Position-dependent activity of α-fetoprotein enhancer element III in the adult liver is due to negative regulation

α-Fetoprotein (AFP) transcription is activated early in hepatogenesis, but is dramatically repressed within several weeks after birth. AFP regulation is governed by multiple elements including three enhancers termed EI, EII, and EIII. All three AFP enhancers continue to be active in the adult liver, where EI and EII exhibit high levels of activity in pericentral hepatocytes with a gradual reduction in activity in a pericentral-periportal direction. In contrast to these two enhancers, EIII activity is highly restricted to a layer of cells surrounding the central veins. To test models that could account for position-dependent EIII activity in the adult liver, we have analyzed transgenes in which AFP enhancers EII and EIII were linked together. Our results indicate that the activity of EIII is dominant over that of EII, indicating that EIII is a potent negative regulatory element in all hepatocytes except those encircling the central veins. We have localized this negative activity to a 340-bp fragment. This suggests that enhancer III may be involved in postnatal AFP repression.

Identification of an additional negative regulatory region for p53 sequence-specific DNA binding

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80–93 and 364–393. The C-terminal residues 364–393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80–93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80–93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80–93 and 364–393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.

Expression of a dominant negative TrkB receptor, T1, reveals a requirement for presynaptic signaling in BDNF-induced synaptic potentiation in cultured hippocampal neurons

We have developed a method to analyze the relative contributions of pre- and postsynaptic actions of a particular gene product in neurons in culture and potentially in slices using adenovirus-mediated gene transfer. A recombinant virus directed the expression of both a GFP reporter protein and TrkB.T1, a C-terminal truncated dominant negative TrkB neurotrophin receptor. When expressed in the presynaptic cell at synapses between embryonic hippocampal neurons in culture, the dominant negative TrkB.T1 inhibited two forms of synaptic potentiation induced by the neurotrophin brain-derived neurotrophic factor (BDNF): (i) greater evoked synaptic transmission and (ii) higher frequency of spontaneous miniature synaptic currents. These inhibition effects are not seen if the transgene is expressed only in the postsynaptic cell. We conclude that BDNF-TrkB signal transduction in the presynaptic terminal leads to both types of potentiation and is therefore the primary cause of synaptic enhancement by BDNF in these neurons.

The negative feedback actions of progesterone on gonadotropinreleasing hormone secretion are transduced by the classical progesterone receptor

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0–12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12–24 hr, P implant removed; 24–36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3α-hydroxy-5α-pregnan-20-one, which operates through the type A γ-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.

The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.

Two forms of replication initiator protein: Positive and negative controls

The pir gene of plasmid R6K encodes the protein, π, a replication and transcription factor. Two translational options for the pir gene give rise to two forms of π protein: a 35.0-kDa form (π35.0) and a shortened 30.5-kDa form (π30.5). Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only π35.0 can bind to a site in the (A⋅T)-rich segment of its γ ori and activate the γ ori in vivo and in vitro. However, unlike π35.0, π30.5can inhibit in vivo and in vitro replication (activated by π35.0). We propose that the two forms of π might have distinct functions in replication. We show that although both forms of π produce dimers, the nature of these dimers is not identical. The N-terminal 37 amino acid residues appear to control the formation of the more stable π35.0 dimers, whereas another, apparently weaker interface holds together dimers of π30.5. We speculate that the leucine zipper-like motif, absent in π30.5, controls very specific functions of π protein.

Negative genetic correlation for adult fitness between sexes reveals ontogenetic conflict in Drosophila

Because of their distinctive roles in reproduction, females and males are selected toward different optimal phenotypes. Ontogenetic conflict between the sexes arises when homologous traits are selected in different directions. The evolution of sexual dimorphism by sex-limited gene expression alleviates this problem. However, because the majority of genes are not sex-limited, the potential for substantial conflict may remain. Here we assess the degree of ontogenetic conflict in the fruit-fly, Drosophila melanogaster, by cloning 40 haploid genomes and measuring their Darwinian fitness in both sexes. The intersexual genetic correlations for juvenile viability, adult reproductive success, and total fitness were used to gauge potential conflict during development. First, as juveniles, where the fitness objectives of the two sexes appear to be similar, survival was strongly positively correlated across sexes. Second, after adult maturation, where gender roles diverge, a significant negative correlation for reproductive success was found. Finally, because of counterbalancing correlations in the juvenile and adult components, no intersexual correlation for total fitness was found. Highly significant genotype-by-gender interaction variance was measured for both adult and total fitness. These results demonstrate strong intersexual discord during development because of the expression of sexually antagonistic variation.

Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells

We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.

RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit

The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t1/2 ≈2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4Δ cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription–activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.