Different combinations of regulatory elements may explain why placenta-specific expression of the glycoprotein hormone alpha-subunit gene occurs only in primates and horses.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitary of all mammals but in placenta of only primates and horses. In humans, two different elements, termed upstream regulatory element (URE) and cAMP response element (CRE), are required for placenta-specific expression of the alpha-subunit gene. The URE binds a protein unique to placenta whereas the CRE binds a ubiquitous protein. Comparative analysis of the promoter-regulatory region of the alpha-subunit gene from a number of mammals indicates that a functional URE has been retained and suggests the potential for placenta-specific expression. Indirect evidence also indicates that the URE-binding protein has been conserved, even in placenta from mammals that fail to express the alpha-subunit gene. Lack of expression of the alpha-subunit gene in placenta of rodents and cattle can be traced to a single nucleotide change that renders the CRE-like sequence of these genes incapable of binding the protein that confers responsiveness to cAMP. In contrast, although expression of the alpha-subunit gene occurs in horse placenta, the promoter-regulatory region lacks a functional CRE but appears to retain a functional URE. This suggests that either a different accessory element and cognate protein interacts with the horse URE to provide placenta-specific expression or that a completely different set of regulatory elements is required for placenta-specific expression in horses.     

Different combinations of regulatory elements may account for expression of the glycoprotein hormone alpha-subunit gene in primate and horse placenta.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitaries of all mammals and in the placentas of primates and horses. In humans, tandem cAMP response elements (CREs), located in the proximal promoter-regulatory region of the alpha-subunit gene, act together with an adjacent upstream regulatory element to confer placenta-specific expression. Here, we report that the alpha-subunit genes of Old World Monkeys contain a single functional CRE. This suggests that tandem CREs are unique to higher primates and humans and are not absolutely required for placenta-specific expression. In contrast, the comparable promoter-regulatory region of the horse alpha-subunit gene lacks a functional CRE but appears to retain a functional upstream regulatory element. This suggests that acquisition of placenta-specific expression of the alpha-subunit gene occurred independently in these distantly related mammals. As a result, different combinations of cis-acting elements may explain why expression of the alpha-subunit gene only occurs in placenta of primates and horses.     

Activation of fibroblast procollagen alpha 1(I) transcription by mechanical strain is transforming growth factor-beta-dependent and involves increased binding of CCAAT-binding factor (CBF/NF-Y) at the proximal promoter.

During normal developmental tissue growth and in a number of diseases of the cardiopulmonary system, adventitial and interstitial fibroblasts are subjected to increased mechanical strain. This leads to fibroblast activation and enhanced collagen synthesis, but the underlying mechanisms involved remain poorly understood. In this study, we have begun to identify and characterize mechanical strain-responsive elements in the rat procollagen alpha 1(I) (COL1A1) gene and show that the activity of COL1A1 promoter constructs, transiently transfected into cardiac fibroblasts, was increased between 2- and 4-fold by continuous cyclic mechanical strain. This was accompanied by an approximately 3-fold increase in the levels of total active transforming growth factor-beta (TGF-beta) released into the medium. Inclusion of a pan-specific TGF-beta neutralizing antibody inhibited strain-induced COL1A1 promoter activation. Deletion analysis revealed the presence of two potential strain response regions within the proximal promoter, one of which contains an inverted CCAAT-box overlapping a GC-rich element. Both mechanical strain and exogenously added TGF-beta1 enhanced the binding activity of CCAAT-binding factor, CBF/NF-Y, at this site. Moreover, this element was sufficient to confer strain-responsiveness to an otherwise unresponsive SV40 promoter. In summary, this study demonstrates that strain-induced COL1A1 promoter activation in cardiac fibroblasts is TGF-beta-dependent and involves increased binding of CCAAT-binding factor at the proximal promoter. Furthermore, these findings suggest a novel and potentially important TGF-beta response element in the rat COL1A1 gene.     

PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells.

The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.     

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.     

Stable expression of a dominant negative mutant of CCAAT binding factor/NF-Y in mouse fibroblast cells resulting in retardation of cell growth and inhibition of transcription of various cellular genes

The heterotrimeric CCAAT-binding factor CBF specifically interacts with the CCAAT motif present in the proximal promoters of numerous mammalian genes. To understand the in vivo function of CBF, a dominant negative mutant of CBF-B subunit that inhibits DNA binding of wild type CBF was stably expressed in mouse fibroblast cells under control of tetracycline-responsive promoter. Expression of the mutant CBF-B but not the wild-type CBF-B resulted in retardation of fibroblast cell growth. The analysis of cell growth using bromodeoxyuridine labeling showed that expression of the mutant CBF-B decreased the number of cells entering into S phase, and also delayed induction of S phase in the quiescent cells after serum stimulation, thus indicating that the inhibition of CBF binding prolonged the progression of S phase in fibroblasts. These results provide direct evidence for the first time that CBF is an important regulator of fibroblast growth. The inhibition of CBF binding reduced expression of various cellular genes including the alpha2(1) collagen, E2F1, and topoisomerase IIalpha genes which promoters contain the CBF-binding site. This result implied that expression of many other genes which promoters contain CBF-binding site was also decreased by the inhibition of CBF binding, and that the decreased expression of multiple cellular genes possibly caused the retardation of fibroblast cell growth.