Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.The a,-fetoprotein (AFP) gene, a member of the albumin gene family, is expressed by fetal or malignant hepatocytes and repressed in normal mature hepatocytes. This forms the basis of a long-exploited model system to study cell differentiation and its impairment in neoplasia (1). Molecular genetics have brought the AFP model to molecular levels of cell differentiation, toward finely discerning how genes respond to or escape from developmental and growth signals (2,40,41). As yet, little is known about how differential regulation is exerted on the tandemly organized AFPalbumin locus and whether the two genes operate under shared elements of control. However, at least one wellcharacterized transcription factor, the glucocorticoid receptor, is known to selectively shut off the AFP gene in the developing liver (4, 21). This hormonal effect reaches into the mechanisms of neoplastic resistance to differentiation, because the AFP gene in malignant cells is generally refractory to glucocorticoids (2).Analyses of rat AFP gene and chromatin structures have identified domains in the 5' region of the locus potentially involved in its liver-specific, developmental stage-dependent, and glucocorticoid-regulated expression. A promoter domain spanning '230 nucleotides comprises a chromatin DNase I-hypersensitive (DH) site (32, 42, 44) that is selectively suppressed by dexamethasone (44), glucocorticoid receptor recognition sequences, and octamer motifs similar to those present in other genes under developmental and growth control (8). A distal domain (-2 to -4 kilobases [kb] relative to the AFP trans...

Genetic analyses have revealed the essential role of the murine Hoxa5 gene for the correct specification of the cervical and upper thoracic region of the skeleton, and for the normal organogenesis and function of the respiratory tract, both structures expressing Hoxa5 during embryogenesis. To understand how the expression domains of the Hoxa5 gene are established during development, we have analyzed the cis‐acting control regions mediating Hoxa5 gene expression using a transgenic approach. Four transcripts are derived from the Hoxa5 locus. The shortest and most abundant one displays a specific spatio‐temporal profile of expression at earlier stages and in more anterior structures along the embryonic axis than the larger forms. We established that an 11.1 kilobase pair (kb) genomic fragment, extending from position −3.8 kb to +7.3 kb relative to Hoxa5 transcription initiation site, was sufficient to reproduce the temporal expression and substantially reconstitute the spatial pattern of the major Hoxa5 transcript. By deletion analyses, we identified a 2.1 kb fragment located downstream of the Hoxa5 gene that possesses mesodermal enhancer activity. Overall, the findings demonstrate that cis‐acting regulatory elements essential for the correct expression of the major Hoxa5 transcript are located both upstream and downstream of the Hoxa5 coding sequences. Dev Dyn 1999;214:127–140.  © 1999 Wiley‐Liss, Inc.

In an attempt to identify proteins that may regulate alpha 1-fetoprotein (AFP) gene expression, we screened a cDNA expression library from neonatal rat liver with two essential cis-elements of the AFP promoter and enhancer. We isolated two cDNAs which were found to correspond to leucine zipper proteins of the CC-AAT/enhancer binding protein (C/EBP) family: C/EBP beta and C/EBP gamma. The three related proteins C/EBP alpha, beta and gamma bind with indistinguishable specificity to multiple DNA sites in the promoter and the enhancer of the AFP gene. In addition, C/EBP beta and C/EBP gamma readily heterodimerize with each other as well as with C/EBP alpha. The mRNAs coding for C/EBP beta and C/EBP gamma are expressed in a wider variety of rat tissues than C/EBP alpha mRNA, including yolk sac and fetal liver. The steady-state levels of C/EBP alpha, beta and gamma mRNAs increase during liver development, in parallel with their respective gene transcriptional rates. The high levels of C/EBP beta and gamma mRNAs in rat yolk sac and fetal liver, where C/EBP alpha is poorly expressed, suggest that C/EBP beta and/or gamma could be preponderant or early regulators of the AFP gene in these tissues.