream of the hGH-N gene. The region sufficient to mediate this activity has recently been sublocalized to a 404-base pair segment of HS I,II (F14 segment). In the present study, we identified multiple binding sites for the pituitary POU domain transcription factor Pit-1 within this segment. Using a transgenic founder assay, these sites were shown to be required for high level, position-independent, and somatotrope-specific expression of a linkedhGH-N transgene. Because the Pit-1 sites in thehGH-N gene promoter are insufficient for such gene activation in vivo, these data suggested a unique chromatin-mediated developmental role for Pit-1 in the hGH LCR.

Characterization of a human cDNA encoding a widely expressed and highly conserved cysteine-rich protein with an unusual zinc-finger motif

A human term placental cDNA library was screened at low stringency with a human prolactin cDNA probe. One of the cDNAs isolated hybridizes to a 1.8 kb mRNA present in all four tissues of the placenta as well as to every nucleated tissue and cell line tested. The sequence of the full-length cDNA was determined. An extended open reading frame predicted an encoded protein product of 20.5 kDa. This was directly confirmed by the in vitro translation of a synthetic mRNA transcript. Based upon the characteristic placement of cysteine (C) and histidine (H) residues in the predicted protein structure, this molecule contains four putative zinc fingers. The first and third fingers are of the C4 class while the second and fourth are of the C2HC class. Based upon sequence similarities between the first two and last two zinc fingers and sequence similarities to a related rodent protein, cysteine-rich intestinal protein (CRIP), these four finger domains appear to have evolved by duplication of a preexisting two finger unit. Southern blot analyses indicate that this human cysteine-rich protein (hCRP) gene has been highly conserved over the span of evolution from yeast to man. The characteristics of this protein suggest that it serves a fundamental role in cellular function.

129X1/SvJ Mouse Strain Has a Novel Defect in Inflammatory Cell Recruitment

Vitamin D-binding protein (DBP) has been reported to contribute to innate immunity. To verify prior in vitro and cell-based observations supporting this role, we assessed the ability of a recently developed DBP-null mouse line to recruit neutrophils and macrophages to a site of chemical inflammation. The interrupted DBP allele had been generated by homologous recombination in 129X1/SvJ embryonic stem cells and these cells were subsequently used to generate a line of DBP−/− (null) mice. Initial studies revealed a marked defect in the ability of these DBP−/− mice to recruit cells to the peritoneum after localized thioglycolate injection. However, progressive outcrossing of the DBP−/− mice to the C57BL/6J strain, conducted to provide a uniform genetic background for comparison of DBP-null and control mice, resulted in a progressive increase in cell recruitment by the DBP−/− mice and a loss in their apparent recruitment defect when compared with the DPB wild-type controls. These data suggested that the observed recruitment phenotype initially attributed to the absence of DBP was not linked to the DBP locus, but instead reflected the underlying genetic composition of the 129X1/SvJ ES cells used for the initial DBP gene disruption. A profound cell recruitment defect was confirmed in the 129X1/SvJ mice by direct analysis. Each of three commonly used inbred lines was discovered to have a distinct level of cell recruitment to a uniform stimulus (C57BL/6J > BALB/c > CD1 > 129X1/SvJ). Thus, this study failed to support a unique role for DBP in cellular recruitment during a model inflammatory response. Instead, the data revealed a novel and profound defect of cell recruitment in 129X1/SvJ mice, the strain most commonly used for gene deletion studies.

Molecular Biology of the Growth Hormone-Prolactin Gene System

Publisher Summary Both have assumed distinct physiological profiles. The expression of each depends on appropriate developmental differentiation of somatotrope and lactotrope cells in the anterior pituitary and of syncytiotrophoblasts in the placenta. Expression of these hormones is controlled primarily at the level of gene transcription. Alternative processing of the GH and Prl RNA transcripts increases the complexity of the encoded protein products. Physiological functions of GH and Prl depend on expression of appropriate cognate receptors on the surfaces of target tissues. The GH and Prl receptors are expressed in a wide range of tissues, and the transcripts are subject to alternative RNA processing. Naturally occurring mutations have been identified that interfere with differentiation of somatotropes and lactotropes that specifically block expression of the GH gene, or that block expression of fuctional GH receptors. The phenotype for Prl deficiency has not yet been described, although children deficient in Prl gene expression have been identified. Thus, the physiological, biochemical, and genetic studies on the GH and Prl gene system have generated an interlocking set of data that forms a firm foundation for future investigations and serves as a prototype for studies of related systems.

VITAMIN D-BINDING PROTEIN GENE TRANSCRIPTION IS REGULATED BY THE RELATIVE ABUNDANCE OF HEPATOCYTE NUCLEAR FACTORS 1ALPHA AND 1BETA

Vitamin D-binding protein (DBP)/Gc-globulin, the major carrier of vitamin D and its metabolites in blood, is synthesized predominantly in the liver in a developmentally regulated fashion. By transient transfection analysis, we identified three regions in the 5′-flanking region of the rat DBP gene, segments F-2, B, and A, that contain tissue-specific transcriptional determinants. Gel mobility shift and DNase I footprinting analyses showed that all three regions contained binding sites for the hepatocyte nuclear factor 1 (HNF1), a transcriptional regulator composed of HNF1α and HNF1β hetero- and homodimers. The activity of the most proximal segment A (coordinates −141 to −43) was DBP promoter-specific, position-dependent, and positively controlled by HNF1α. In contrast, the two more distal determinants (segments F-2 and B; coordinates −1844 to −1621 and −254 to −140, respectively) acted as classical enhancers in transfected hepatocyte-derived HepG2 cells; their activities were promoter- and orientation-independent, and disruption of their respective HNF1-binding sites resulted in marked loss of DBP gene expression. Remarkably, the activities of these two distal elements depended upon the relative levels of HNF1α and HNF1β; HNF1α had a major stimulatory effect, whereas HNF1β acted as a trans-dominant inhibitor of HNF1α-mediated enhancer activity. These results suggested that the net expression of the DBP gene reflected a balance between the two major HNF1 species; the relative abundance of HNF1α and HNF1β proteins in a cell may thus play a critical role in determining the pattern of gene expression.

Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Human α-globin gene expression is silenced by terminal truncation of chromosome 16p beginning immediately 3′ of the ζ-globin gene

SummaryThe high level expression of the human α globin genes in erythroid tissue appears to require a set of DNaseI hypersensitive sites located upstream of the human α-globin gene cluster. These sequences, termed the locus control region (LCR), include two erythroid specific and a number of less restricted DNaseI hypersensitive sites. In this report we describe an individual with α-thalassemia associated with a truncation of the short arm of chromosome 16 that removes the LCR region and inactivates the adjacent intact α-globin genes. This genetic study supports the critical role of the LCR in the transcriptional activation of the human α-globin gene cluster and substantiates the importance of LCR deletions in the etiology of α-thalassemia.

High-level production of human α- and β-globins in insect cells

High-level production of hunan α - and β -globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70–140 mg protein/liter of cell culture or 5–10% of the total cellular protein. Two recombinant baculoviruses for α -globin, H α and H βα , differ in their construction in that the 5′-untranslated region of the β -globin gene is inserted 5′ to the α -globin mRNA coding region in H βα . This insertion results in a 40% increase in yield of α -globin over that of H α . Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both α - and β -globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.

Gestational Physiology of the Growth Hormone Gene Family

The human growth hormone (hGH) gene family, located in a cluster spanning 48 kb on chromosome 17q22–24 (1), contains 5 genes: the pituitary GH gene (hGH-N) and 4 placentally expressed genes, chorionic somatomammotropin-like (hCS-L), hCS-A, hGH-variant (hGH-V), and hCS-B (2). These genes evolved by a series of duplication events, the most ancient one giving rise to the distantly related and unlinked (3) single prolactin (PRL) gene (4, 5). The linked GH and CS genes, presumed to be generated by 3 recent duplication events, now share >90% nucleotide sequence identity. These genes are expressed in a highly tissue-specific manner; hGH-N is expressed solely in pituitary somatotropes/somatolactotropes, and the hCS and hGH-V genes are expressed exclusively in the syncytiotrophoblastic layer of the placenta (6, 7). In contrast, hPRL is expressed in both pituitary lactotropes/somatolactotropes and placental decidua (8).